Metrics details Piwi-interacting RNAs (piRNAs) are essential for maintaining genome integrity and fertility in various organisms piRNA genes are encoded in heterochromatinized genomic clusters The molecular mechanisms of piRNA transcription remain intriguing Through small RNA sequencing and chromatin editing we discovered that spatial aggregation of piRNA genes enhances their transcription in nematodes The facultative heterochromatinized piRNA genome recruits the piRNA upstream sequence transcription complex (USTC; including PRDE-1 TOFU-4 and TOFU-5) and the H3K27me3 reader UAD-2 which phase-separate into droplets to initiate piRNA transcription We searched for factors that regulate piRNA transcription and isolated the SUMO E3 ligase GEI-17 as inhibiting and the SUMO protease TOFU-3 as promoting piRNA transcription foci formation Our study revealed that spatial aggregation of piRNA genes phase separation and deSUMOylation may benefit the organization of functional biomolecular condensates to direct piRNA transcription in the facultative heterochromatinized genome Prices may be subject to local taxes which are calculated during checkout A distinct small RNA pathway silences selfish genetic elements in the germline A novel class of small RNAs bind to MILI protein in mouse testes A germline-specific class of small RNAs binds mammalian Piwi proteins Characterization of the piRNA complex from rat testes A novel class of small RNAs in mouse spermatogenic cells piRNAs can trigger a multigenerational epigenetic memory in the germline of C and evolution of Caenorhabditis elegans piRNAs Emerging roles and functional mechanisms of Piwi-interacting RNAs Intrinsically disordered proteins in cellular signalling and regulation PRDE-1 is a nuclear factor essential for the biogenesis of Ruby motif-dependent piRNAs in C CapSeq and CIP-TAP identify Pol II start sites and reveal capped small RNAs as C Promoters recognized by forkhead proteins exist for individual 21U-RNAs Comparative epigenomics reveals that RNA polymerase II pausing and chromatin domain organization control nematode piRNA biogenesis The USTC co-opts an ancient machinery to drive piRNA transcription in C elegans SNAPc component SNPC-4 coats piRNA domains and is globally required for piRNA abundance A chromodomain protein mediates heterochromatin-directed piRNA expression Casein kinase II promotes piRNA production through direct phosphorylation of USTC component TOFU-4 The upstream sequence transcription complex dictates nucleosome positioning and promoter accessibility at piRNA genes in the C piRNA production requires heterochromatin formation in Drosophila Heterochromatin-dependent transcription of satellite DNAs in the Drosophila melanogaster female germline Co-dependent assembly of Drosophila piRNA precursor complexes and piRNA cluster heterochromatin A heterochromatin-specific RNA export pathway facilitates piRNA production A heterochromatin-dependent transcription machinery drives piRNA expression Transgenerationally inherited piRNAs trigger piRNA biogenesis by changing the chromatin of piRNA clusters and inducing precursor processing The Rhino–Deadlock–Cutoff complex licenses noncanonical transcription of dual-strand piRNA clusters in Drosophila The Drosophila HP1 homolog Rhino is required for transposon silencing and piRNA production by dual-strand clusters Specialization of the Drosophila nuclear export family protein Nxf3 for piRNA precursor export The nucleolus as a multiphase liquid condensate Stress granule assembly is mediated by prion-like aggregation of TIA-1 Proteomic analysis of interchromatin granule clusters Liquid phase condensation in cell physiology and disease Biomolecular condensates: organizers of cellular biochemistry Biomolecular phase separation in stress granule assembly and virus infection Sequence variations of phase-separating proteins and resources for studying biomolecular condensates A phase separation model for transcriptional control Phase separation of TAZ compartmentalizes the transcription machinery to promote gene expression Phase separation in gene transcription control PML nuclear bodies: from architecture to function Functional domains of NEAT1 architectural lncRNA induce paraspeckle assembly through phase separation O.Signalling mechanisms and cellular functions of SUMO SUMO: glue or solvent for phase-separated ribonucleoprotein complexes and molecular condensates Compositional control of phase-separated cellular bodies Sumoylation of the human histone H4 tail inhibits p300-mediated transcription by RNA polymerase II in cellular extracts Protein SUMOylation and phase separation: partners in stress Panoramix SUMOylation on chromatin connects the piRNA pathway to the cellular heterochromatin machinery HDAC1 SUMOylation promotes Argonaute-directed transcriptional silencing in C PIE-1 SUMOylation promotes germline fates and piRNA-dependent silencing in C The SUMO Ligase Su(var)2-10 controls hetero- and euchromatic gene expression via establishing H3K9 trimethylation and negative feedback regulation Su(var)2-10 and the SUMO pathway link piRNA-guided target recognition to chromatin silencing Large-scale sequencing reveals 21U-RNAs and additional microRNAs and endogenoussiRNAs in C Assembly of the synaptonemal complex is a highly temperature-sensitive process that is supported by PGL-1 during Caenorhabditis elegans meiosis SNPC-1.3 is a sex-specific transcription factor that drives male piRNA expression in C piRNAs initiate transcriptional silencing of spermatogenic genes during C Reprogramming the piRNA pathway for multiplexed and transgenerational gene silencing in C Dual sgRNA-directed gene knockout using CRISPR/Cas9 technology in Caenorhabditis elegans rRNA intermediates coordinate the formation of nucleolar vacuoles in C A genome-wide RNAi screen identifies factors required for distinct stages of C Dynamic reorganization of the genome shapes the recombination landscape in meiotic prophase The mechanisms of PML-nuclear body formation Targeted chromosomal rearrangements via combinatorial use of CRISPR/Cas9 and Cre/LoxP technologies in Caenorhabditis elegans RdRP-synthesized antisense ribosomal siRNAs silence pre-rRNA via the nuclear RNAi pathway Ingestion of bacterially expressed dsRNAs can produce specific and potent genetic interference in Caenorhabditis elegans Systematic characterization of chromodomain proteins reveals an H3K9me1/2 reader regulating aging in C Download references elegans Gene Knockout Consortium and the National Bioresource Project for providing the strains Some strains were provided by the Caenorhabditis Genetics Center which is funded by the National Institutes of Health Office of Research Infrastructure Programs (P40 OD010440) This work was supported by grants from the National Natural Science Foundation of China (32230016 to S.G. and 32090044 and 31971128 to S.X.) and the National Key R&D Program of China (2022YFA1302700 to S.G This work was supported by the Research Funds of Center for Advanced Interdisciplinary Science and Biomedicine of IHM (QYPY20230021 to S.G.) This study was supported in part by the Fundamental Research Funds for the Central Universities This work was also supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (CAS; XDB 37040202 to S.X.) and the CAS Project for Young Scientists in Basic Research (YSBR-068 to S.X.) These authors contributed equally: Chengming Zhu Ministry of Education Key Laboratory for Membraneless Organelles & Cellular Dynamics Hefei National Research Center for Physical Sciences at the Microscale Center for Advanced Interdisciplinary Science and Biomedicine of IHM University of Science and Technology of China CAS Center for Excellence in Molecular Cell Science contributed analytic tools and performed the bioinformatics analysis The authors declare no competing interests Nature Structural & Molecular Biology thanks Wen Tang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available in collaboration with the Nature Structural & Molecular Biology team Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Images showing the subcellular localization of TOFU-4::GFP and LacI::tagRFP in the nuclei of pachytene germ cells from wild-type animals Images showing the subcellular localization of UAD-2::GFP and LacI::tagRFP in the nuclei of pachytene germ cells from animals with 256xLacO insertions Images showing the subcellular localization of TOFU-4::GFP and LacI::tagRFP in the nuclei of pachytene germ cells from animals with 256xLacO insertions Images displaying the subcellular localization of UAD-2 and USTC components alongside the chromatin marker mCherry::H2B in the spermiogenesis region of the germline in L4 stage hermaphrodite animals Images displaying the subcellular localization of UAD-2 and USTC components alongside the chromatin marker mCherry::H2B of the germline in adult male animals Heatmap of ChIP-seq binding profiles of UAD-2 around type I and type II piRNA transcription start sites (TSSs) Heatmap of ChIP-seq binding profiles of TOFU-4 around type I and type II piRNA transcription start sites (TSSs) Heatmap of ChIP-seq binding profiles of TOFU-5 around type I and type II piRNA transcription start sites (TSSs) Genome-wide distribution of piRNA genes (n = 25883) in C Boxplots presenting log2(mean of piRNA reads from two biological replicates) for Cbr-PRG-1-associated piRNAs across Cluster 1 The box itself represented the interquartile range between 5% and 95% thereby encompassing the middle 90% of the data set The central horizontal line and the adjacent numeric value within the box represent the mean value The star within the box represents the median value Error bars represent the mean ± 1.5 standard deviations (SD) indicating the variability of the data around the mean Outliers are individually marked as points representing data points that fall outside the range of mean±1.5 SD Statistical significance was determined using a two-sample t test Graphs presenting the fractional distribution of piRNA genes and their corresponding expression reads (normalized as reads per million from two biological replicates) across Cluster 1 Source data are provided as a Source Data file Source data Diagram showing sequence details of intrachromosomal inversion in LG IV Graphs presenting the expression levels of total piRNAs from Cluster I of the indicated animals based on average reads from two biological replicates Scatter plots displaying the expression levels of Cluster I and out-Cluster piRNAs in the wild-type (x-axis) and inversion strain (y-axis) based on the log2(average piRNA reads of two biological replicates) Boxplot revealing log2(average piRNA reads of two biological replicates) for both the wild-type and inversion strains across Cluster I Statistical significance was determined using a paired-sample t test Statistical analysis of the focus area for UAD-2::GFP and TOFU-5::GFP in pachytene cells Quantification of size data from n = 3 independent animals error bars represent the mean ± 1.5 standard deviations (SD) The images reflect the subcellular localization of the mentioned proteins in pachytene cells under the indicated culture conditions Images displaying the subcellular localization of PRDE-1::GFP and the chromatin marker mCherry::H2B in the germline from wild-type animals and uad-2(ust200) mutants and out-Cluster piRNAs in the wild type (x-axis) and uad-2(ust200) mutants (y-axis) based on the log2(reads per million+1) from a single biological replicate Boxplots revealing total piRNA reads per million of the indicated adult animals from a single biological replicate Boxplots revealing log2(piRNA reads per million+1) of the indicated adult animals form a single biological replicate Graphs presenting the relative fluorescence unit intensity (RFU) of mCherry::PRDE-1 for wild-type animals and the indicated UAD-2 variants The fluorescence intensity threshold is set to 8–255 Graphs presenting the relative fluorescence unit intensity (RFU) of wild-type animals and the indicated UAD-2 variants Images showing the subcellular localization of the indicated UAD-2::GFP variants and TOFU-4:mCherry in pachytene cells Scatter plots displaying the second biological replicate expression levels of Cluster I and out-Cluster piRNAs in the wild-type (x-axis) and indicated mutants (y-axis) Boxplots revealing log2(piRNA reads per million+1) of the indicated adult animals of the second biological replicate Boxplots illustrating log2(pre-piRNA reads per million+1) for WT worms and gei-17 mutants at 25 °C Images displaying GFP::FLAG::Degron::GEI-17 (green) and mCherry::H2B (magenta) in embryos and larval animals Complementation test of two alleles of tofu-3 at 20 °C Images of pachytene cells of the indicated adult animals grown at 20 °C mCherry::TOFU-3 rescued UAD-2::GFP foci formation in tofu-3(tm3068) cells Sequence alignment of the TOFU-3 (ULP-5) protein Subcellular localization of UAD-2::GFP in pachytene cells in eri-1(mg366);UAD-2::GFP worms fed the indicated RNAi bacteria Deep sequencing of total small RNAs of the indicated adult animals from one biological replicate Scatter plots depicting the expression levels of Cluster I and out-Cluster piRNAs in the wild-type (x-axis) and tofu-3 mutant strains (y-axis) from one biological replicate Expression levels of total piRNAs from LG IV of the indicated adult animals cultured at 20 °C normalized as reads per million+1 from one biological replicate Subcellular localization of UAD-2::GFP and mCherry::H2B in pachytene cells from WT tofu-3(ust235) and tofu-3(tm3068) mutant worms qRT-PCR analysis of tofu-3 mRNA in wild-type animals and mCherry::TOFU-3 animals Quantification of qRT-PCR data from n = 3 independent animals Boxplots illustrating log2(pre-piRNA reads per million+1) for wild-type worms and mCherry::TOFU-3 animals at 25 °C from one biological replicate Fluorescence recovery after photobleaching (FRAP) of UAD-2::GFP under L4440 and smo-1 RNAi was conducted using a Zeiss LSM980 confocal microscope All images are representative of 6 animals Graphs presenting the relative fluorescence unit intensity (RFU) of the control area and bleached area of UAD-2 under L4440 and smo-1 RNAi Quantification of FRAP data from n = 6 independent animals Images showing germline nuclei expressing UAD-2::GFP the germline was imaged within 5 minutes using a Leica Thunder imaging system Germlines were treated with 10% 1,6-hexanediol to prohibit phase separation Sequences of sgRNAs for CRISPR–Cas9-mediated gene editing and primers for RT–qPCR a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such 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Volume 12 - 2024 | https://doi.org/10.3389/fcell.2024.1495035 PIWI-interacting RNAs (piRNAs) are small non-coding RNAs that bind to the PIWI subclass of the Argonaute protein family and are essential for maintaining germline integrity crucial for regulating gene expression and genome stability by suppressing transposable elements (TEs) Recent insights revealed that piRNAs and PIWI proteins known for their roles in germline maintenance translation and retrotransposon silencing in both stem cells and bodily tissues we explore the multifaceted roles of piRNAs and PIWI proteins in numerous biological contexts emphasizing their involvement in stem cell maintenance we discussed the up-and-coming animal models beyond the classical fruit fly and earthworm systems for studying piRNA-PIWIs in self-renewal and cell differentiation our review offers new insights and discusses the emerging roles of piRNA-dependent and independent functions of PIWI proteins in the soma especially the mRNA regulation at the post-transcriptional level and cardiovascular and neurodegenerative diseases PIWI proteins and their functional domains in the piRNA-PIWI complex and the overview of the piRNA pathway in Drosophila (A) Phylogenetic tree of the Argonaute (AGO) protein family with each class of AGOs specific to the type of small non-coding RNAs (B) (left) The MID domain of PIWI proteins binds to the 5′phosphate group of piRNA (shown in blue) and the PAZ domain binds to the 2′-O-methylated 3′end of piRNA (right) The target RNA is captured by piRNA through base-pair complementarity (C) The piRNA biogenesis in Drosophila: First the piRNA cluster on the chromosome is transcribed by the RNA Pol II to produce piRNA precursors with the assistance of proteins The piRNA precursors are transported out of the nucleus by proteins such as Nxf3 and Bootlegger (Boot) and their secondary structure is removed by Armitage (Armi) and cut by Zucchini to form piRNA intermediates after being trimmed by Nibbler (Nbr) and methylated at the 3′end by Hen1 bind to Piwi (PIWIL1 homolog) to form a Piwi-piRNA complex (i) Other piRNA intermediates bind to Aub (PIWIL2 homolog) and then enter the ping-pong cycle dominated by Aub and Ago3 (PIWI4 homolog) (ii) silences target RNAs (such as TE transcripts) and amplifies piRNAs transcribed by RNA Pol II is exported from the nucleus to the cell cytoplasm the mature piRNA cleaves the target TE transcript act as a template and facilitate the processing of the piRNA precursor bound by the Aub protein and cleaved with the help of the protein Trimmer enters the cycle to continue the silencing of complementary transcripts and the generation of piRNAs Nomenclature of the PIWI proteins in different organisms and the auxiliary proteins involved in this cycle are yet to be confirmed we explored the roles of piRNAs and PIWI proteins in stem cells other than germline we discussed the emerging roles of piRNA and PIWI proteins both dependent and independent of each other in regulating the expression of TE transcripts and mRNAs along with their translation in somatic cells and the development of numerous human diseases The functions of PIWI proteins in planarian adult pluripotent stem cells (aPSCs) and mouse adult neural progenitor cells (aNPCs) The SMEDWI-2 complex binds to TEs that are being transcribed For other TE transcripts that successfully enter the cytoplasm the SMEDWI-1 complex silences them through the ping-pong cycle poorly translated transcripts are recognized and silenced by the SMEDWI-1 complex facilitated by its interaction with the small ribosomal subunit (B) Knockdown of DNAJA1 in neoblasts reduced the abundance of SMEDWI-2 protein but had little effect on the expression levels of SMEDWI-2 transcripts (C) The effects of SMEDWI-1 and SMEDWI-3 on mRNA in neoblasts: (i) SMEDWI-1 and SMEDWI-3 assist germinal histone H4 (gH4) transcripts in localizing to chromatoid bodies cleave mRNAs that code for all the histone proteins The cleaved mRNAs become templates for the newly generated piRNA-SMEDWI-1 complexes (iii) The piRNA-SMEDWI-3 complexes bind other mRNAs no changes were observed in the mRNA levels and this association’s outcome is yet to be determined (denoted as ?) (D) The piRNA-MILI complex is involved in the preservation of mouse aNPC pluripotency (left) Artificial knockout of MILI causes aNPCs to differentiate into unhealthy astrocytes (right) The piRNA-MILI complex inhibits protein synthesis in aNPCs by silencing tRNA SINEB1 and mRNAs encoding ribosomal proteins thereby slowing down their differentiation piRNA-PIWI-mediated mRNA regulation in Drosophila adult stem cells (A) Aub activating mRNA translation in the early Drosophila embryo: The piRNA-Aub complex binds to complementary regions within mRNAs bound by the poly(A)-binding protein Wispy thereby initiating the process of translation (B) Role of JAK/STAT pathway and PIWI proteins in Drosophila ISCs: The JAK/STAT pathway activates ISC proliferation and induces the expression of Drosophila Piwi protein which silences transposons and ensures gene stability Knocking down Piwi led to the accumulation of multiple TE transcripts knocking down or deleting PIWIL1 did not impact transposon transcript levels indicating that the piRNA pathway was not functional in this setting These findings highlight the importance of careful analysis to confirm true piRNA activity and avoid misinterpreting aberrant gene expression in cancer piRNA-specific and independent function of PIWIs in stem cell differentiation (A) PIWIL1 overexpression in glioblastoma stem cells promotes self-renewal and tumorigenic potential Knockdown of PIWIL1 upregulates tumor suppressors inhibiting GSC self-renewal and proliferation while promoting differentiation and senescence (B) piRNAs in the differentiation of BMSCs: piR-36741 promotes the differentiation of BMSCs into osteoblasts (C) piRNAs in exosomes/microvesicles have anti-viral roles Mouse NSCs excrete Ex/Mvs carrying piRNAs out of the cells via exocytosis These Ex/Mvs specific piRNAs bind and degrade target viral RNAs of HIV and SARS-Cov-2 its role in the piRNA-guided antiviral functions is yet to be demonstrated (denoted as ?) piRNA and PIWI functions in emerging models of adult stem cells in animals the SMEDWI-1-piRNA complex binds to the small ribosome subunit to cleave rRNAs The cleaved RNA fragments will form new SMEDWI-1-piRNA complexes and continue to participate in target RNA cleavage the expression of PIWI-1 and TSPAN-1 has been detected to increase simultaneously the connection between them has not been fully established in terms of their expression changes and the downstream effects on neoblast differentiation into a variety of progenitor cells (C) High piwi-1 expression was detected during the differentiation of neoblasts expressing the histone variant H3.3 Tfap2 and Piwi-1 jointly induce i-cells to differentiate into germ cells with little information known in terms of how Piwi-1 controls Tfap2 expression We will provide a comparable and contrasting view of piRNA functions and PIWI proteins in TE silencing and mRNA regulation in the soma PIWI proteins form a complex with piRNAs, namely piRNA-induced silencing complex (piRISC), to mediate both transcriptional and post-transcriptional gene silencing (Loubalova et al., 2023). Not all PIWI proteins operate similarly or at the same level in the regulation of gene expression. While MILI/PIWIL2 and MIWI/PIWIL1 are involved at the post-transcriptional level, MIWI2/PIWIL4 primarily engages in transcriptional gene silencing (Loubalova et al., 2023) we will specifically discuss the role of the piRNA-PIWI complexes in the regulation of mRNAs and retrotransposon silencing at the post-transcriptional level These interactions ensure effective TE silencing in somatic tissues demonstrating the piRNA pathway’s essential role in protecting the genome beyond germ cells piRNA-PIWI-mediated regulation of mating-induced germline hyperactivity and somatic collapse in the aging of C (i) Mating initiates hyperactivity in the germline (ii) This germline hyperactivity leads to the downregulation of piRNAs (iii) The downregulation of piRNAs results in the de-silencing and activation of Hedgehog-like ligands (iv) The activation of these Hedgehog-like ligands enhances Hedgehog signalling which impacts somatic cells through specific receptors (v) The increased Hedgehog signalling in somatic cells triggers a cascade of events leading to somatic collapse characterized by a decline in somatic cell function and health ultimately accelerating aging and reducing lifespan The independent roles of PIWI proteins outside their traditional association with piRNAs have been explored mainly in the context of human diseases. The expression and function of PIWIs and potential piRNAs along with the downstream targets responsible for the disease phenotype are detailed in Table 3 their expression and function in human diseases This underscores the complexity of distinguishing bona fide piRNAs from other small RNAs in somatic tissues and the importance of robust in silico analysis for accurate identification Such methodological precision is critical for understanding the diverse roles of piRNAs in diseases as their functions extend beyond canonical germline processes to influence mRNA regulation and transposon silencing in somatic contexts (A) (i) PIWIL1 downregulates tumor suppressor mRNAs (VCL TPM2) by recruiting the NMD machinery (SMG1 (ii) PIWIL1’s role in upregulating oncogene mRNAs (CCND3 is associated with the progression of hepatocellular carcinoma (HCC) (ii) Downregulation of PIWIL1/HIWI along with piR-017724 prevents the development of HCC (C) PIWIL1/HIWI in pancreatic cancer: (i) The upregulation of piR-017061 the downregulation of piR-017061 resulted in the increased expression of EFNA5 an oncogene implicated in the progression of pancreatic cancer (D) PIWIL2 in lung cancer: PIWIL2 induces the expression of CDK2 and Cyclin A which inhibit apoptosis and prevent G2/M cycle arrest Inhibition of PIWIL2 leads to apoptosis and G2/M cycle arrest Dysregulated expression and functions of piRNA and PIWIs in other human diseases (A) Dysregulated piRNAs and PIWI proteins in cardiovascular neurodegenerative and respiratory tract diseases (B) piRNA HNEAP in cardiomyocyte necroptosis: (i) HNEAP recruits DNMT1 to Atf7 mRNA which results in increased Atf7 mRNA transcription and protein expression Whether HNEAP forms a complex with PIWIL2 and PIWIL4 is yet to be determined (denoted as ?) (ii) Elevated Atf7 expression inhibits the transcription of CHMP2A leading to increased cardiomyocyte necroptosis (iii) Knockdown of Atf7 reduces cardiomyocyte necroptosis induced by pathological stimuli such as hypoxia/reoxygenation (H/R) exposure. indicating Atf7’s role as a pro-necroptotic transcription factor We believe future studies on the mechanisms underlying the changes in PIWI expression and the targets namely protein-coding genes might shed light on the pivotal information necessary for developing accurate and effective piRNA-PIWI-based treatment strategies for ILD and TB miRNAs) to compensate for piRNA-PIWI contributions to maintaining gene expression in these cells Since their discovery in Drosophila, the majority of work on the piRNA pathway has been conducted in classical animal models, including C. elegans, and to some extent, in mice (Ernst et al., 2017; Kim et al., 2018; Huang and Wong, 2021; Ramat and Simonelig, 2021). Since piRNA sequence conservation is low across species (Parhad and Theurkauf, 2019) it is conceivable that piRNA function in gene silencing varies across model systems that facilitate the study of stem cell development and the pathogenesis of human diseases These variations underscore the need to explore this pathway critically to system specificity and whether common features exist between experimental systems that could be closely applied to human biology and pathology it is worthwhile analyzing sequence similarities among piRNAs between systems that control the binding pattern of PIWIs and/or their interacting partners to these small RNAs and the target transcripts Insights from these analyses might shed some light on the underlying causes of piRNA-independent functions of PIWIs where stage and tissue-dependent expression of piRNA is minimal or null prompting PIWIs to go solo owing to their interaction with other RBPs or high affinity for specific motifs in target transcripts Despite these knowledge gaps surrounding the peculiar functions of the piRNA pathway the growing body of evidence indicates that PIWI proteins can operate independently of piRNAs where they regulate oncogenic pathways through mRNA degradation and translational inhibition This dual functionality underscores the versatility of PIWI proteins in diverse cellular processes and disease pathogenesis Thus targeting the piRNA pathway has become a promising avenue for therapeutic interventions But it would only be possible from a better understanding of i) the precise mechanisms underlying PIWI protein interactions with other cellular components during the development of tumors ii) the molecular details of PIWI-independent functions in the development of tissue-specific cancers and iii) the stage-dependent expression of specific piRNAs and/or PIWIs and their targets in the healthy vs Understanding how piRNAs contribute to the molecular mechanisms underlying these diseases could lead to novel therapeutic approaches aimed at mitigating the effects of aging and promoting healthy longevity Despite the advancements in these disease contexts several gaps persist in our comprehensive understanding of the piRNA-PIWI axis The interaction between piRNAs and other small RNAs is not well characterized in somatic cells The potential crosstalk between these pathways could reveal new layers of regulatory complexity with implications for understanding cellular homeostasis and disease states The variability in PIWI protein functions across species and cell types complicates the translation of findings from model organisms to humans as well as within and across the tissue/cell types Addressing these issues will require a concerted effort to standardize methodologies and establish cross-species comparisons revealing that these protein functions are regulated similarly across various cell types or differ significantly the piRNA-PIWI pathway represents a versatile and essential regulatory system with broad implications for stem cell biology Its roles in processes ranging from transposon silencing to mRNA regulation and its emerging significance in somatic tissues underscore the need for continued research Understanding these mechanisms will be crucial for developing targeted therapies that harness the regulatory potential of the piRNA-PIWI pathway offering new hope for treating a range of conditions from cancer to neurodegenerative and cardiovascular diseases The author(s) declare that financial support was received for the research This work was supported by the Natural Sciences and Engineering Research Council (NSERC) of Canada (Discovery Grant: RGPIN - 2022-03780) and the Dean of Science Startup Funds from Memorial University of Newfoundland (MUN) (to PKK) YJ’s graduate fellowship is partly supported by the Dept We thank all the researchers who have advanced the field and apologize to those whose work was not cited due to space constraints We thank all the members of 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fibrosis PubMed Abstract | Google Scholar Jiang Y and Kakumani PK (2024) Somatic piRNA and PIWI-mediated post-transcriptional gene regulation in stem cells and disease Received: 11 September 2024; Accepted: 25 November 2024;Published: 09 December 2024 Copyright © 2024 Patel, Jiang and Kakumani. 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Metrics details Deletion of hnRNPK in mouse spermatogonia leads to male sterility due to arrest permatogenesis yet the underlying molecular mechanisms remain elusive This study investigated the testicular proteome on postnatal day 28 (P28) to elucidate the infertility associated with Hnrnpk deficiency identifying 791 proteins with altered expression: 256 were upregulated Pathway enrichment analysis demonstrated that the downregulated proteins are primarily involved in spermatogenesis key proteins essential for piRNA metabolism Mechanistic studies employing RNA immunoprecipitation (RIP) and fluorescence in situ hybridization/immunofluorescence (FISH/IF) assays demonstrated that hnRNPK directly interacts with the 3’UTR of piRNA pathway transcripts These results establish that Hnrnpk deficiency disrupts the piRNA pathway by diminishing the expression of essential regulatory proteins thereby impairing piRNA production and spermatogenesis Our findings elucidate a novel molecular basis for infertility linked to hnRNPK dysfunction and advance understanding of post-transcriptional regulation in male germ cell development Although the functional mechanism of these proteins in piRNA production is well understood a complete understanding of their expression regulation during spermatogenesis remains elusive In-depth investigations into the regulation of their expression are necessary to integrate these proteins into a comprehensive regulatory pathway thereby systematically elucidating the mechanisms by which the PIWI/piRNA complex facilitates piRNA processing Such research will ultimately enhance our understanding of the piRNA pathway as well as its roles and mechanisms of action in spermatogenesis hnRNPK deficiency disrupts spermatogenesis at the pachytene stage—a phase marked by robust piRNA activity This temporal overlap suggests hnRNPK may orchestrate piRNA pathway components though the mechanistic basis remains unknown we aimed to investigate the underlying molecular mechanisms responsible for the meiotic cell cycle arrest in Hnrnpk-deficient spermatogonia utilizing tandem mass tag (TMT) labeling quantitative proteomics technology Further analyses revealed that perturbed expression of the piRNA pathway protein played a significant role in Hnrnpk-deficient mice we have successfully identified the mRNA of piRNA pathway-related genes as interaction partners of hnRNPK in the testis This identification was achieved through the utilization of RNA immunoprecipitation (RIP) and Fluorescent in situ hybridization coupled with immunofluorescence (FISH/IF) staining techniques it can be inferred that hnRNPK directly binds to the mRNA of these piRNA metabolic pathway-related genes thereby regulating the translation of multiple transcripts these genes collectively exert a significant regulatory influence on piRNA production during the pachytene stage thereby playing a crucial role in the process of spermatogenesis our work uncovers hnRNPK as a critical upstream regulator of the piRNA pathway offering new insights into the molecular basis of pachytene-stage spermatogenesis Hnrnpk flox/flox mice were generated with the aid of Cyagen Biosciences Inc China) in a C57BL/6 genetic background using TurboKnockout® technology Wide-type (WT) and Hnrnpk cKO mice were sacrificed by cervical dislocation at P28 and the testes were harvested to be measured and weighed the peptide was labeled using TMT 6-plex reagents (Thermo Fisher Scientific) according to the manufacturer’s recommendations each TMT reagent was dissolved in 88 µL anhydrous acetonitrile and added to different peptide mixtures Three independent biological repeats were performed while Hnrnpk cKO samples were labeled with TMT 129 peptides from each TMT sample were mixed in a vacuum concentrator for further analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and followed with HRP-labeled Goat Anti-Mouse IgG or HRP-labeled Goat Anti-Rabbit IgG as secondary antibodies at a dilution of 1:1000 and detected by the enhanced chemiluminescence system the blot was imaged using a FluorChem M multicolor fluorescence imaging system (ProteinSimple) The obtained protein band intensities were analyzed by Image J Software sections were washed and incubated with Alexa Fluor 488 and 555-conjugated secondary antibodies for 2 h at room temperature and DAPI Staining Solution (Beyotime) were used to visualize the nucleus sections were washed and incubated with HRP-labeled Goat Anti-Mouse IgG or HRP-labeled Goat Anti-Rabbit IgG as secondary antibodies and then stained the tissues using the DAB Horseradish Peroxidase Color Development Kit (Beyotime) The images were visualized using a Nikon 80i with NIS-Elements system (Nikon) Total RNA was extracted from P28 WT and Hnrnpk cKO testes as described above 2 ug total RNA was used as the starting material for the construction of the Small RNA sequencing library Small RNA (18–40 nt) was isolated from total RNA samples using 15% PAGE gel and recovered by gel cutting Then the isolated small RNA was enriched by ethanol precipitation and centrifugation the small RNA samples were subjected to library preparation according to the manufacturer’s protocol of TruSeq Small RNA Sample Preparation Kit (Illumina) The library preparations were sequenced on an Illumina Novaseq6000 platform and paired-end reads were generated Mm9 was used as the mouse genome reference sequence The lengths of miRNAs and piRNAs were defined as 20–24 and 24–45 nt testes collected from P18 WT and Hnrnpk cKO were cut into 1–2 mm3 pieces and fixed in cacodylate buffer at 4 °C overnight After three washes in 0.1 M cacodylate buffer the samples were incubated in 1% OsO4 for 1 h at room temperature and stained in 2% uranyl acetate for 30 min dehydration was done through consecutive incubation in sequentially ethanol solutions (30 Then samples were sequentially embedded in an Eponate mixture for polymerization about 24 h at 60 °C Ultrathin sections (~ 70 nm) were cut on an ultra-microtome and these sections were subsequently re-stained with uranyl acetate and lead citrate the sections were photographed using an H7000 electron microscope (Hitachi) at 120 kV Imprint® RNA Immunoprecipitation Kit (Sigma) was applied for RIP assay and the operation steps were carried out according to the instructions after removing the tunica albuginea of the testis the seminiferous tubule samples were cracked in the RIP lysis buffer RIP buffer including magnetic beads with hnRNPK antibody and normal mouse IgG were incubated at 4 °C for 4 h immunoprecipitation RNA was extracted and RT-qPCR analysis was performed and Mael containing hnRNPK binding motif ‘UCCC’ and corresponding mutation binding site were amplified by PCR and cloned into the downstream of the Renilla luciferase gene in psi-CHECK2 vector to construct wild-type 3’UTR and mutants reporter vectors of Piwil1 Aszl and Mael (The 3’UTR sequence information see in Supplemental materials 1) The pcDNA3.1-Hnrnpk and the above-mentioned reporter vector were co-transfected into HEK-293T cells using Lipofectamine 3000 Fireflies and renin luciferase activities in cell lysates were detected using a dual luciferase reporting kit after 48 h of transfection R-Luc activity normalized with F-Luc activity was set to 1 RNA FISH and antibody staining were performed on dissected P28 testes. For RNA FISH experiments, the procedure was carried out according to the instructions of Fluorescent In Situ Hybridization Kit (RiboBio). The Piwil1 probe labeled with FITC dye (125 nM) was used in this study and sequences of custom probe sets are listed in Table S2 All hybridizations were done overnight in the dark at 37 °C in a humidifying chamber All the experiments were carried out at least in triplicate and the values were shown as mean ± SEM Statistical significances were measured based on a t-test using the SPSS18.0 software P ≤ 0.05 (*) was considered significant difference and p ≤ 0.01 (**) was considered a very significant difference between the two groups Whole proteome analysis of Hnrnpk cKO mice testis (a) PCA analysis of TMT-proteoome in each group (b) Volcano plot of the differentially expressed proteins in Hnrnpk cKO testes compared with the WT control at P28 Green dots represent significantly downregulated proteins red dots represent significantly upregulated proteins (p-adj value < 0.05 and gray dots represent unchanged proteins (c) The heatmap of differentially expressed proteins (d) Validation of proteins expression by western blot analysis (E) Statistical analysis of the results shown in D (n = 3) Proteins involved in spermatogenesis are influenced by hnRNPK (a) Bubble graph of gene ontology (GO) analysis results from upregulated DEPs enriched in biological processes Gradation from pink to red indicates an increasing p-value (decreasing significance) the Bubble size represents the number of proteins in that term and the ratio of proteins is in the category over total downregulated proteins three MCODE complexes automatically identified in Metascape Their functional labels are generated based on the top-three functional enriched terms (c) Bubble graph of gene ontology (GO) analysis results from downregulated DEPs enriched in biological processes we suggest that hnRNPK is linked to piRNA production in pachytene spermatocytes and may regulate piRNA biogenesis by influencing genes related to piRNA metabolism The level of piRNA-related proteins was reduced in the testes of Hnrnpk cKO mice and MAEL expression in WT and Hnrnpk cKO mouse testes (b) Statistical analysis of the results shown in A (n = 3) (c) Immunofluorescence validation of PIWIL1 expression in WT and Hnrnpk cKO mouse testes (d) Immunohistochemistry detection of PIWIL2 expression in WT and Hnrnpk cKO mouse testes (e) Immunofluorescence detection of ASZ1 and γH2A.X expression in WT and Hnrnpk cKO mouse testes (f) Immunofluorescence detection of MAEL and γH2A.X expression in WT and Hnrnpk cKO mouse testes (g) Immunohistochemistry detection of DDX4 expression in WT and Hnrnpk cKO mouse testes the stages of spermatogenic epithelial cycle Red arrow points to the leptotene spermatocyte wight arrow points to the pachytene spermatocyte This reduction suggests a decrease in total piRNA levels in response to hnRNPK deletion Hnrnpk depletion reduces the piRNA populations (a) Length distribution of small RNAs in WT and Hnrnpk cKO testes (b) Distribution of the 1st and 10th nucleotides of piRNAs (24–35 nt in length) in WT and Hnrnpk cKO testes (c) The expression of thirteen piRNAs was selected to verify by RT-qPCR analysis Electron micrographs of WT and Hnrnpk cKO pachytene spermatocytes from 18 d testes Red arrow points to the typical mitochondria The area within the white dashed box is the zoomed part of the image hnRNPK interacts with the 3’UTR of piRNA metabolic process-related genes and regulates their translation (a) RIP RT-qPCR assay was carried out to detect the interaction between piRNA metabolic process-related genes and hnRNPK in testis (n = 3) (b) FISH/IF images showing colocalization of Piwil1 (green) and hnRNP K (red) in pachytene spermatocyte (PS) and round sperm; scale bars The results are representative of three biologically independent samples (c) RT-qPCR was used to detect the expression of piRNA metabolic process-related DEPs (Piwil1 (d-i) Luciferase activity of wild-type (WT) or mutant (MUT) Piwil1 (d) Piwil2 (h) and Aszl (i) 3’ UTR in 293T cells after co-transfection with pcDNA3.1 or pcDNA3.1-Hnrnpk (j) Luciferase assays were performed by co-transfection of Piwil1 and Mael 3’ UTR WT and MUT plasmids with pcDNA3.1-Hnrnpk in 293T cells Data were expressed as mean ratio of relative luciferase activities (Renilla Luciferase / Firefly Luciferase) and normalized to that in cells transfected with pcDNA3.1 plasmid These findings indicate that hnRNPK directly interacts with the 3’UTR of transcripts such as Piwil1 and Mael to modulate their gene expression at the translational level hnRNPK negatively regulates the expression of Piwil2 while positively regulating the expression of the other aforementioned genes it is noteworthy that hnRNPK may not engage in direct interaction with Aszl 3’UTR in spermatocytes despite the identification of Aszl enrichment through hnRNPK-RIP analysis the observed down-regulation of PIWIL2 expression in the testis of Hnrnpk cKO mice implies the intricate nature of PIWIL2 regulation at the translational level This regulation is not solely governed by hnRNPK but potentially involves the influence of other factors the preliminary findings suggest that hnRNPK exerts its influence on gene expression at the post-transcriptional level by binding to mRNA and modulating the expression of Piwil1 Subsequent phenotypic analyses unveiled that the loss of hnRNPK in male germ cells disrupts their development and causes significant abnormalities during the pachytene stage of meiosis These results strongly suggest that hnRNPK plays a crucial regulatory role and is indispensable for the proper development of male reproductive structures the deletion of Hnrnpk in germ cells significantly diminished the signals of these piRNA-related proteins potentially accounting for the severe phenotype observed in Hnrnpk cKO mice The marked reduction of these proteins in Hnrnpk cKO mice may impede piRNA biogenesis and function during meiosis ultimately resulting in defects in spermatogenesis and disruption of gene expression This hypothesis was partially corroborated by subsequent small RNA-Seq and TEM photomicrographs which revealed a significant decrease in the proportion of piRNAs and the mature piRNA 1U signature as well as the absence of the IMC structure in pachytene spermatocytes in Hnrnpk cKO mice these findings demonstrate that hnRNPK deficiency disrupts the piRNA pathway ultimately leading to spermatogenesis failure and infertility Hnrnpk cKO testes exhibited widespread protein dysregulation implying transcriptional and post-transcriptional roles in spermatogenesis gene networks future studies should employ large-scale sequencing (e.g. ChIP-seq) to map hnRNPK’s RNA/DNA targets in testicular tissues thereby clarifying its multifunctional contributions to germ cell development Proposed action mechanism underlying hnRNPK-mediated piRNA metabolic process-related proteins regulation in spermatogenesis the interaction between piRNA metabolic process-related proteins (such as Ddx4 Tdrd7 and Mael) mRNA 3′UTR and hnRNPK is disrupted which results in decrease of these proteins expression hnRNPK affects piRNA production in pachytene spermatocytes and plays an important role in the meiotic process of spermatogenesis meiosis The mass spectrometry raw data have been deposited to the ProteomeXchange Consortium (https://proteomecentral.proteomexchange.org) via the iProXpartner repository with the dataset identifier PXD056125 The other original data and methods of all studies included in the article can be directly contacted with the corresponding author (Yongjie Xu xyj@xynu.edu.cn) if you want to further query All data generated or analyzed during this study are included in this published article The molecular evolution of spermatogenesis across mammals PIWI-interacting RNAs: small RNAs with big functions PiRNAs in sperm function and embryo viability Two modes of targeting transposable elements by PiRNA pathway in human testis Intact PiRNA pathway prevents L1 mobilization in male meiosis Metabolic stress activates an ERK/hnRNPK/DDX3X pathway in pancreatic beta cells C19ORF84 connects piRNA and DNA methylation machineries to defend the mammalian germ line PiRNAs and their diverse roles: a transposable element-driven tactic for gene regulation PIWI-specific insertion module: a newly identified regulatory element essential for longer PiRNAs loading and male fertility Polyubiquitin gene Ubb is required for upregulation of Piwi protein level during mouse testis development Tudor domain containing 12 (TDRD12) is essential for secondary PIWI interacting RNA biogenesis in mice Disruption of PiRNA machinery by deletion of ASZ1/GASZ results in the expression of aberrant chimeric transcripts in gonocytes Building RNA-protein germ granules: insights from the multifaceted functions of DEAD-box helicase Vasa/Ddx4 in germline development An essential role for PNLDC1 in PiRNA 3’ end trimming and male fertility in mice Mouse MOV10L1 associates with Piwi proteins and is an essential component of the 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pathway in cancer HnRNP K is a haploinsufficient tumor suppressor that regulates proliferation and differentiation programs in hematologic malignancies is required for optic axon regeneration in Xenopus laevis HnRNP K post-transcriptionally co-regulates multiple cytoskeletal genes needed for axonogenesis Expression and localization of heterogeneous nuclear ribonucleoprotein K in mouse ovaries and preimplantation embryos The multifunctional RNA-binding protein HnRNPK is critical for the proliferation and differentiation of myoblasts Deletion of heterogeneous nuclear ribonucleoprotein K in satellite cells leads to inhibited skeletal muscle regeneration in mice a mature Liver-Specific MicroRNA required for hepatitis C virus replication Comparative proteomics and phosphoproteomics analysis reveal the possible breed difference in Yorkshire and duroc Boar spermatozoa iProX in 2021: connecting proteomics data sharing with big data Metascape provides a biologist-oriented resource for the analysis of systems-level datasets meiosis and undergo premature chromosome condensation Identification and verification of potential PiRNAs from domesticated Yak testis Disruption of sphingolipid metabolism elicits apoptosis-associated reproductive defects in Drosophila Mitochondria associated germinal structures in spermatogenesis: PiRNA pathway regulation and beyond ADAD2 functions in spermiogenesis and PiRNA biogenesis in mice HENMT1 and PiRNA stability are required for adult male germ cell transposon repression and to define the spermatogenic program in the mouse PROX1 promotes breast cancer invasion and metastasis through WNT/beta-catenin pathway via interacting with HnRNPK Incomplete cre-mediated excision leads to phenotypic differences between Stra8-iCre; Mov10l1(lox/lox) and Stra8-iCre; Mov10l1(lox/Delta) mice FIGNL1-FIRRM is essential for meiotic recombination and prevents DNA damage-independent RAD51 and DMC1 loading Requirement for CCNB1 in mouse spermatogenesis Mitochondrial regulation during male germ cell development HNRNPK maintains epidermal progenitor function through transcription of proliferation genes and degrading differentiation promoting mRNAs Hnrnpk is essential for embryonic limb bud development as a transcription activator and a collaborator of insulator protein Ctcf Hnrnpk maintains chondrocytes survival and function during growth plate development via regulating Hif1alpha-glycolysis axis The cancer-testis LncRNA lnc-CTHCC promotes hepatocellular carcinogenesis by binding HnRNP K and activating YAP1 transcription Download references This research was funded by the National Natural Science Foundation of China (31972537 and 32202654) the Department of Science and Technology in Henan Province (242102111015 and 222102110013) the Natural Science Foundation of Henan Province (242300421337 and 242300420509) the Key Research Projects of Higher Education Institutions in Henan Province (24A230015) and the Nanhu Scholars Program of Xinyang Normal University The APC was funded by the Department of Science and Technology in Henan Province (242102111015) Haixia Xu and Jiahua Guo contributed equally to this work Institute for Conservation and Utilization of Agro-Bioresources in Dabie Mountain All authors have read and approved the final manuscript The animal study protocol was approved by the Ethics Committee of Xinyang Normal University (protocol code XYEC-2021-011 and January 1 and conducted according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals The study was carried out in compliance with the ARRIVE guidelines version 2.0 Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Below is the link to the electronic supplementary material Download citation DOI: https://doi.org/10.1038/s41598-025-91081-1 Metrics details The prevailing assumption is that all the molecular events required for piRNA-directed DNA methylation occur after the engagement of MIWI2 We find that SPIN1 expression precedes that of both SPOCD1 and MIWI2 H3K9me3 and SPIN1 before the initiation of piRNA-directed DNA methylation We generated a Spocd1 separation-of-function allele in the mouse that encodes a SPOCD1 variant that no longer interacts with SPIN1 We found that the interaction between SPOCD1 and SPIN1 is essential for spermatogenesis and piRNA-directed DNA methylation of young LINE1 elements We propose that piRNA-directed LINE1 DNA methylation requires a developmentally timed two-factor authentication process The first authentication is the recruitment of SPIN1–SPOCD1 to the young LINE1 promoter and the second is MIWI2 engagement with the nascent transcript independent authentication events underpin the precision of piRNA-directed LINE1 DNA methylation piRNA-directed transposon methylation requires precision Failing to methylate every active transposon would be detrimental to the genomic integrity of the germline but aberrant off-target methylation could result in germline-transmitted epimutations piRNAs endow MIWI2 with the specificity to identify active transposon loci and whether other mechanisms contribute to identifying active transposons and the exacting precision of the pathway remains unknown AlphaFold2 co-folding prediction of the interaction between SPIN1 (Q61142) and SPOCD1 (B1ASB6; only amino acids 326–348 are shown) Crosslinking mass spectrometry of mouse SPOCD1 fragment 1b (amino acids 203-409) with mouse SPIN1 (amino acids 49–262) Phylogenetic tree from ray-finned fishes to mammals showing the presence of SPOCD1 and SPIN1 in the indicated animal clades AlphaFold2 prediction of SPOCD1 from Anolis carolinensis (an anole lizard XP_031752218.1) and Latimeria chalumnae (coelacanth TFIIS-M domain and SPIN1-interacting β-hairpin are highlighted Multiple sequence alignment of the SPOCD1 SPIN1-interacting β-hairpin region from different species Numbering for mouse SPOCD1 is shown above the sequences and secondary-structure elements of mouse SPOCD1 are shown below Sequences are coloured according to sequence identity Representative Coomassie gel image of n = 3 co-precipitation experiments with the indicated recombinant SPOCD1 from different species with mouse SPIN1 SPIN1 (green) and DAPI (blue) staining of wild-type fetal testis sections from the indicated developmental time points Images are representative of n = 3 biological replicates Volcano plot showing enrichment (log2(mean label-free quantification ratio of anti-HA immunoprecipitates from Spocd1HA/HA/wild-type)) and statistical confidence (−log10(P-value of two-sided Student’s t-test)) of proteins co-purifying with HA-SPOCD1 from E14.5 fetal testes; n = 3 H3K9me3 and SPIN1 from E14.5 fetal germ cells H3K9me3) and three (SPIN1) biological replicates metaplot and heatmaps of signal over elements of different transposon families (e) are shown as well as young and old copies in the L1Md_T (f) and L1Md_A (g) families Columns adjacent to the heatmaps show statistically significant peaks called for SPIN1 and the indicated histone modifications the overlap of H3K4me3 and H3K9me3 peaks with SPIN1 peaks is significant for L1Md_A (P = 0.0099 Z-score = 2,007) by one-tailed permutation tests enrichment of overlapping H3K4me3 and H3K9me3 peaks with SPIN1 peaks is significantly different between young and old L1Md_A (adjusted P < 2.2 × 10−16) and L1Md_T (adjusted P < 2.2 × 10−16) copies as observed by two-tailed Fisher’s exact test charts show overlap analysis of H3K4me3 and H3K9me3 peaks (h) and SPIN1 peaks (i) with the indicated genomic features P-values and Z-scores from one-tailed permutation tests to assess the statistical significance of overlaps of CUT&TAG peaks with LINE1 elements are shown a, Representative western-blot analyses of n = 3 immunoprecipitations of the mouse wild type and eight SPOCD1 alanine mutations (8 Ala mut) with SPIN1 in HEK 293 T cells. For whole-blot source data, see Supplementary Fig. 1 Representative Coomassie gel image of n = 3 co-precipitation experiments with the indicated recombinant proteins Representative images of E16.5 gonocytes from n = 3 wild-type (WT) and Spocd1ΔSPIN1 mice stained for DNA (blue) and SPOCD1 (c) Number of embryos per plug fathered by studs with the indicated genotype mated to wild-type females from n = 6 wild-type (15 plugs in total) and n = 6 Spocd1ΔSPIN1 studs (12 plugs) Testis weight of adult mice with the indicated genotype from n = 8 wild-type and n = 8 Spocd1ΔSPIN1 mice a representative image of testes from wild-type (left) and Spocd1ΔSPIN1 (right) mice P-values in f and g were determined by unadjusted two-sided Student’s t-test Representative images of PAS and haematoxylin-stained testes sections of wild-type and n = 5 Spocd1ΔSPIN1 adult mice with different types of spermatogenic arrest observed in the tubules of the Spocd1ΔSPIN1 testes indicated The percentage of each type of tubule is noted alongside Adult testis sections stained for the DNA damage marker γH2AX (red) (i) and apoptotic cells (red) by TUNEL assay (j) from wild-type and Spocd1ΔSPIN1 mice (representative of n = 3 mice per genotype for γH2AX and n = 2 wild-type plus n = 3 Spocd1ΔSPIN1 mice for TUNEL) Representative testis sections of n = 3 wild-type Spocd1ΔSPIN and Spocd1−/− mice stained red for the LINE1 ORF1p (a) or IAP GAG protein (b) RNA-seq heat maps showing fold changes in expression relative to wild type for the ten most upregulated LINE1 and ERVK transposable elements in Spocd1−/− P20 testes (n = 3 from each genotype) ***P < 0.001 of Bonferroni-corrected two-sided Wald’s test assuming n-binominal distribution Only significant differences (P < 0.05) are shown Genomic CpG methylation analysis of P14 undifferentiated spermatogonia from wild-type (n = 6) Spocd1ΔSPIN (n = 4) and Spocd1−/− mice (n = 3) Percentages of CpG methylation levels of the indicated genomic features (with genic promoter and CpG island (CGI) regions defined as those not overlapping transposable elements and intergenic regions as those not overlapping transposable elements or genes) or transposable elements (not overlapping genes) are shown as box plots Boxes represent interquartile range from the 25th to the 75th percentile and whiskers show the data range of the median ± twice the interquartile range Significant differences (P < 0.05 of Bonferroni-corrected two-tailed Student’s t-tests) of Spocd1ΔSPIN (n = 4) and Spocd1−/− (n = 3) samples to wild-type (n = 6) are indicated Metaplots of mean CpG methylation over the indicated transposable element **P = 0.01–0.001 and ***P < 0.001 for Bonferroni-corrected two-tailed Student’s t-tests comparing the average CpG methylation of the promoter region to wild type for Spocd1ΔSPIN1 (red) and Spocd1−/− (blue) Correlation analysis of mean CpG methylation loss relative to the wild type for individual transposable elements of the indicated LINE1 and ERVK families in relation to their divergence from the consensus sequence in Spocd1ΔSPIN spermatogonia How SPOCD1 is recruited to IAPs remains unknown but we speculate that another SPOCD1-associated protein could mediate this recruitment through the recognition of a distinct chromatin signature or sequence motif The different mechanisms in LINE1 and IAPs reveals an unexpected complexity in the pathway The prevailing notion is that all the molecular events required for piRNA-directed DNA methylation occur after the engagement of the piRNA–MIWI2 ribonucleoprotein complex with the nascent transcript we demonstrate that multiple independent and developmentally choreographed events are required for LINE1 piRNA-directed DNA methylation Our revised model posits that the recruitment of SPIN1–SPOCD1 through chromatin modification to young LINE1 elements constitutes a first licensing step The engagement of MIWI2 with the nascent transcript is the second licensing event and triggers DNA methylation we propose that a two-factor authentication system ensures the precision of LINE1 piRNA-directed methylation Male fertility was assessed by mating studs to Hsd:ICR (CD1) wild-type females and counting the number of pups born for each plugged female animal tissue samples were collected from one or more litters and allocated to groups according to genotype No further randomization or blinding was applied during data acquisition and analysis Animals were maintained at the University of Edinburgh in accordance with the regulation of the UK Home Office or at the Institute for Molecular Biology in Mainz in accordance with local and European animal-welfare laws Ethical approval for the UK mouse experimentation has been given by the University of Edinburgh’s Animal Welfare and Ethical Review Body and the work done under licence from the UK Home Office Animal experiments done in Germany were approved by the ethical committees on animal care and use of the federal states of Rheinland-Pfalz Duke University) 1:500; anti-γH2AX (Bethyl Laboratories) 1:500; anti-MIWI2 (a gift from R Université de Genève) 1:500; anti-SPOCD1 rabbit serum rb175 1:500 (O’Carroll laboratory antibody); anti-SPIN1 (Cell Signaling Technologies) 1:500 (of a custom preparation of 1.1 μg μl−1 in PBS) Images were taken on a Zeiss Observer or Zeiss LSM880 with an Airyscan module Images acquired using the Airyscan module were deconvoluted with the Zeiss Zen software ‘Airyscan processing’ with settings 3D and a strength of 6 ImageJ and Zeiss Zen software were used to process and analyse the images cells were washed twice with PBS and resuspended in 1 ml lysis buffer (IP buffer: 150 mM KCl supplemented with 1× protease inhibitors (cOmplete ULTRA EDTA-free Roche) with 37 units per ml benzonase (Millipore)) and lysed for 30 min The lysate was cleared by centrifugation for 10 min at 21,000g Cleared lysate (800 μl) was incubated with 20 μl of anti-HA beads (Pierce) that had been calibrated in lysis buffer and incubated for 1 h at 4 °C on a rotating wheel The beads were washed four times with lysis buffer Immunoprecipitates were eluted at 50 °C for 10 min in 20 μl 0.1% sodium dodecyl sulphate (SDS) Lysates and eluates were run on a 4–12% bis–tris acrylamide gel (Invitrogen) and blotted onto a nitrocellulose membrane (Amersham Protran 0.45 NC) according to standard laboratory procedures The membrane was blocked with blocking buffer (4% (w/v) skimmed milk powder (Sigma-Aldrich) in PBS-T (phosphate buffered saline 0.1% Tween-20)) and subsequently incubated for 1 h with primary antibodies (anti-HA (C29F4s anti-SPOCD1 rabbit serum rb175 (O’Carroll laboratory antibody) 1:500 or anti-α-Tubulin (T9026 Sigma-Aldrich) 1:1,000) in blocking buffer The anti-α-tubulin staining was used as loading control on the same blot as the experimental staining the membrane was incubated with secondary antibodies (IRDye 680RD donkey anti-rabbit or IRDye 800CW donkey anti-mouse It was washed three times for 10 min in PBS-T and imaged on a LI-COR Odyssey CLx system Exposure of the entire images was optimized in Image Studio Lite (LI-COR) and areas of interest were cropped for presentation GST-tagged mouse SPOCD1 fragments (amino acids 203–409) Latimeria SPOCD1 fragments (XP_014348336.1 amino acids 510–1009) and His-tagged SPIN1 (amino acids 49–262) were cloned in a pET-based backbone Proteins were expressed in Escherichia coli BL21 (DE3) Bacteria were grown in 2xTY media at 37 °C until an optical density of 0.8 was reached the bacteria were induced with 1 mM IPTG and grown for another 14–16 h Cells were collected and pellets were stored at −80 °C until purification The pellets were resuspended in 50 ml lysis buffer (20 mM Tris-HCl Roche cOmplete EDTA-free Protease Inhibitor Cocktail 0.01 mg ml−1 DNaseI (Sigma) and 2 mM AEBSF (Pefabloc) for SPIN1 0.01 mg ml−1 DNaseI (Sigma) and 2 mM AEBSF (Pefabloc) for SPOCD1) and cells were lysed with the Constant systems 1.1 kW TS cell disruptor at 25 kPSI The cleared lysate was used to load on a cOmplete His-Tag Purification Column (Roche) for SPIN1 or incubated with 7 ml glutathione sepharose high-performance beads (Cytiva) for SPOCD1 calibrated in the respective buffer Elution from column/beads with increasing (2.5–500 mM) imidazole gradient for SPIN1 or GST elution buffer containing 20 mM reduced glutathione for SPOCD1 The fractions of interest were pooled and dialysed overnight in 20 mM Tris-HCl The SPIN1 construct was cleaved with GST–3C protease (made in our lab) overnight The SPOCD1 constructs were concentrated and stored at −80 °C until used SPIN1 was further purified by ion exchange with a gradient of 100–1,000 mM NaCl (Resource Q Cytiva) and size-exclusion chromatography (HiLoad 16/600 Superdex 200 pg the protein was concentrated and stored at −80 °C until used starting from H3Δ1–31T32C C110A constructs that also contained the required H3X and H3Y mutations H3X was used for H3K4me3 and H3Y for H3K9me3 A biotinylated 209-bp DNA fragment containing the 601 nucleosome positioning sequence was generated by PCR and purified by ion-exchange chromatography on a HiTrap Q column followed by ethanol precipitation Mononucleosomes were then assembled from histone octamers and 601 DNA by gradient dialysis Nucleosome assembly was verified by native gel electrophoresis on 6% acrylamide gels in 0.5× TGE buffer (12.5 mM Tris After incubation with recombinant proteins beads were washed three times with high-salt pull-down buffer (as above but with 350 mM NaCl) for 5 min Nucleosomes and bound proteins were eluted by boiling in 1.5× SDS sample buffer (95 mM Tris HCl Binding was analysed by western blotting with antibodies against His tag (Sigma H1029 Antibodies against histone H3 (Abcam ab176842 H3K4me3 (Cell Signaling) 1:2,000 and H3K9me3 (Abcam ab176916) 1:1,000 were used to verify nucleosome loading and modification state For analytical size-exclusion chromatography 125 μg SPIN1 and/or 500 μg mouse GST–SPOCD1-F1b were used for each run Proteins were diluted in 250 μl size-exclusion chromatography buffer (20 mM HEPES 1 mM DTT) and injected on a Superdex 200 10/300 GL column loaded on an SDS–PAGE gel and visualized by Coomassie staining IP-MS of SPOCD1–HA from Spocd1HA/+ E14.5 fetal testis using 50 μl of anti-HA beads (Pierce, 88837) was done as previously described4 with a reduced number of 25 testes per replicate Wild-type fetal testes were used as controls To purify foetal germ cells for CUT&Tag analysis, E14.5 testes were dissected from embryos carrying the Oct4eGFP allele34 A single cell suspension was obtained by sequential treatment with 100 µl collagenase solution at 37 °C for 8 min (10 units of collagenase A (Sigma-Aldrich 10103578001); 2× NEAAs (Gibco); 2× Na-pyruvate (Gibco); 25 mM HEPES–KOH pH 7.5) and 200 µl TryPLE Express (Gibco) at 37 °C for 5 min with gentle flicking and pipetting of the solution to aid dissociation Digestion was neutralized by 70 µl prewarmed FBS and cells were collected by spinning at 600g for 4 min at room temperature followed by two washes in FACS buffer (1× PBS; 2 mM EDTA 10% FBS; 2 µg ml−1 DAPI) and filtering (Corning Cell sorting was done on an Invitrogen Bigfoot using a 100 μm nozzle and gating for DAPI-negative (live) OCT4–eGFP-positive (germ cells) populations into collection tubes containing 100 µl 1× PBS For EM-seq, CD9+ spermatogonia were sorted from P14 testes as described previously52 using Fc block (eBioscience lot 2297433) 1:50; biotin-conjugated anti-CD45 (eBioscience and biotin-conjugated anti-CD51 (Biolegend lot B308465) 1:100 anti-CD9APC (eBioscience Cells were sorted into DMEM media on a BD Aria II sorter pelleted for 5 min at 500g and snap frozen in liquid nitrogen For gating strategies, see Supplemental Fig. 2 using pA–Tn5 at a 1:400 dilution (Diagenode C01070001) and 15 PCR cycles of library amplification Libraries were cleaned up by magnetic bead-based solid-phase separation and assessed on a Tapestation (Agilent) Antibodies and dilutions used for CUT&Tag were rabbit IgG control (Abcam and guinea pig anti-rabbit IgG (Antibodies Online Pooled libraries were sequenced using paired-end 150 bp on a NextSeq 2000 instrument (Illumina) using the following parameters: -bs 1 --normalizeUsing CPM —exactScaling --ignoreForNormalization MT Log2 enrichment profiles of CUT&Tag samples over IgG controls were generated with deepTools bamCompare using the following parameters: -bs 1 --normalizeUsing CPM --exactScaling --ignoreForNormalization MT --scaleFactorsMethod None Monomers associated with inert promoters (subtypes 6 and 2) were removed from the analysis The central regions of repetitive elements were length-normalized to 5 kb with flanking regions ±2 kb from the start and end positions Heatmaps and profile plots show data in consecutive 10b bins with regions subdivided by elements and arranged in descending order of total enrichment across all samples Histology experiments on mouse samples were done as previously described4 TUNEL assay experiments were done as previously described4 two-tailed Student’s t-tests were used to compare the differences between groups and adjusted for multiple testing using Bonferroni correction where indicated Averaged data are presented as mean ± s.e.m. No statistical methods were used to predetermine the sample size The experiments were not randomized and the investigators were not blinded to allocation during experiments and outcome assessment Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article The scripts used for the EM-seq and RNA-seq analysis are available from github at https://github.com/rberrens/SPOCD1-piRNA_directed_DNA_met, and the scripts used for ChIP and CUT&Tag analysis are available from github at https://github.com/swebb1/heep-et-al_2024 The endonuclease activity of Mili fuels piRNA amplification that silences LINE1 elements TEX15 is an essential executor of MIWI2-directed transposon DNA methylation and silencing SPOCD1 is an essential executor of piRNA-directed de novo DNA methylation Zoch, A. et al. C19ORF84 connects piRNA and DNA methylation machineries to defend the mammalian germ line. Mol Cell https://doi.org/10.1016/j.molcel.2024.01.014 (2024) Structural mechanism of bivalent histone H3K4me3K9me3 recognition by the Spindlin1/C11orf84 complex in rRNA transcription activation Identification of autonomous IAP LTR retrotransposons mobile in mammalian cells Jr A novel active L1 retrotransposon subfamily in the mouse Developmentally regulated piRNA clusters implicate MILI in transposon control Meiotic catastrophe and retrotransposon reactivation in male germ cells lacking Dnmt3L MIWI2 is essential for spermatogenesis and repression of transposons in the mouse male germline Transcription of IAP endogenous retroviruses is constrained by cytosine methylation The diverse roles of DNA methylation in mammalian development and disease A piRNA pathway primed by individual transposons is linked to de novo DNA methylation in mice Two waves of de novo methylation during mouse germ cell development Nucleolar protein Spindlin1 recognizes H3K4 methylation and stimulates the expression of rRNA genes Distinct mode of methylated lysine-4 of histone H3 recognition by tandem tudor-like domains of Spindlin1 Active genes are tri-methylated at K4 of histone H3 Selective recognition of methylated lysine 9 on histone H3 by the HP1 chromo domain Methylation of histone H3 lysine 9 creates a binding site for HP1 proteins Highly accurate protein structure prediction with AlphaFold Highly accurate protein structure prediction for the human proteome Broad heterochromatic domains open in gonocyte development prior to de novo DNA methylation Role of the Dnmt3 family in de novo methylation of imprinted and repetitive sequences during male germ cell development in the mouse Efficient low-cost chromatin profiling with CUT&Tag A tudor domain protein SPINDLIN1 interacts with the mRNA-binding protein SERBP1 and is involved in mouse oocyte meiotic resumption The DNA methyltransferase DNMT3C protects male germ cells from transposon activity encoding a DNA methyltransferase homolog required for meiosis and transposon repression in the mouse male germline DNA methylation of retrotransposon genes is regulated by Piwi family members MILI and MIWI2 in murine fetal testes A transit-amplifying population underpins the efficient regenerative capacity of the testis One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome engineering Oct4 expression is not required for mouse somatic stem cell self-renewal A MILI-independent piRNA biogenesis pathway empowers partial germline reprogramming G9a co-suppresses LINE1 elements in spermatogonia ColabFold: making protein folding accessible to all PyMOL Molecular Graphics System v.1.8 (Schrodinger scalable generation of high-quality protein multiple sequence alignments using Clustal Omega Jalview Version 2−a multiple sequence alignment editor and analysis workbench ChromID identifies the protein interactome at chromatin marks A synthetic biology approach to probing nucleosome symmetry The histone H3.1 variant regulates TONSOKU-mediated DNA repair during replication Trimmomatic: a flexible trimmer for Illumina sequence data Li, H. Aligning sequence reads, clone sequences and assembly contigs with BWA-MEM. Preprint at https://arxiv.org/abs/1303.3997 (2013) The Sequence Alignment/Map format and SAMtools deepTools2: a next generation web server for deep-sequencing data analysis Subtype classification and functional annotation of L1Md retrotransposon promoters SeqPlots – interactive software for exploratory data analyses pattern discovery and visualization in genomics Wickham, H. Welcome to the Tidyverse. J. Open Source Softw. https://doi.org/10.21105/joss.01686 (2019) Defective germline reprogramming rewires the spermatogonial transcriptome The nf-core framework for community-curated bioinformatics pipelines regioneR: an R/Bioconductor package for the association analysis of genomic regions based on permutation tests Gaspar, J. M. Improved peak-calling with MACS2. Preprint at bioRxiv https://doi.org/10.1101/496521 (2018) Software for computing and annotating genomic ranges pyGenomeTracks: reproducible plots for multivariate genomic datasets The Perseus computational platform for comprehensive analysis of (prote)omics data Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2 Download references This research was supported by funding from Wellcome to D.O’C (095021 and 200885); the Wellcome Centre for Cell Biology (203149 and multi-user equipment grants 108504 and 092076); funding for the Wellcome Discovery Research Platform for Hidden Cell Biology (226791); and support from the microscopy proteomics and bioinformatics cores of the Wellcome Discovery Research Platform for Hidden Cell Biology was funded by a German Research Foundation fellowship (DFG award ZO 376/1-1); J.B were funded by a German Research Foundation collaborative research centre grant (DFG grant SFB 1361 Work in P.V.’s lab was supported by Wellcome (104175/Z/14/Z) and the UK Biotechnology and Biological Sciences Research Council (BBS/E/B/000C0421) are funded by the Darwin Trust of Edinburgh This work used the University of Edinburgh Protein Production Facility (EPPF) the Wellcome Centre for Cell Biology’s Centre Optical Instrumentation Laboratory (COIL) proteomics and bioinformatics core platforms and the Centre for Regenerative Medicine’s FACS facility We also thank staff at the EMBL GeneCore facility in Heidelberg for preparing the methyl-seq libraries and sequencing all libraries; S Nick at IMB flow-cytometry core facility for assistance with operating the Bigfoot cell sorter instrument (project number 511658729); and M Heinen at the IMB protein production core facility for the recombinant Tn5 protein fusions These authors contributed equally: Ansgar Zoch Zhejiang University-University of Edinburgh Institute (ZJU-UoE Institute) execution and analysis of most of the experiments did the immunofluorescence and IP-MS experiments did the CUT&RUN and CUT&Tag experiments under the guidance of J.B did the bioinformatic analysis of the EM-seq generated site-specifically modified histones and designed and performed the nucleosome pull-down experiments did the molecular-biology and histology experiments analysed the crosslinking-mass spectrometry data contributed to analyses and experimental design reviewer(s) for their contribution to the peer review of this work HA (red) and DAPI (blue) staining of E16.5 foetal testis sections from Spocd1HA/+ mice treated with PBS or RNase A prior to fixation HA (red) and DAPI (blue) staining of E16.5 foetal testis sections from E16.5 Miwi2−/−;Spocd1HA/+ and Miwi2+/−;Spocd1HA/+ mice SPIN1 (green) and DAPI (blue) staining of E16.5 Miwi2+/− and Miwi2−/− E16.5 foetal testis sections (c) shows a zoom-in of the cell highlighted with a dashed rectangle in (d) Images of (a-d) are representative of n = 3 biological replicates sequences are coloured according to sequence identity Numbering above according to mouse sequence Panels show H3K4me3 (a) and H3K9me3 (b) ChIP-seq signal in reads per million (RPM) over young and old elements within the indicated LINE1 family Metaplot and heat maps of indicated CUT&Tag signal of H3K4me3 H3K9me3 and SPIN1 over young and old L1MD_F elements Columns adjacent to the heatmaps show peaks called for SPIN1 and the indicated histone modifications Data depicts element plus adjacent 2 kb for each of the transposon families indicated Genome snapshots showing datatracks of CUT&Tag signal of H3K4me3 H3K9me3 and SPIN1 over selected genome regions containing a young L1Md_A Enrichment of overlapping H3K4me3 and H3K9me3 peaks with SPIN1 peaks is not significantly different between young and old L1Md_F copies as observed by a two-tailed Fisher’s exact test Representative images of sections from n = 3 wild-type foetal testis stained for SPIN1 (green) and DAPI (blue) from indicated timepoints Cell shown in (a) is highlighted with a white box in (b) a, Representative western blot analyses of n = 3 anti-HA immunoprecipitations of the HA epitope-tagged mouse wild-type, SPOCD1 8 alanine mutated proteins or GFP control with FLAG-tagged DNMT3L in HEK 293 T cells. For whole blot source data, see Supplementary Fig. 1 Schematic representations of the mouse Spocd1 locus and encoded 1015 amino acid protein are shown sgRNA used for generation of the Spocd1ΔSPIN1 allele and adjacent PAM site are indicated Schematic of CRISPR targeting strategy showing the location of single-stranded oligo DNA donor (ssODN) and homology arms (HA) used and sequencing trace of the part of Spocd1ΔSPIN1 exon 4 harbouring the mutation sites a 30 bp sequence creating the 8 alanine mutation is highlighted in red Representative image of genotyping result for n = 3 Spocd1+/+ Representative images of E16.5 gonocytes from n = 3 Spocd1ΔSPIN1 and wild-type control mice stained for SPOCD1 (e) Supplementary Figures containing uncropped scans of the western-blot experiments shown in Figs and the FACS gating strategy for sorting fetal germ cells and undifferentiated spermatogonia (Supplementary Fig Further data relating to crosslinking mass spectrometry data Download citation DOI: https://doi.org/10.1038/s41586-024-07963-3 Sorry, a shareable link is not currently available for this article. 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Volume 1 - 2021 | https://doi.org/10.3389/fmmed.2021.791931 This article is part of the Research TopicEpigenetic Mechanisms in Cardiovascular DiseaseView all 4 articles The relationship regarding non-coding genomes and cardiovascular disease (CVD) has been explored in the past decade there remains a lack of sensitive and specific genomic biomarkers in the diagnosis and prognosis of CVD Piwi-interacting RNA (piRNA) is a group of small non-coding RNA (ncRNA) which associated with Piwi proteins There is an emerging strong body of evidence in support of a role for ncRNAs This article reviews the current evidence for piRNA-regulated mechanisms in CVD which could lead to the development of new therapeutic strategies for prevention and treatment Transcriptomics refer to the total amount of RNA transcribed by a specific tissue or cell in a certain stage or functional state and includes protein-coding messenger RNAs (mRNAs) and non-coding RNAs (ncRNAs) (Suzuki and Sugano, 2006) The functional significance of non-coding RNAs is particularly evident for small regulatory RNAs With its rapid development, omics has launched the molecular understanding of disease phenotypes into a new era. While the analysis of RNA Atlas data have expanded the catalog and their roles in regulating protein-coding genes and pathways (Lorenzi et al., 2021) integration of transcriptome profiling enabled analyses toward ncRNA for functional evidence is still being explored PIWI protein-interacting RNAs (piRNAs) have been identified as an important class of small regulatory RNAs together with microRNAs (miRNAs) and short-interfering RNAs (siRNAs) which corresponds piRNA to form piRNA/PIWI complexes that are associated with transposon silencing the development of high-throughput technologies over the past decades provided us an initial understanding of noncoding genomes The crucial roles of the piRNA/PIWI pathway reflected in mediating the regulation of mRNA and satellite RNA homeostasis by transposons and pseudogenes via transcriptional or posttranscriptional mechanisms piRNAs also play the protective role in germline genome integrity and stability by transposon silencing and epigenetic regulation (Qian et al., 2021; Rosenkranz et al., 2021) Cytoplasmic piRNA/PIWI complex can fulfill its function in transposon silencing through multiple pathways including: Hsp90-HOP to influence canalization; interact with translational initiators to induce inhibit polysomes and subsequent protein translation; piRNAs sequence-specific silencing to maintain genomic integrity and produce antiviral immune memories; piRNA-induced silence compounds via mitochondria to suppress transposons Cardiovascular disease (CVD) remains the leading cause of death worldwide. Over 17 million people die of cardiovascular disease worldwide each year, similar to the death rate from all cancers combined (Batki et al., 2019). In China, due to population aging and changes in dietary structure, the morbidity and mortality of CVDs including hypertension, coronary heart disease, and congestive heart failure have shown upward trends (Hu et al., 2020) which imposes additional social and financial burdens The prevention and treatment of CVDs is still a major task of modern medicine Exploring the multifaceted functions of these RNAs in the pathogenesis of CVD may fulfill a promising clinical applications as diagnostic biomarkers and therapeutic targets Description of piRNAs and CVD literature articles They constructed CHAPIR knockout (CHAPIR KO) mice with reduced cardiac fibrosis and hypertrophy after transverse aortic constriction (TAC) surgery the similar function was also found in Ang-II-induced hypertrophic growth of cardiomyocytes The CHAPIR - METTL3-PARP10-NFATC4 signal axis may be considered as therapeutic target used to treat pathological hypertrophy and maladaptive cardiac remodeling in the future CVD: cardiovascular disease; piRNA: PIWI-interacting RNAs; mRNA: messenger RNA; lncRNA: long non-coding RNA these mechanisms may shed new light on the understanding regulatory mechanism of piRNAs in CVD Strict thermodynamic parameters and binding energy thresholds can be applied to predict potential targets that are complementary to piRNAs Promoting the adequate proliferation of cardiomyocytes may be a new treatment target to prevent abnormal hypertrophy and pathological fibrosis To determine the molecular basis of pluripotent stem cells it could provide an important understanding of the characteristics in cardiovascular system at the RNA level and connect the existing evidence of reference genes Further research along these directions regarding the function of stem cells and piRNA/PIWI proteins pathway can supplement our knowledge in cardiovascular therapy There is much to be discovered about the existence and function of PIWI protein in somatic cells variety of pathological heart conditions and the related mechanisms have not been elucidated and the roles of these piRNA/PIWI proteins therein are not conclusive yet Conjoint assays of in-depth small RNA sequencing and m6A-seq and other technologies piRNAs and the corresponding mechanism of transposon silencing and epigenetic regulation could be unveiled the exploration of piRNAs involved in stem cell-derived cardiomyocytes is of great promoting value to the molecular mechanisms underlying cardiac repair Integrating research focused on these areas will provide the potential applications of piRNAs in the clinical diagnosis and therapeutic strategies for cardiovascular diseases IC and QZ contributed to the major writing of the manuscript SL and XL contributed to the conception and design of the study All authors contributed to manuscript revision Small RNA Profiling Reveals Deregulated Phosphatase and Tensin Homolog (PTEN)/Phosphoinositide 3-Kinase (PI3K)/Akt Pathway in Bronchial Smooth Muscle Cells from Asthmatic 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This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use *Correspondence: Xinli Li, eGlubGkzMjY3QHllYWgubmV0 Metrics details piRNAs are crucial for transposon silencing we report on the genetic landscape of piRNA dysfunction in humans and present 39 infertile men carrying biallelic variants in 14 different piRNA pathway genes the testicular phenotypes differ from those of the respective knockout mice and range from complete germ cell loss to the production of a few morphologically abnormal sperm A reduced number of pachytene piRNAs was detected in the testicular tissue of variant carriers LINE1 expression in spermatogonia links impaired piRNA biogenesis to transposon de-silencing and serves to classify variants as functionally relevant These results establish the disrupted piRNA pathway as a major cause of human spermatogenic failure and provide insights into transposon silencing in human male germ cells in which disrupted piRNA biogenesis has been tightly linked to male-specific infertility the role of the piRNA pathway in spermatogenic failure in men remains largely unexplored we shed light on the impact of disrupted piRNA biogenesis on human spermatogenesis by presenting 39 infertile men carrying rare putative pathogenic variants in 14 different genes encoding proteins of the piRNA pathway the observed testicular phenotypes repeatedly differ from those of the respective knockout mice we show that the dysfunction of piRNA pathway proteins in the human adult testis not only leads to a reduced amount of pachytene piRNAs but is also associated with a gene-specific increase of transposon expression in spermatogonia These analyses can serve as readout for the functional relevance especially of the identified missense variants a Schematic representation and AlphaFold2 structure predictions of PIWIL1 The schematic depicts both novel (red) and known (black) homozygous variants with amino acids affected by new variants highlighted in the protein structure in red b Periodic acid-Schiff (PAS) staining of testicular tissue of men with normal spermatogenesis (control) Representative tubules showing the most advanced stage of spermatogenesis observed in three independent sections are shown Immunohistochemical staining (IHC) for round spermatid marker protein Cyclic AMP Element Modulator (CREM) and spermatocyte marker protein γH2AX round spermatids were detected as most advanced germ cells rarely pachytene spermatocytes with γH2AX positive sex bodies but no haploid germ cells were observed c IHC staining for PIWIL1 and GTSF1 in controls and variant carriers demonstrating absence of PIWIL1 in M2006 due to homozygous stop-gain variant p.(Arg230*) and absence of GTSF1 in M2243 with homozygous frameshift variant p.(Arg74Lysfs*4) Representative tubules showing the staining pattern observed in independent sections (control: N = 3 a Localization of variants in schematic of MOV10L1 and HENMT1 structure with protein domains colored and newly identified biallelic variants (red bold for homozygous) as well as previously described homozygous variants (black) indicated Pairs of compound heterozygous variants are indicated by identical symbols (*,#) in superscript DNA2/NAM7; CAF1 chromatin assembly factor 1 domain (yellow); GPAT/DHAPAT acetyltransferase and methyltransferase domains (blue) b Periodic acid-Schiff (PAS) staining of representative testicular tissue of variant carriers demonstrating SCO in M2803 [PLD6 p.(His377Arg)/(Arg49His)] and presence of haploid germ cells (round/elongated spermatids) in M1125 [PNLDC1 c Immunohistochemical (IHC) staining for GPAT2 in testicular tissue with full spermatogenesis (control) and GPAT2 variant carriers with meiotic arrest GPAT2 is expressed in perinuclear structures in spermatocytes and this staining pattern is absent in M690 and M2556 d IHC for MAEL in testicular tissue with full spermatogenesis (control) and M2435 with compound heterozygous presence of two MAEL LoF variants p.(Arg267*)/p.(Cys283_Ala303del) MAEL is expressed in perinuclear structures in spermatocytes and distinct condensed structures in round spermatids and this staining pattern is absent in the variant carrier several members of the TDRD gene family have been linked to piRNA biogenesis We identified rare homozygous variants in TDRD1 and TDRD12 that matched our filtering criteria a Schematic representation and AlphaFold2 structure predictions of TDRDs with amino acids affected by new variants highlighted in the protein structures in red MYND-ZF MYND-type zinc finger domain (gray) b Periodic acid-Schiff (PAS) staining of testicular tissue of variant carriers demonstrating meiotic arrest in M1648 [TDRD1 presence of elongated spermatids in M800 [TDRD9 p.(Asn198Ser)] and round spermatid arrest in M2227 [TDRD12 the open reading frame is shifted resulting in the synthesis of a truncated protein p.(Asp289Alafs*3) if the mutant transcript is not degraded by NMD and/or TE expression in 14 variant carriers (10 biallelic LoF 4 homozygous missense) supported the pathogenicity of the respective variants a For each piRNA factor gene described in this study the reproductive phenotype of the male knockout mice is compared with the phenotypes observed in novel and known human infertile biallelic variant carriers testicular phenotypes of human variant carriers differ from those described for the respective knockout mice b Venn diagram depicting overlap between reproductive phenotypes of infertile men affected by biallelic variants in the same gene TDRD9) a phenotypic overlap could be observed for missense and LoF variant carriers LoF variant carriers are in most cases not affected by a more severe phenotype than missense variant carriers ES+ elongated spermatids present in testicular tissue Through comprehensive exploration of biallelic variants in exome/genome data of >2000 infertile men genes encoding proteins involved in piRNA biogenesis are a major previously underexplored contributor to human spermatogenic failure this study also reports homozygous potentially pathogenic missense variants in TDRD1 and DDX4 both of which are highly intolerant to genetic variations the gene-specific testicular phenotype can be used to aid assessment of the variant’s pathogenicity TDRD12 variant carriers exhibited highly variable phenotypes ranging from SCO to even a few sperm in the ejaculate with at least four different biallelic variants (including several LoF variants) identified per gene in this and other studies and TDRD9 are excellent candidates to be included in the diagnostic workup of infertile men it has not been fully proven that the variants lead to a complete loss of protein function further data is needed to draw firm conclusions whether human spermatogenesis may be less stringently controlled and progresses further despite disrupted piRNA biogenesis It remains to be determined whether the round spermatids/sperm produced in some men are actually suitable for procreation due to the limited amount of testicular tissue from variant carriers available for analysis of piRNA factor expression the data presented here are a first indication of a co-dependency and it cannot be ruled out that different expression profiles are at least in part also related to different germ cell compositions of the testicular sections in contrast to the highly conserved piRNA biogenesis genes the pachytene piRNA loci themselves are highly divergent between mice and men the dysfunctions in spermatogenesis might not result from harmful transposon expression but could be a consequence of transcriptional dysregulation genetic variants concordantly result in LINE1 de-repression in spermatogonia the encoded proteins might also be involved in biogenesis of pre-pachytene piRNAs that are mainly loaded to PIWIL2 which is expressed at all stages of male germ cell maturation including spermatogonia impaired biogenesis of pre-pachytene piRNAs might lead to de-silencing of transposons in spermatogonia resulting in expression of LINE1 ORF1p the function of this protein in human piRNA biogenesis and fertility still needs to be elucidated this study provides extensive data linking disrupted piRNA biogenesis to human spermatogenic failure demonstrates that piRNA pathway genes are a major target for scrutinizing genetic causes of male infertility and suggests that safeguarding of the genome during spermatogenesis is in some instances less stringent in men than in mice The detailed characterization of pathogenic human variants provides insight into the molecular function of the factors involved in piRNA biogenesis and piRNA-mediated transposon silencing This opens the possibility to investigate key protein domains and to assess the pathogenicity of gene variants samples were prepared and enriched following the manufacturer’s protocols of either Illumina’s Nextera DNA Exome Capture kit or Twist Bioscience’s Twist Human Core Exome Kit and sequencing was performed on the Illumina NovaSeq 6000 Sequencing System Only variants with a MAF ≤ 0.01 (gnomAD database Patients in which additional candidate variants were identified were excluded from further analysis In case that no parental DNA was available for analysis biallelic occurrence of heterozygous variants was determined by long-read sequencing using long-range PCR products encompassing both genomic regions of interest amplified from variant carriers as template for library generation 1 µg of PCR products was used for subsequent preparation of MinION sequencing library Barcoding and sequencing was carried out according to manufacturer’s instructions (MinION quality control and variant calling phasing of variants on same/different alleles was determined To analyze the TDRD12 c.963+1G>T variant the region encompassing exon 8–10 of TDRD12 was amplified and subcloned into pcDNA3.1 and for MOV10L1 c.2179+3A>G into pSPL3B A transient transfection with mutant and wild-type Minigene constructs was performed using Human Embryonic Kidney cells (HEK293T Lenti-X Total RNA was extracted using the RNeasy Plus Mini Kit (QIAGEN®) and reverse-transcribed into cDNA with the ProtoScript® II First Strand cDNA Synthesis Kit (New England Biolabs GmbH®) Amplification of the region of interest was performed and RT-PCR products were separated on a 2% agarose gel Wild-type and mutant constructs were verified by Sanger sequencing Membranes were washed with TBST and incubated for 1 h with respective HRP-bound secondary antibodies After washing with TBST membranes were imaged using the ChemiDoc MP Imaging system (Bio-Rad) Images of protein structures were generated with Pymol (v.2.5.4 6 μm sections were deparaffinized and rehydrated as follows: 2 × 10 min xylene Tissue sections were stained with hematoxylin for 8 min and washed in running tap water for 10 min Slides were subsequently rinsed in dH2O followed by 10 dips in 95% EtOH tissue sections were counterstained with eosin for 1 min and dehydrated the slides were cleared for 2 × 5 min in xylene and mounted with Pertex® mounting medium (Histolab #00801) sections were washed with 1x TBS and incubated with a corresponding biotinylated secondary antibody in 5% BSA/TBS for 1 h sections were incubated with streptavidin-horseradish peroxidase (#189733 sections were washed with TBS and incubated with 3,3’-Diaminobenzidine tetrahydrochloride (DAB Germany) for visualization of antibody binding Staining was validated by microscopical acquisition and stopped with aqua bidest Counterstaining was conducted using Mayer’s hematoxylin (#109249 dehydrated with increasing ethanol concentrations and mounted using M-GLAS® mounting medium (#103973 sections from testicular tissue with full spermatogenesis were included as positive controls as well as omission and IgG controls In case the proband testicular staining pattern for a respective antibody differed from the staining pattern in the positive control the the experiment was repeated at least once Slides were evaluated and documented using a PreciPoint O8 Scanning Microsocope Olympus BX61VS Virtual Slide System Axioskop (Zeiss or an Olympus BX61 microscope with an attached Retiga 400R camera (Olympus USA) and integrated CellSens imaging software (Olympus RNA from snap-frozen testicular tissues of three controls with full spermatogenesis and infertile men with biallelic variants in PIWIL1 (M2006) M2548) was extracted using Direct-zol RNA Microprep kit (Zymo Research The quantity and quality of the isolated RNA were assessed with Qubit RNA High Sensitivity kit (Invitrogen #Q32852) and Agilent RNA Nano kit (Agilent Shapiro-Wilk test for normality of the data and Mann-Whitney U test was used for comparing the expression changes in piRNA quantities of different lengths (26-31 nt) Statistical comparisons between two groups were performed by Student’s t test or Mann-Whitney U test Experimental replicates were performed as indicated in the respective Figure legends All putative pathogenic variants were validated by Sanger Sequencing The Investigators were not blinded to allocation during experiments and outcome assessment Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article Evolutionarily conserved pachytene piRNA loci are highly divergent among modern humans A translation-activating function of MIWI/piRNA during mouse spermiogenesis Miwi catalysis is required for piRNA amplification-independent LINE1 transposon silencing Sun, Y. H., Lee, B. & Li, X. Z. The birth of piRNAs: how mammalian piRNAs are produced, originated, and evolved. Mamm. Genome https://doi.org/10.1007/s00335-021-09927-8 (2021) MITOPLD is a mitochondrial protein essential for nuage formation and piRNA biogenesis in the mouse germline piRNA-associated germline nuage formation and spermatogenesis require MitoPLD profusogenic mitochondrial-surface lipid signaling GASZ is essential for male meiosis and suppression of retrotransposon expression in the male germline MOV10L1 is necessary for protection of spermatocytes against retrotransposons by Piwi-interacting RNAs MVH in piRNA processing and gene silencing of retrotransposons Whole-exome sequencing improves the diagnosis and care of men with non-obstructive azoospermia Mutation in TDRD9 causes non-obstructive azoospermia in infertile men Diverse monogenic subforms of human spermatogenic failure The piRNA-pathway factor FKBP6 is essential for spermatogenesis but dispensable for control of meiotic LINE-1 expression in humans Genetic architecture of azoospermia-time to advance the standard of care Mili and Miwi target RNA repertoire reveals piRNA biogenesis and function of Miwi in spermiogenesis GTSF1 accelerates target RNA cleavage by PIWI-clade Argonaute proteins DNA recognition by an RNA-guided bacterial Argonaute The RNA helicase MOV10L1 binds piRNA precursors to initiate piRNA processing Zucchini consensus motifs determine the mechanism of pre-piRNA production in piRNA biogenesis in germline stem cells PNLDC1 is essential for piRNA 3’ end trimming and transposon silencing during spermatogenesis in mice HENMT1 and piRNA stability are required for adult male germ cell transposon repression and to define the spermatogenic program in the mouse Expression of MAEL in nuage and non-nuage compartments of rat spermatogenic cells and colocalization with DDX4 Cytoplasmic compartmentalization of the fetal piRNA pathway in mice Reduced pachytene piRNAs and translation underlie spermiogenic arrest in Maelstrom mutant mice Structure and function of eTudor domain containing TDRD proteins a gene encoding a protein with two copies of a CHHC Zn-finger motif is involved in spermatogenesis and retrotransposon suppression in murine testes The mouse homolog of Drosophila Vasa is required for the development of male germ cells The TDRD9-MIWI2 complex is essential for piRNA-mediated retrotransposon silencing in the mouse male germline encodes a cytoplasmic protein essential for spermatogenesis is essential for male germ-cell differentiation and nuage/germinal granule formation in mice An essential role for PNLDC1 in piRNA 3’ end trimming and male fertility in mice is essential for spermatogenesis and transposon repression in meiosis Meiotic recombination: insights into its mechanisms and its role in human reproduction with a special focus on non-obstructive azoospermia Ubiquitination-deficient mutations in human piwi cause male infertility by impairing histone-to-protamine exchange during spermiogenesis Lack of evidence for a role of PIWIL1 variants in human male infertility Generation of an iPSC line (HUSTi002-A) from fibroblasts of a patient with Sertoli cell-only syndrome carrying c.731_732delAT in PIWIL2 gene Whole-exome sequencing in patients with maturation arrest: a potential additional diagnostic tool for prevention of recurrent negative testicular sperm extraction outcomes A role for Fkbp6 and the chaperone machinery in piRNA amplification and transposon silencing An ancient transcription factor initiates the burst of piRNA production during early meiosis in mouse testes Multiple epigenetic mechanisms and the piRNA pathway enforce LINE1 silencing during adult spermatogenesis MIWI N-terminal RG motif promotes efficient pachytene piRNA production and spermatogenesis independent of LINE1 transposon silencing The non-redundant functions of PIWI family proteins in gametogenesis in golden hamsters piRNA-directed cleavage of meiotic transcripts regulates spermatogenesis The piRNA pathway is essential for generating functional oocytes in golden hamsters World Medical Association Declaration of Helsinki: ethical principles for medical research involving human subjects Krausz, C. et al. Genetic dissection of spermatogenic arrest through exome analysis: clinical implications for the management of azoospermic men. Genet. Med. https://doi.org/10.1038/s41436-020-0907-1 (2020) Oud, M. S. et al. A de novo paradigm for male infertility. Nat. Commun. https://doi.org/10.1038/s41467-021-27132-8 (2022) Cutadapt removes adapter sequences from high-throughput sequencing reads Fast and accurate long-read alignment with Burrows-Wheeler transform The Genome Analysis Toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data A single-cell type transcriptomics map of human tissues Gene ontology: tool for the unification of biology PANTHER: making genome-scale phylogenetics accessible to all Protocol update for large-scale genome and gene function analysis with the PANTHER classification system (v.14.0) REVIGO summarizes and visualizes long lists of gene ontology terms CirGO: an alternative circular way of visualising gene ontology terms Somalier: rapid relatedness estimation for cancer and germline studies using efficient genome sketches Rapid generation of splicing reporters with pSpliceExpress Identification of male germ cells undergoing apoptosis in adult rats DDX3Y is likely the key spermatogenic factor in the AZFa region that contributes to human non-obstructive azoospermia Comparative marker analysis after isolation and culture of testicular cells from the immature marmoset Ultrafast and memory-efficient alignment of short DNA sequences to the human genome DASHR 2.0: integrated database of human small non-coding RNA genes and mature products SciPy 1.0: fundamental algorithms for scientific computing in Python Download references Open Access funding enabled and organized by Projekt DEAL David MacKenzie MacLeod & Dónal O’Carroll institut de génétique médicale d’Alsace IGMA Instituto de Investigaciones Biomédicas Sant Pau Antoni Riera-Escamilla & Csilla Krausz Centre of Reproductive Medicine and Andrology Department of Clinical and Surgical Andrology Department of Gynecology and Obstetrics Novafertil IVF Center Department of Andrology Novafertil IVF Center The Newcastle upon Tyne Hospitals NHS Foundation Trust Department of Experimental and Clinical Biomedical Sciences “Mario Serio” conceived and designed the experiments and wrote the manuscript provided critical feedback and helped shape the research and manuscript All authors revised and approved the final version of the manuscript reviewers for their contribution to the peer review of this work Download citation DOI: https://doi.org/10.1038/s41467-024-50930-9 Metrics details Stem cells preferentially use glycolysis instead of oxidative phosphorylation and this metabolic rewiring plays an instructive role in their fate; however the underlying molecular mechanisms remain largely unexplored PIWI-interacting RNAs (piRNAs) and PIWI proteins have essential functions in a range of adult stem cells across species we show that piRNAs and the PIWI protein Aubergine (Aub) are instrumental in activating glycolysis in Drosophila female germline stem cells (GSCs) Higher glycolysis is required for GSC self-renewal and aub loss-of-function induces a metabolic switch in GSCs leading to their differentiation Aub directly binds glycolytic mRNAs and Enolase mRNA regulation by Aub depends on its 5′UTR mutations of a piRNA target site in Enolase 5′UTR lead to GSC loss These data reveal an Aub/piRNA function in translational activation of glycolytic mRNAs in GSCs and pinpoint a mechanism of regulation of metabolic reprogramming in stem cells based on small RNAs very little is known regarding the regulatory mechanisms underlying metabolic remodeling in stem cells despite the importance of this remodeling in stem cell fate Aub binding to glycolytic mRNAs is statistically significant p = 1.0e-5 using the Generalized hypergeometric test except the oocyte that is in purple (GSC: germline stem cell; CB: cystoblast) The spectrosome acquires an elongated form during GSC division Differentiation proceeds from anterior (left) to posterior (right) 2 and 3 contain mitotically active germ cells c–e’ Confocal images of wild-type (wt) germaria immunostained with an anti-Ald (c White arrows indicate GCSs and white arrowheads indicate the region containing germline cysts where glycolytic enzyme levels were quantified Eno (g) and PyK (h) protein levels in GSCs and differentiating cyst cells using immunostaining experiments shown in c–e’ Fluorescence intensity was measured in arbitrary units using the ImageJ software Horizontal bars represent the mean and standard deviations ****p-value <0.0001 using the paired two-tailed Student’s t test p = 1.84e−7 in (f); 5.99e−7 in (g); 8.76e−10 in (h) i–k’ Confocal images of mosaic germaria containing control (i k’) clonal cells stained with anti-GFP (green) (lack of GFP indicates clonal cells) and the yellow dashed line (j–k’) indicate mutant clonal differentiating cysts in contact with the niche Heterozygous Ald1EY13155 females in which mitotic clones were induced are not viable after 17 days l Relative percentages of germaria with at least a clonal GSC at 7 The number of germaria analyzed is indicated Data are presented as mean values with standard deviations Quantifications were from two independent experiments except for Enof07543 for which it was from one experiment showing that Ald1 and Eno mutant GSCs did not undergo apoptosis These results show that glycolytic enzymes accumulate at higher levels in GSCs than in differentiating cysts and that these higher levels of glycolytic enzymes are required for GSC self-renewal by preventing their differentiation a Scheme of Pyruvate Dehydrogenase (PDH) complex regulation by Pyruvate Dehydrogenase Phosphatase (PDP) and Pyruvate Dehydrogenase Kinase (PDK) b–f Confocal images of germaria from 7 day old-females stained with anti-Vasa (green) The white dashed line indicates two GSCs in a control nos-Gal4/+ germarium (b) and the yellow dashed lines indicate two GSCs in a nos-Gal4/UAS-Pdp-RNAi germarium (c) and a single GSC or differentiating cysts in the niche in nos-Gal4/UAS-Pdk-RNAi (d) nos-Gal4/spargelEY05931 (e) and ewgEY05137/+; nos-Gal4/+ (f) germaria g Quantification of germaria showing GSC loss (0-1 GSC) 7 and 14 days after eclosion The number of scored germaria (n) is indicated p = 0.34 between wt and Pdp RNAi; 2.4e−6 between wt and Pdk RNAi; 1.0e−5 between wt and spargelEY05931; 1.0e-5 between wt and ewgEY05137 p = 0.16 between wt and Pdp RNAi; 1.0e−6 between wt and Pdk RNAi; 1.0e−5 between wt and spargelEY05931; 1.0e−5 between wt and ewgEY05137 d–e’ Confocal images of immunostaining of wt and aubQC42/HN2 germaria with anti-Eno (green) and DAPI (blue) (a–b’) or anti-PyK (green) and anti-Hts (red) (d–e’) White and yellow arrows indicate GCSs in wt and aub mutant germaria White and yellow arrowheads indicate the region containing germline cysts where glycolytic enzyme levels were quantified f Quantification of Eno and PyK protein levels in wt and aubQC42/HN2 mutant GSCs and differentiating cyst cells using immunostaining experiments shown in (a–b’ Ratios of fluorescence intensity of one GSC to one cyst cell per germarium were plotted ****p-value < 0.0001 using the unpaired two-tailed Student’s t test g Schematic representation of the FRET Laconic sensor The binding of lactate to the ligand-binding domain leads to a conformational change that increases the distance between the donor (mTFP) and the acceptor (Venus) FRET Laconic sensor efficiency inversely correlates with lactate concentration i FRET ratio images in control nos-Gal4/UASz-Laconic (h) and aub mutant aubQC42/HN2; nos-Gal4/UASz-Laconic (i) anterior-most region of germaria Control and aub mutant GSCs are indicated with white and yellow dashed lines The rainbow colormap indicates the FRET efficiency levels j Quantification of FRET Laconic sensor efficiency in control and aub mutant GSCs from three day old-females based on acceptor photobleaching using the unpaired two-tailed Student’s t test p = 7.14e−5 between control and aubQC42/HN2 and 1.72e−4 between control and aubQC42/g1 These reduced amounts of lactate indicated lower glycolysis and/or higher oxphos in aub mutant GSCs consistent with the proposed role of Aub in activating glycolysis in GSCs Together these results show that Aub plays a key role in increasing glycolysis in GSCs expression of glycolytic enzymes is reduced in GSCs and this is accompanied by a switch in energy metabolism towards oxphos with higher expression of ATP synthase that might contribute to mitochondrial maturation Mean of two biological replicates for Hex-A and Pfk mRNAs and three biological replicates for the other mRNAs in (d) f–i Genetic interaction between aub and either Ald1 or Eno for GSC self-renewal Confocal images of immunostaining of aubHN2/+ (f) aubHN2/+; Def(Ald1)/+ (g) and aubHN2/EnoKG01162 (h) germaria with anti-Vasa (green) The white dashed line indicates two GSCs in a control aubHN2/+ germarium (f) and the yellow dashed lines indicate differentiating cysts in the niche in aubHN2/+; Def(Ald1)/+ (h) and aubHN2/EnoKG01162 (g) germaria i Quantification of germaria showing GSC loss (0-1 GSC) in heterozygous and double heterozygous mutant females of the indicated genotypes compared to the sum of GSC loss from the two corresponding single heterozygous p = 0.0084 for aubHN2/+; Df(Ald1)/+; 0.021 for aubQC42/+; Df(Ald1)/+; 0.58 aubg1/+; Df(Ald1)/+; 0.31 for aubHN2/EnoKG01162; 0.0015 for aubQC42/EnoKG01162; 0.0067 for aubg1/EnoKG01162 in lines with a positive regulation of Ald1 and Eno by Aub these results confirm the direct binding of Aub to glycolytic mRNAs and show the requirement of Aub loading for this binding genetic data provide functional evidence that Aub positively regulates glycolytic mRNAs through this interaction The sequence and occurrences of GSC Quasimodo piRNAs potentially targeting Eno are indicated Nt in blue are non-complementary to the Eno sequence Dashes and stars indicate deleted and modified nt A part of the protein sequence of Eno long isoform is shown; the modified nt do not change the coding sequence Boxed nt indicate the donor and new donor splice sites b–e Confocal images of immunostaining of wt (b) EnoΔpi12 (d) and Enopimut1 (e) germaria with anti-Vasa (green) The white and yellow dashed lines indicate GSCs in wt (b) and mutant (c-e) germaria g Quantification of germaria showing GSC loss (0–1 GSC) in wt and mutant females p = 2.0e−4 between wt and EnoΔpi11; 1.0e−4 between wt and EnoΔpi11/f07543; 0.0012 between wt and EnoΔpi12; 0.0017 between wt and EnoΔpi12/f07543 p = 8.52e−5 between wt and EnoΔpi11; 6.99e−5 between wt and EnoΔpi11/f07543; 6.0e−4 between wt and EnoΔpi12; 0.0088 between wt and EnoΔpi12/f07543 p = 0.0021 between wt and Enopimut1; 0.015 between wt and Enopimut1/f07543 p = 0.004 between wt and Enopimut1; 0.047 between wt and Enopimut1/f07543 a Schemes of Eno reporter transgenes; open boxes: nos promoter; orange boxes: Eno UTRs; green boxes: GFP coding sequence; blue box: SV40 3′UTR b–e Confocal images of immunostaining of germaria expressing Eno reporter transgenes nosP-5′Eno-GFP-3′Eno (b White and yellow arrows point to GSCs in wt and mutant germaria White and yellow arrowheads indicate the region containing germline cysts where GFP was quantified in wt and mutant germaria g Quantification of GFP levels in wt and aubQC42/g1 mutant GSCs and differentiating cyst cells using immunostaining experiments shown in (b–e) ****p-value < 0.0001 using the unpaired two-tailed Student’s t test with Welch’s correction (p = 2.36e−5) in (f) and the two-tailed Mann–Whitney test (p = 2.34e−8) in (g) these data demonstrate that Eno mRNA activation by Aub in GSCs depends on piRNA targeting and is required for GSC self-renewal They further show that Eno mRNA regulation by Aub involves its 5′UTR and likely occurs at the level of translation These data provide functional evidence that Aub-dependent activation of glycolysis is a key contribution to Aub function in GSC self-renewal the use of a different energetic pathway might favor this key selection process without affecting cell fate This indicates a role of SMEDWI-3 in mRNA regulation that might extend to metabolic mRNAs Here we show that metabolic rewiring in stem cells is regulated by Aub and piRNAs thus revealing a crosstalk between energy metabolism and the piRNA pathway Understanding how this crosstalk impacts on piRNA biology constitutes a major challenge for future studies All Drosophila stocks used in this study are listed in Supplementary Table 2 Drosophila were raised at 25 °C on standard medium The same numbers of flies were used for control and experimental crosses and kept to a maximum of 12 females and 6 males per vial Crosses were transferred to fresh vials every three days All crosses involving nos-Gal4 were performed using nos-Gal4 females The PCR fragments were amplified with Q5 High-Fidelity DNA Polymerase or Phusion® High-Fidelity DNA Polymerase and the resulting plasmids were validated by sequencing Transgenic lines were generated using PhiC31-based integration into the attP2 landing site on chromosome III PCR fragments were amplified with Q5 High-Fidelity DNA Polymerase or Phusion® High-Fidelity DNA Polymerase and the resulting plasmids were validated by sequencing The donor plasmid and pCCD6-Eno-gRNA that also produces the Cas9 enzyme were co-injected into the w1118 stock at the Madrid Drosophila Transgenesis Facility (Centro de Biología Molecular Severo Ochoa) Injected flies were individually crossed to produce independent lines and these lines were screened by PCR using a primer corresponding to the mutant sequence in Eno 5′UTR A CRISPR edited line containing this mutant sequence was not recovered but two lines containing short deletions in this region were identified (EnoΔpi11 and EnoΔpi12) Eno sequence overlapping the donor plasmid was validated in these lines The Enopimut1 allele was obtained as follows A new donor plasmid pBS-Eno-Mutpi-DsRed was generated by inserting the scarless-DsRed cassette (PBac[3xP3-DsRed]) into an TTAA site in the right homology arm of pBS-Eno-Mutpi using the NEBuilder HiFi DNA Assembly Cloning Kit (NEB) pBS-Eno-Mutpi-DsRed was co-injected with pDCC6-Eno-gRNA into the Act5c-Cas9 DNAlig4(169) stock (BL #58492) by BestGene Inc and the CRISPR mutants were selected on the basis of DsRed fluorescent eyes by BestGene Inc The DsRed cassette was removed by crosses with the PBac Transposase source stock (BL #8285) The mutant sequence was validated by sequencing the Eno region overlapping the donor plasmid The Flipase under a heat shock promoter was used to induce mitotic clones in the germline of adult females Clone induction was performed in adults to analyze to role of Ald1 and Eno in GSCs during adult stages Enof07543 and aubHN2 mutant germline clones were induced in 3 day-old females by two 37 °C heat shocks of 1 h spaced by an 8 h-recovery period at 25 °C The flies were then maintained at 25 °C until ovarian dissection 14 or 21 days following the last heat shock Ovaries were dissected in PBS followed by a fixation in 4% paraformaldehyde in PBT (PBS supplemented with 0.1% Tween 20) for 20 min at room temperature in rotation Ovaries were then rinsed for 10 min in PBT blocked with 10% bovine serum albumin (BSA) in PBT for 1 h and incubated with primary antibodies in PBT supplemented with 1% BSA overnight at 4 °C in rotation The following primary antibodies were used at the indicated dilutions: mouse anti-Hts (1B1 clone rabbit anti-Vasa (Santa Cruz Biotechnology rabbit anti-cleaved Caspase 3 (Cell Signaling and mouse anti-Atp5A (Santa Cruz Biotechnology the ovaries were washed three times for 30 min in PBT-1% BSA and then incubated with fluorescent secondary antibodies diluted in PBT-0.1% BSA for 4 h at room temperature in rotation the ovaries were incubated in 0.1 μg/mL DAPI in PBT to stain DNA They were then mounted in Vectashield mounting medium Images were acquired using a Confocal Leica SP8 and analyzed with the ImageJ software the fixation was with 5% formaldehyde in PBS for 25 min at room temperature in rotation Ovaries were then washed with PBS followed by a permeabilization step with 1% Triton X-100 in PBS for 2 h Confocal images of germaria were analyzed using ImageJ The cytoplasm of one GSC and one cyst cell in a same plane were delineated using the Freehand selections tool The GFP fluorescence intensity was measured for each cell and this quantification was replicated three times The mean of these three measurements was then calculated The ratio: intensity in GSC/intensity in cyst cell was calculated by dividing the mean intensity value obtained for the GSC by the one obtained for the cyst cell in the same plane The graphs were produced using the GraphPad Prism software The number of foci counted was divided by the cytoplasm area to obtain the number of mRNA foci/surface The graphs were produced using the GradPad Prism software The ovaries were dissected in PBS pH 7.4 and immediately fixed in 2.5% Glutaraldehyde in PHEM buffer (60 mM PIPES 4 mM MgSO4·7 H20) at room temperature for 1 h Ovaries were post-fixed with 1% osmium tetroxide for 1 h at 4 °C and then en bloc stained with 1% uranyl acetate in double-distilled H2O at 4 °C for 1 h Dehydration series were carried with out with successive ethanol baths at 30% To preserve mitochondrial crista structure dehydration steps were limited to 5 min each Ovaries were processed in a standard manner and embedded in Epoxy resin 700 nm semi-thin sections were stained with 0.1% toluidine blue to evaluate the area of interest mounted on formvar coated slotted copper grids and stained with 1.5% uranyl acetate in 70% ethanol and lead citrate Stained grids were examined under a Tecnai G2 20 S-TWIN Transmission Electron Microscope Ovaries were rinsed twice in 75% ethanol and once in Schneider’s Insect Medium (Sigma) in a glass block for disinfection They were then dissected in clean Schneider’s Insect Medium using forceps The epithelial sheath was removed from several ovarioles using needles during no more than 20 min and ovarioles were then transferred to a drop of 20 μL of 10S oil on a high-resolution microscope slide Confocal images and FRET efficiencies were acquired in vivo using a Confocal Leica SP8 microscope Quantification of FRET efficiency was based on mTFP fluorescence before and after Venus photobleaching The microscope settings were the following: for mTFP excitation was at 458 nm and the detection window between 460 and 510 nm; for Venus excitation was at 514 nm and the detection window between 560 and 620 nm The bleaching was performed at 514 nm for 2 min FRET efficiency was calculated as follows: (mTFP fluorescence intensity after Venus photobleaching)—(mTFP fluorescence intensity before Venus photobleaching)/(mTFP fluorescence intensity after Venus photobleaching) Fluorescence intensity was measured using ImageJ Similar quantifications were obtained using the LEICA FRET-AB application Ovaries were dissected in Schneider’s Insect Medium (Sigma) They were stained with Red CMX Ros MitoTracker (Invitrogen M7512) at 500 nM for 30 min at room temperature with rotation ovaries were fixed in 4% paraformaldehyde in PBS for 20 min at room temperature the ovaries were incubated in 0.1 μg/mL DAPI in PBT for 10 min at room temperature The ovaries were washed three times for 10 min in PBS and mounted in Vectashield mounting medium Images were acquired using a Leica SP8 Confocal microscope and analyzed using the ImageJ software The cytoplasm of GSCs was delineated using the Freehand selections tool The MitoTracker fluorescent signal over the threshold of 10% identified mitochondria and was defined as ROI; its total surface was calculated in µm2 using the Measure tool They were stained with Deep Red FM MitoTracker (ThermoFisher Scientific) at 500 nM during 15 min at room temperature with rotation TMRM (Biotium) was then added at 100 nM to the previous mix for 15 min at room temperature ovarioles were mounted in PBS on glass slides and immediately imaged using a Leica SP8 confocal microscope Acquired images were analyzed using ImageJ Ratiometric images were generated as follows After inverting the colors for each channel and changing them to gray Ratiometric images were then obtained by dividing the TMRM channel by the MitoTracker channel using the Image Calculator function of ImageJ The color of the resulting image was changed for Rainbow RGB A scale bar and calibration bar were added Quantification of mitochondrial membrane potential was performed as follows The cytoplasm of one or two GSCs per germarium was delineated using the Freehand selections tool The fluorescence intensity of the two channels were measured and the value obtained for the TMRM channel was divided by the value obtained for the MitoTracker channel Transcript quantification was done using --validateMappings option with the raw data on Drosophila melanogaster genome (FlyBase release 6.28) Each RNA-seq dataset was analyzed independently and glycolytic mRNAs were filtered using thresholds of minimum expression of 10 and 100 Transcripts Per Million (TPM) in all replicates The remaining reads were then filtered by size and the pool was cleaned by collapsing identical sequences and attributing a unique identifier to each along with the number of times a read had been retrieved in the whole pool We got a library of 4,370,902 sequences of putative piRNAs The library was then annotated by independent mappings against transposable elements and piRNA clusters (from piPipes) and Drosophila transcriptome (from FlyBase) with the following bowtie’s options (transposons: -v 3 -a --best --strata; piRNA clusters: -v 0 -m 1; transcriptome: -v 0 -m 1 --norc) annotations were next merged with sequence headers alongside the unique identifier Unannotated piRNAs were kept and their annotation field complemented with a « 0 » Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article Somatic oxidative bioenergetics transitions into pluripotency-dependent glycolysis to facilitate nuclear reprogramming Reprogramming of human primary somatic cells by OCT4 and chemical compounds The senescence-related mitochondrial/oxidative stress pathway is repressed in human induced pluripotent stem cells Metabolic oxidation regulates embryonic stem cell differentiation Coordinated changes of mitochondrial biogenesis and antioxidant enzymes during osteogenic differentiation of human mesenchymal stem cells Energy metabolism in the acquisition and maintenance of stemness The distinct metabolic profile of hematopoietic stem cells reflects their location in a hypoxic niche Mitochondria and the dynamic control of stem cell homeostasis Metabolic remodeling in early development and cardiomyocyte maturation Regulation of mitochondrial function and cellular energy metabolism by protein kinase C-lambda/iota: a novel mode of balancing pluripotency Functions of PIWI proteins in gene regulation: new arrows added to the piRNA Quiver A novel class of evolutionarily conserved genes defined by piwi are essential for stem cell self-renewal is required for germ line stem cell self-renewal and appears to positively regulate translation Aubergine and piRNAs promote germline stem cell self-renewal by repressing the proto-oncogene Cbl Uniting germline and stem cells: the function of Piwi proteins and the piRNA pathway in diverse organisms The piRNA pathway in planarian flatworms: new model Couples piRNA Amplification in Nuage to Phased piRNA Production on Mitochondria Daedalus and Gasz recruit Armitage to mitochondria bringing piRNA precursors to the biogenesis machinery The genetic makeup of the Drosophila piRNA pathway Glycolytic enzymes localize to ribonucleoprotein granules in Drosophila germ cells bind Tudor and protect from transposable elements Green fluorescent protein tagging Drosophila proteins at their native genomic loci with small P elements ATP synthase promotes germ cell differentiation independent of oxidative phosphorylation Electron transport chain biogenesis activated by a JNK-insulin-Myc relay primes mitochondrial inheritance in Drosophila The BDGP gene disruption project: single transposon insertions associated with 40% of Drosophila genes The FLP recombinase of yeast catalyzes site-specific recombination in the Drosophila genome Mitochondrial remodelling is essential for female germ cell differentiation and survival Ectopic expression of the Drosophila Bam protein eliminates oogenic germline stem cells Regulation of PDH activity and isoform expression: diet and exercise Dynein light chain interacts with NRF-1 and EWG structurally and functionally related transcription factors from humans and drosophila Transcriptional paradigms in mammalian mitochondrial biogenesis and function The Drosophila PGC-1 homologue Spargel coordinates mitochondrial activity to insulin signalling Mitochondrial dynamics in the Drosophila ovary regulates germ stem cell number Upregulated energy metabolism in the Drosophila mushroom body is the trigger for long-term memory A genetically encoded FRET lactate sensor and its use to detect the Warburg effect in single cancer cells Photoconversion of YFP into a CFP-like species during acceptor photobleaching FRET experiments Live imaging of the Drosophila ovarian niche shows spectrosome and centrosome dynamics during asymmetric germline stem cell division Defining the expression of piRNA and transposable elements in Drosophila ovarian germline stem cells and somatic support cells A satellite repeat-derived piRNA controls embryonic development of Aedes Control of glycolysis through regulation of PFK1: old friends and recent additions Metabolic regulation of stem cell function in tissue homeostasis and organismal ageing UCP2 regulates energy metabolism and differentiation potential of human pluripotent stem cells Ramat, A. et al. The PIWI protein Aubergine recruits eIF3 to activate translation in the germ plasm. Cell Res. https://doi.org/10.1038/s41422-020-0294-9 (2020) Kinetic coupling of the respiratory chain with ATP synthase drives ATP production in cristae membranes Cristae formation-linking ultrastructure and function of mitochondria The expression profile of purified Drosophila germline stem cells Mitochondrial fragmentation drives selective removal of deleterious mtDNA in the germline Efficient expression of genes in the Drosophila germline using a UAS promoter free of interference by Hsp70 piRNAs Imaging translation dynamics in live embryos reveals spatial heterogeneities Efficient CRISPR/Cas9 plasmids for rapid and versatile genome editing in Drosophila Salmon provides fast and bias-aware quantification of transcript expression piPipes: a set of pipelines for piRNA and transposon analysis via small RNA-seq BEDTools: a flexible suite of utilities for comparing genomic features Download references This work was supported by the CNRS-University of Montpellier UMR9002 FRM (Equipe FRM EQU202303016322) and MSDAvenir PRR held a salary from CNRS and VI PPIT-University of Seville JC held a salary from ANR and LabUM Montpellier CG held a salary from ANR and MSDAvenir; and AR held a salary from ANR These authors contributed equally: Aymeric Chartier performed experiments and analyzed the data wrote the manuscript with inputs from all authors Nature Communications thanks the anonymous Download citation DOI: https://doi.org/10.1038/s41467-024-52709-4 Metrics details Heart regeneration after tissue injury depends on the proliferation of existing cardiomyocytes Manipulating the non-coding transcriptome holds promise for the therapeutic induction of cardiomyocyte proliferation in the damaged human heart A study now demonstrates that piRNAs have key roles in this regenerative process Ma, W. et al. Nat. Cardiovasc. Res. https://doi.org/10.1038/s44161-024-00592-z (2025) Download references School of Cardiovascular and Metabolic Medicine & Sciences British Heart Foundation Centre of Research Excellence Reprints and permissions Download citation DOI: https://doi.org/10.1038/s44161-024-00590-1 Metrics details The Developmental Origins of Health and Disease (DOHaD) concept explores the link between exposure to adverse conditions during fetal and early childhood development and the onset of chronic non-communicable diseases Changes in epigenetics that control gene expression have been identified as potential contributors to the developmental origin of PCa control transposable elements (TEs) and maintain genome integrity in germ cells stress-induced deregulation of TEs warrants investigating the role of piRNAs in the prostate gland from the DOHaD perspective This study aimed to detect and characterize piRNA expression in the ventral prostate (VP) of Sprague Dawley rat offspring at 21 postnatal days (PND21) and PND540 The rats were subjected to maternal protein restriction during pregnancy and lactation to understand its impact on prostate development and aging Histological analyses showed that the gestational and lactation low-protein diet (GLLP) group experienced a delay in prostate gland development with increased stromal and epithelial compartments and decreased luminal compartments during early life Aging in this group resulted in decreased luminal compartments and increased stromal areas Epithelial atrophy was observed in both groups with an increased incidence of carcinoma in situ in the GLLP group Small RNA sequencing from control and restricted groups (at PND21 and PND540) identified piRNA clusters in both young and aged animals We also detected the expression of PIWI genes (Riwi Our data highlight the key role of maternal malnutrition in modulating piRNA expression in the offspring’s VP with the potential to influence prostate developmental biology and the risk of prostatic disorders with aging which may interfere with health or disease during aging Although most studies describe the role of epigenetic mechanisms associated with embryonic development and cancer biology we employed small RNA sequencing (sRNAseq) to investigate the presence of piRNA pathways during early life and aging in prostate rats subjected to MPR Our objective was to characterize piRNAs in the VP of male Sprague Dawley rats exposed to MPR during pregnancy We confirmed the expression of piRNA sequences in the rat prostate and linked this mechanism to dysregulation associated with aging These findings provided a foundation for pioneering research into the role of piRNA clusters in the prostate of rats exposed to MPR The male offspring were euthanized on a postnatal day (PND) 21 (weaning) (n = 12/group) and PND 540 (n = 12/group) had free access to a normal protein diet after weaning until the end of the experiment The animals were euthanized by an overdose of anesthesia (ketamine/xylazine) followed by decapitation and weighing and the blood and ventral prostate (VP) were collected and processed by a different analysis as described below and hormonal levels were analyzed using a Student t-test and statistical differences were considered when p < 0.05 housing and use of animals were performed accordingly with the appropriate guidelines and regulations Efforts were made to minimize suffering and to reduce the animal numbers used in the experiments The acquisition and description of data followed the recommendations set out in the ARRIVE guidelines stained with hematoxylin–eosin (HE) for an overview of glandular morphology RNA extraction was performed with Trizol (Ambion Four samples from the CTR group and 4 samples from the GLLP group were used for RNA sequencing The samples were quantified using the Nanodrop and had their integrity inferred by the Bioanalyzer keeping only samples with RIN (RNA Integrity Number) values greater than 8 An aliquot of unfractionated total RNA was submitted for library construction and sequencing Ribo-Zero was used during library preparation for rRNA depletion Purification of small RNAs was carried out using the TruSeq Standard mRNA Sample Preparation Kit (Illumina) and TruSeq Small RNA Library Preparation Kit (Illumina) and construction of the libraries followed the manufacturer’s specifications Sequencing was performed with the HiSeq Sequencing System Macrogen (Seoul-South Korea) processed the entire library preparation and sequencing process The sequencing data is deposited in the Gene Expression Omnibus (GEO DataSets) under accession code GSE180674 (small non-coding RNAs) RT-qPCR reactions were performed in duplicates for each target gene on the QuantStudio 12 K flex Real-Time PCR System (Applied Biosystems USA) in 96 wells per plate and normalized by the ratio between the reference genes (Gapdh and Gusb) the appropriate statistical analyses were carried out Statistical analyses were performed in the RStudio® development environment (RStudio for Ubuntu version 9.3-2024) and using the ggplot2 package26 The results were subjected to the normalization test (Shapiro–Wilk) and the others were subjected to the Mann–Whitney test Data were expressed as mean ± Standard Deviation (SD) and differences were considered statistically significant when p < 0.05 This study was performed in line with the principles of ethics Approval was granted by the Biosciences Institute/UNESP Ethics Committee for Animal Experimentation (Protocol #1178) Representative histological sections of the VP lobes from the CTR and GLLP groups on PND 21 and 540 (A,C) Glandular growth in the GLLP group on PND 21 was impaired compared to the CTR the carcinoma in situ was highlighted by the dashed circle piRNA clusters expressed in prostate from R (A) The UpsetPlot represents the intersections between the clusters found in the prostate with the testis reference data (B) Number of reads (standardized in Log10) of the sRNAs by the sequence length distribution this comparison showed that there was no overlap between miRNA from the same RNAseq meaning that the sequences predicted in the cluster have a profile more similar to piRNA Known piRNAs sequences that presented similarity with the clusters predicted in the prostate The overlapping means that the known piRNA is found simultaneously in more than one cluster from the different groups The numbers represent the number of piRNAs shared or unique to each condition The chromosome where a certain piRNA is located is also represented by the side column Distribution of TEs present in the piRNA clusters expressed in testis and prostate The diameter of the circles indicates the abundance of each TE in the piRNA clusters expressed in the samples The squares highlight a lesser abundance of piRNA derived from retrotransposons L1 and ERVK expressed in older (PND 540) animals (A) Riwi (PIWI1) expression at ages 21 and 540 days (B) Rili (PIWI2) expression at PND 21 and 540 (C) Rili2 (PIWI4) expression at PND 21 and 540 Fold change is indicated by the lateral vertical bars The asterisk indicates the statistical difference between samples and was applied to Student’s t-test (p < 0.05) The central bars indicate the standard deviation (SD) We analyzed RNAseq data of the ventral prostate to provide new insights into the association of maternal malnutrition with the deregulation of piRNAs in male offspring rats we identified the differential expression profile of the VP piRNAs between young and old offspring These analyses revealed that both aging and maternal malnutrition altered epigenetic markers involved in prostate developmental biology and aging contributing to the increased incidence of prostate disorders in old male offspring rats There is an extensive discussion about how exposure to adverse conditions during the first stages of life can increase the propensity for the development of chronic diseases in offspring in adulthood Our results demonstrated an increased incidence of carcinoma in situ in the GLLP group demonstrating the potential of gene expression and epigenetic modulating mechanisms involved Compiling epigenomic analyses of VP in early life and aging we found a profile of unique and differentially found piRNA clusters in the prostate of young and old rats exposed to MPR characterizing piRNAs as potential modulators of prostate carcinogenesis These results show the epigenetic potential of maternal exposure to nutritional adversities in addition to providing the characterization of piRNA in prostate tissue being the first study to highlight the correlation of this pathway between prostate and MPR our findings highlight the emerging role of piRNAs in somatic tissues The nine clusters commonly found between the prostate and testis indicate that these are specifically essential for regulatory cell functions as they were not affected independently by diet or aging in the prostate and are present in germinal tissues (testis) Both prostate and testis overlapped in the analysis which in addition to differing in miRNAs also confirmed that they were piRNAs being expressed about reads in the association of a prostate CTR 21 cluster in common with the testis the number of reads was statistically higher in the prostate again emphasizing that the role of piRNAs is essentially characterized in gonads but is differentially regulated in somatic cells we presented the piRNA expression profile in rat VP within the context of the DOHaD concept The young rats had more piRNA clusters than old ones and diet influenced these cluster expressions the number of transposable elements in piRNA clusters varied with age and the dam’s diet these data highlighted the key role of maternal malnutrition in altering epigenetic mechanisms involved in prostate developmental biology early in life with long-lasting consequences for the incidence of prostate disorders in old offspring rats The data that support the findings of this study are openly available in The Gene Expression Omnibus (GEO) (https://www.ncbi.nlm.nih.gov/gds) on accession number(s) GSE180674 and SRP309332 and disability in older adults—Present status and future implications Statin safety and associated adverse events: A scientific statement from the American Heart Association blood pressure in childhood and adult life Developmental origins of health and disease: Brief history of the approach and current focus on epigenetic mechanisms The role of the prostate in male fertility The inflammatory microenvironment and microbiome in prostate cancer development Maternal low-protein diet impairs prostate growth in young rat offspring and induces prostate carcinogenesis with aging Impairment of microvascular angiogenesis is associated with delay in prostatic development in rat offspring of maternal protein malnutrition Impact of long-term high-fat diet intake on kidney morphology and function in gestational protein-restricted offspring PIWI-interacting RNAs: Small RNAs with big functions The biogenesis and functions of piRNAs in human diseases PIWI-interacting RNA: Its biogenesis and functions piR-001773 and piR-017184 promote prostate cancer progression by interacting with PCDH9 Streptozotocin-induced maternal hyperglycemia increases the expression of antioxidant enzymes and mast cell number in offspring rat ventral prostate Identification of potential molecular pathways involved in prostate carcinogenesis in offspring exposed to maternal malnutrition Increased oxidative stress and cancer biomarkers in the ventral prostate of older rats submitted to maternal malnutrition FastQ screen: A tool for multi-genome mapping and quality control Protract—A software for probabilistic piRNA cluster detection BEDTools: A flexible suite of utilities for comparing genomic features Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method A greedy algorithm for aligning DNA sequences ggplot2: Elegant Graphics for Data Analysis (Springer Early-life origin of prostate cancer through deregulation of miR-206 networks in maternally malnourished offspring rats MicroRNA regulation of the proliferation and apoptosis of Leydig cells in diabetes Roles of transposable elements in the regulation of mammalian transcription In utero exposure to maternal low protein diets induces hypertension in weanling rats independently of maternal blood pressure changes Small RNAs as biomarkers to differentiate benign and malign prostate diseases: An alternative for transrectal punch biopsy of the prostate Piwi-interacting RNAs: A new class of regulator in human breast cancer Conserved piRNA expression from a distinct set of piRNA cluster loci in eutherian mammals piRNA PROPER suppresses DUSP1 translation by targeting N6-methyladenosine-mediated RNA circularization to promote oncogenesis of prostate cancer Beyond transposons: The epigenetic and somatic functions of the Piwi-piRNA mechanism The emerging roles of PIWI-interacting RNA in human cancers Monitoring the interplay between transposable element families and DNA methylation in maize Transposable element regulation and expression in cancer Integrated transcriptome and proteome analysis indicates potential biomarkers of prostate cancer in offspring of pregnant rats exposed to a phthalate mixture during gestation and lactation Non-gonadal somatic piRNA pathways ensure sexual differentiation piRNA-DQ722010 contributes to prostate hyperplasia of the male offspring mice after the maternal exposed to microcystin-leucine arginine piRNAs and PIWI proteins as diagnostic and prognostic markers of genitourinary cancers Download references This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior—Brazil (CAPES)—Finance Code 001; Sao Paulo State Research Foundation FAPESP (2022/04339-1 and 2022/03990-0); Conselho Nacional de Desenvolvimento Científico e Tecnológico (311481/2021-3) These authors contributed equally: Jordana I Department of Structural and Functional Biology All authors read and approved the final manuscript and J.L.A designed the experiment and wrote the manuscript B.H.S and P.L.M.F performed the data analysis J.L.A and O.J.I.N supervised all the processes Download citation DOI: https://doi.org/10.1038/s41598-024-77901-w Metrics details The discovery of diverse functions and mechanisms in cancer has underscored the significance of emerging non-coding RNAs (ncRNAs) such as PIWI-interacting RNAs (piRNAs) and circular RNAs (circRNAs) Understanding their role in clear cell renal cell carcinoma (ccRCC) is imperative and necessitates comprehensive investigation This study aims to further explore the diagnostic potential of piRNAs and circRNAs for ccRCC The dysregulated piRNAs and circRNAs in ccRCC were identified using small RNA (sRNA) high-throughput sequencing technology while their expression in clinical samples was assessed by RT-qPCR A paired t-test was performed to compare the expression levels of piRNAs and circRNAs between ccRCC and adjacent tissues ROC curve analysis was conducted to evaluate the diagnostic specificity and area under the curve (AUC) of piRNAs and circRNAs High-throughput sequencing revealed a significant downregulation of 17 piRNAs and 694 circRNAs in ccRCC tissues accompanied by a significant upregulation of 5 piRNAs and 490 circRNAs RT-qPCR analysis demonstrated markedly lower expression levels of piR-has-150997 and uniq-84737 in the ccRCC group compared to the adjacent tissue group (p < 0.05) When considering the combined detection of piR-hsa-150997 the diagnostic AUC for ccRCC was found to be high at an approximate value of AUC = 0.878 The diagnostic performance of piR-has-150997 and circARID1B_037 demonstrates promise for ccRCC and circARID1B_037 could serve as an ideal diagnostic marker system with significant clinical utility The clinical tissues utilized in this study were obtained from patients who underwent surgical tumorectomies at the Department of Urology First Affiliated Hospital of Jinan University the tissues were immediately immersed in RNA-later for preservation and subsequently refrigerated at 4 °C overnight the samples were transferred to liquid nitrogen for long-term storage Histological and pathological diagnoses of the clinical samples were confirmed by experienced clinical pathologists as per the study protocol all patients provided written informed consent for their tissue utilization in research and the study protocol was approved by the Ethical Committee of the First Affiliated Hospital of Jinan University in accordance with the Declaration of Helsinki Statistical analyses were performed using SPSS 26.0 (IBM Corp. USA) and GraphPad Prism 7.0 (GraphPad Software The differences between ccRCC tissues and their paired adjacent tissues were evaluated using the Student’s t-test Statistical significance was defined as p values < 0.05 (A) Predicted distribution of the first nucleotide of piRNAs (B) Base distribution of predicted piRNAs at each position To characterize the genomic regions where piRNAs are distributed, putative piRNAs were aligned to transposon and gene sequences (Fig. 1D) Analysis revealed that the read count of piRNAs originating from repeat sequence regions exceeded that from gene regions (37,128; 0.42%) examination of the unique piRNA sequence distribution across the genome indicated that 15.84% (14,803 reads) aligned with repeat sequence regions while only 1.34% (1255 reads) mapped to gene regions (A) Differential piRNA volcano plot and heatmap (B) Volcano plot for differential expression analysis of piRNA clusters (C) Cluster_heatmap of differential piRNA cluster expression levels B) Expression of differential piRNAs in clinical samples (n = 68) Heatmap and volcano plot of differentially expressed circRNAs and the distribution of circRNAs on chromosomes shown in a circos plot Consistent with the sequencing data, RT-PCR analysis demonstrated significantly upregulated expression levels of circABCC1 and circNETO2_006, while downregulation was observed in the expression level of circARID1B_037 in the adjacent tissue group (p < 0.05) (Fig. 5). Expression of circRNAs in clinical samples (n = 68) (A) Diagnostic value of differential piRNAs and their parental gene expression in ccRCC (n = 68) (B) Diagnostic value of differential circRNAs and their parental gene expression in ccRCC (n = 68) The diagnostic value of differential circRNAs and differential piRNAs expression in ccRCC (n = 68) elucidating the role and mechanism by which circRNAs function in kidney cancer is an urgent topic necessitating further investigation circRNAs can be internalized by distant cells exerting influence on crucial biological pathways in these recipient cells and potentially facilitating tumor metastasis circRNAs exhibit immense potential as diagnostic and prognostic biomarkers as well as appealing therapeutic targets for renal cancer Our study aims to investigate the roles and mechanisms of circRNAs and piRNAs in ccRCC This involves utilizing high-throughput sequencing to detect aberrantly expressed circRNAs and piRNAs in ccRCC followed by screening for differentially expressed ones and validating them using clinical samples we analyze the diagnostic value of circRNAs and piRNAs in ccRCC Key findings include the identification of 17 upregulated and 5 downregulated piRNAs in tumor tissues compared to normal tissues as well as 694 downregulated and 490 upregulated circRNAs in ccRCC tissues significant reductions were observed in specific piRNA species such as piR-has-150997 and uniq-84737 when comparing ccRCC samples with normal tissues elevated expression levels were detected for circABCC1 and circNETO2_006 while decreased expression was found for circARID1B_037 in ccRCC tissues We evaluated the diagnostic performance of these identified circRNAs and piRNAs using AUC values which indicated good diagnostic potential a combined diagnostic model based on differentially expressed circRNAs and piRNAs demonstrated even higher AUC values suggesting its potential use as a superior diagnostic marker system for ccRCC The main objective of this study was focused on detection and verification of pathological specimens it should be noted that the evidence provided here is restricted to only one source with a limited number of samples In order to augment the significance attributed to piRNAs and circRNAs in both diagnosis as well as treatment approaches for renal cell carcinoma (ccRCC) expanding sample sizes for further validation is highly recommended there exists a research gap pertaining to variations in expression levels among these biomarkers within urine as well as serum when considering ccRCC diagnosis or treatment options Future research endeavors should strive towards addressing this knowledge void through refinement of research plans along with exploration into new investigative pathways thus contributing towards an enhanced comprehension surrounding potential diagnostic capabilities alongside therapeutic roles played by piRNAs and circRNA molecules within ccRCC management practices The ultimate goal of these endeavors is to develop pioneering strategies that efficiently handle patients with ccRCC Abnormal expression of piRNAs in ccRCC included 17 significantly downregulated and 5 significantly upregulated piRNAs as well as 2 downregulated and 1 upregulated piRNA clusters in tumor tissues and uniq-84737 were significantly lower in the ccRCC group compared to the normal tissue group all showing excellent diagnostic performance for ccRCC Abnormal expression of circRNAs in ccRCC included significant downregulation of 694 circRNAs and significant upregulation of 490 circRNAs in tumor tissues The expressions of circABCC1 and circNETO2_006 were significantly higher than those in the adjacent tissue group while circARID1B_037 was significantly lower Combining specific piRNAs improved the diagnostic performance for ccRCC with an AUC of 0.878 for the combined diagnostic model including piR-hsa-150997 uniq_84737,circABCC1,circNETO2_006,andcircARID1B_037 and circARID1B_037 also had a high AUC of 0.872 These two models may serve as effective clinical diagnostic markers Date is provided within the manuscript or contact this e-mile (bolyheng@126.com) for more date information Renal cell carcinoma: An overview of the epidemiology Trends in cause of death among patients with renal cell carcinoma in the United States: A SEER-based study Insights into the genetic and epigenetic mechanisms governing X-chromosome-linked-miRNAs expression in cancer; A step-toward ncRNA precision In silico analysis and comprehensive review of circular-RNA regulatory roles in breast diseases; A step-toward non-coding RNA precision A comprehensive insight and in silico analysis of CircRNAs in hepatocellular carcinoma: A step toward ncRNA-based precision medicine Shaker, F. H. et al. piR-823 tale as emerging cancer-hallmark molecular marker in different cancer types: a step-toward ncRNA-precision. Naunyn Schmiedebergs Arch. Pharmacol. https://doi.org/10.1007/s00210-024-03308-z (2024) Comprehensive profling of circRNAs and the tumor suppressor function of circHIPK3 in clear cell renal carcinoma Epigenetic loss of the PIWI/piRNA machinery in human testicular tumorigenesis piRNA-14633 promotes cervical cancer cell malignancy in a METTL14-dependent m6A RNA methylation manner Overexpression of piRNA pathway genes in epithelial ovarian cancer piRNA: A promising biomarker in early detection of gastrointestinal cancer Senescent neutrophils-derived exosomal piRNA-17560 promotes chemoresistance and EMT of breast cancer via FTO-mediated m6A demethylation piRNA-823 contributes to tumorigenesis by regulating de novo DNA methylation and angiogenesis in multiple myeloma Deep sequencing of small RNA transcriptome reveals novel non-coding RNAs in hepatocellular carcinoma migration and invasion in colorectal cancer Identification of novel piRNAs in bladder cancer is aberrantly expressed in human cancer cells Small RNA guides for de novo DNA methylation in mammalian germ cells Role for piRNAs and noncoding RNA in de novo DNA methylation of the imprinted mouse Rasgrf1 locus Detection of circulating tumor cells in peripheral blood from patients with gastric cancer using piRNAs as markers demonstrates in vitro and in vivo tumor suppressive activity in human gastric cancer cells Mitochondrial PIWI-interacting RNAs are novel biomarkers for clear cell renal cell carcinoma G9a-mediated irreversible epigenetic inactivation of Oct-3/4 during early embryogenesis Altered MiRNA expression in gastric cancer: A systematic review and meta-analysis The regulatory function of piRNA/PIWI complex in cancer and other human diseases: The role of DNA methylation Effect of vitamin D supplementation on primary dysmenorrhea: A systematic review and meta-analysis of randomized clinical trials Role of microRNA 21 in mesenchymal stem cell (MSC) differentiation: a powerful biomarker in MSCs derived cells miRNA-24 and miRNA-466i-5p controls inflammation in rat hepatocytes Download references Funding was provided by the Medical Research Fund of Guangdong Province (Grant No A2019571) and Basic Research Fund of Central universities (Grant No Yingzhi Zhang and Jing Luo contributed equally to this work The First Affiliated Hospital of Jinan University The First College of Clinical Medical Science The Sixth Affiliated Hospital of Jinan University conceptualized and designed the study; Y.Z contributed to formal analysis of data; Y.X. was responsible for project ad-ministration; Y.X. All samples were obtained by protocols approved by the Ethics Committee of the First Affliated Hospital of Jinan University in accordance with the Declaration of Helsinki Informed consent was obtained from each patient Download citation DOI: https://doi.org/10.1038/s41598-025-90874-8 Sign up for the Nature Briefing: Cancer newsletter — what matters in cancer research, free to your inbox weekly. Volume 9 - 2021 | https://doi.org/10.3389/fcell.2021.641052 This article is part of the Research TopicCancer Stem Cells and their Regulation by Non-Coding GenomesView all 11 articles Cancer stem cells (CSCs) are believed to be the main source of cancer relapse and metastasis PIWI-interacting small non-coding RNAs (piRNAs) have been recently recognized to be relevant to cancer biology Whether and how piRNAs regulate human CSCs remain unknown upregulation of piR-823 was identified in tested luminal breast cancer cells especially in the luminal subtype of breast CSCs Enforced expression or targeted knockdown of piR-823 demonstrated its oncogenic function in regulating cell proliferation and colony formation in MCF-7 and T-47D breast cancer cells piR-823 induced ALDH (+) breast CSC subpopulation promoted the expression of stem cell markers including OCT4 Tail vein injection of magnetic nanoparticles carrying anti-piR-823 into the mammary gland of tumor-burdened mice significantly inhibited tumor growth in vivo DNA methyltransferases (DNMTs) including DNMT1 and DNMT3B were demonstrated to be the downstream genes of piR-823 which regulate gene expression by maintaining DNA methylation promoted DNA methylation of gene adenomatous polyposis coli (APC) thereby activating Wnt signaling and inducing cancer cell stemness in the luminal subtype of breast cancer cells The current study not only revealed a novel mechanism through which piRNAs contribute to tumorigenesis in breast cancer by regulating CSCs but also provided a therapeutic strategy using non-coding genomes in the suppression of human breast cancer These piRNAs showed altered expression in cancer cells Although piRNAs are supposed to regulate tumorigenesis and tumor progression by epigenetic regulation at the genome DNA level and/or gene translation at the message RNA level the mechanisms regulating CSCs are yet to be determined Targeting piRNAs in suppressing CSCs may provide a novel strategy to treat cancer patients especially in the prevention of CSC-induced drug resistance and cancer relapse overexpression of piRNA-823 was first observed in the luminal subtype of breast cancer and the tumor regenerative abilities of both MCF-7 and T-47D breast cancer cells piRNA-823 induced stemness and expansion of breast CSCs by regulating DNA methylation and activating Wnt signaling A nanoparticle-based gene therapy targeting piRNA-823 efficiently inhibited tumorigenesis and tumor growth in a mice xenograft model transplanted with MCF-7 cells Human breast cancer samples were collected from Tongji University Shanghai East Hospital All the procedures were approved by the Institutional Review Board (IRB) of Tongji University Shanghai East Hospital All patients were provided with a written informed consent form Four to six-week-old female nude mice were purchased from the Silaike Animal Company (Shanghai All animal procedures were approved by the Institutional Animal Care and Use Committee of the Tongji University School of Medicine originally obtained from the American Type Culture Collection (ATCC) and cultured at 37°C with 5% CO2 in Dulbecco’s Modified Eagle’s Medium (DMEM) medium supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin–streptomycin All oligos for piRNA mimics or antisense inhibitors were synthesized by GenScript (Nanjing The sequence for the piR-823 mimic is: 5′ r(AGCGUUGGUGGUAUAG UGGUGAGCAUAGCUGC)dTdT 3′ (double-strand); for anti-piR-823 is: 5′ G∗C∗AGCUAUGCUwCACCACUAUACCACC AAC∗G∗C∗U∗ (2-O-Methyl to all bases) Oligo transfection was performed using RNAiMAX (Invitrogen) following the manufacturer’s instructions A final concentration of 30 nM was used in all in vitro assays RNA extraction, small RNA reverse transcription, and piRNA real-time PCR analysis were performed following the procedure described in our previous publication (Lü et al., 2020) The sequence for the piR-823 primer is: 5′ AGCGTTGGTGGTATAGTGGT 3′ Canada) was used for ALDH analysis in breast cancer cells following the manufacturer’s instructions trypsinized single cells were suspended in the buffer containing the ALDEFLUOR substrate incubated for 30 min at 37°C with or without the aldehyde dehydrogenase inhibitor DEAB flowed by FACS analysis using a FACScan flow cytometer (BD Biosciences Data were analyzed with the FlowJo software Cancer stem cells were seeded into a 6-well ultra-low attachment plate (Corning United States) with a density of 3,000 cells/well and cultured in DMEM/F12 containing 1xB27 supplement (Invitrogen) 20 ng/mL of human epidermal growth factor (EGF; Sigma) and 20 ng/mL of human basic fibroblast growth factor (bFGF; R&D Systems) for 7–10 days The mammospheres with diameter greater than 40 μm were counted under a microscope for quantitative analysis The procedures for western blot analysis were the same as the procedure described by our previous publication (Xu et al., 2020) The primary antibodies (1:2,000) we used include: OCT4 (2750S Cell Signaling Technology) were used as secondary antibodies (1:5,000) The TOP-LUC/FOP-LUC reporter structures were described previously (Wang et al., 2018) MCF-7 cells were seeded on 12-well plates at a density of 1 × 105 cells/well cells were co-transfected using lipofectamine 2000 (Invitrogen) with 0.5 μg of TOP or FOP reporter vector and 0.1 μg of pTK-RL plasmid the activities of firefly and Renilla luciferase were detected by AutoLumat using the Dual-Luciferase Reporter Assay kit (Promega United States) following the manufacturer’s instructions DNA was prepared from cells using a FastPure Cell/Tissue DNA Isolation Mini Kit (Vazyme Conversion of methylated cytosine to uracil was mediated by bisulfite treatment using the DNA Bisulfite Conversion Kit (Tiangen Methylation-specific PCR (MSP) primers were designed using Methyl Primer Express Software v1.0 (Epigentek Group Inc. PCR was performed using a T100TM Thermal Cycler (Bio-Rad The MethylFlashTM Global DNA Methylation (5 mC) ELISA Kit (EpiGenie United States) was used to quantify the global DNA methylation status of cell samples The fluorescence signals represents the methylation of DNA Matrigel was mixed with 2 × 106 MCF-7 cells and injected into the fat pat of the fourth mammary gland of a nude mouse (n = 16) The mice were separated randomly into two groups (n = 8 for each group) and tail vein-injected with magnetic nanoparticles carrying either anti-piR-823 mimics or anti-NC oligo (0.25 mg/kg body weight per dose every 2 days) The magnetic nanoparticles were prepared with the modified Zn0.4Fe2.6O4@SiO2 nanoparticles and the anti-piR-823 mimic oligo or anti-NC control oligo (2: 1 at room temperature) a piece of magnet was placed near the fourth mammary gland for 1 h to enrich the nanoparticles into the tumor tissues The volume of tumors was measured every 5 days until day 30 after cell transplantation when all the mice were sacrificed Tumors were separated and subjected to further analysis Data are presented as mean ± SEM unless otherwise stated Statistical significance was determined by a standard two-tailed Student’s t-test and one-way ANOVA followed by least-significant difference (LSD) P < 0.05 was considered statistically significant Upregulation of piRNA-823 in ALDH + breast cancer stem cells (B) piR-823 showed upregulation in the ALDH + subpopulation of MCF-7 cells (C) piR-823 expression analysis in different subtypes of breast cancer cell lines (D) Upregulation of piR-823 in the tumor samples from luminal breast cancer patients compared to the matched normal tissue from the same patient (n = 9) (E) ALDH + CSC isolation from breast cancer patients (F) piR-823 expression analysis in ALDH + CSC from breast cancer patients (n = 15) (G) Statistical analysis of piR-823 expression levels in (F) Data are presented as mean ± SEM These results suggest a proliferation-promoting role of piR-823 during tumorigenesis in the tested luminal subtype of breast cancer cells piR-823 promoted cell proliferation in MCF-7 and T-47D luminal subtype of breast cancer (A) Validation of piR-823 knockdown in MCF-7 cells (B,C) CCK8 assay (B) and colony formation assay (C) showing decreased cell proliferation by piR-823 knockdown in MCF-7 cells (D) Validation of piR-823 overexpression in MCF-7 cells (E,F) CCK8 assay (E) and colony formation assay (F) showing promoted cell proliferation in piR-823-overexpression in MCF-7 cells Data are presented as mean ± SEM (N = 3) piR-823 promoted cancer cell stemness in MCF-7 and T-47D luminal subtype of breast cancer (A) piR-823 knockdown in MCF-7 cells decreased ALDH + CSC subpopulation (C) Mammosphere formation assays using MCF-7 cells with or without knockdown of piR-823 (D,E) Quantitative analysis of (C) showing decreased sphere number (D) and size (E) by piR-823 knockdown in MCF-7 cells (F–H) Gene expression analyses showing positive regulation of stemness genes including OCT4 and h-TERT at the mRNA (F,H) and protein (G) levels by piR-823 Knockdown or overexpression of piR-823 was applied in MCF-7 cells for the analyses (I) piR-823 overexpression in MCF-7 cells promoted ALDH + CSC subpopulation (K) Mammosphere formation assays using MCF-7 cells with or without overexpression of piR-823 (L,M) Quantitative analysis of (K) showing increased sphere number (L) and size (M) after piR-823 overexpression in MCF-7 cells In order to further validate the CSC-promoting function of piR-823, another luminal subtype of breast cancer cell line T-47D was assessed in breast cancer stemness assays including ALDH analysis and a mammosphere formation assay (Supplementary Figures S3A–E). The stemness genes OCT4, SOX2, KLF4, NANOG, and h-TERT were downregulated by anti-piR-823 and upregulated by piR-823 overexpression in T-47D cells (Supplementary Figures S3F,G) The results further showed that piR-823 participates in acquiring stem cell-like properties and/or maintaining CSC characteristics in the luminal subtype of breast cancer cells All these data support the potential of piR-823 to serve as a therapeutic target to treat the luminal subtype of breast cancer Tumor-targeted delivery of nanoparticles carrying anti-piR-823 suppressed breast tumor growth in a xenograft model (A) Schematic representation of the procedure for the tail vein injection of Zn0.4Fe2.6O4@SiO2 magnetic nanoparticles carrying either anti-piR-823 or negative control anti-NC to the breast tumor-burdened mice transplanted with MCF-7 cells (B) Tumor images isolated from the mice in (A) Data are presented as mean ± SEM (N = 8) piR-823 activated Wnt signaling through regulating DNA methylation of APC promoter in MCF-7 breast cancer and DNMT3B by piR-823 at the mRNA levels in MCF-7 cells and DNMT3B by piR-823 at the protein levels in MCF-7 cells (C) Increased global DNA methylation in MCF-7 cells by overexpression of piR-823 DNMT inhibitor 5-Aza-2’-deoxycytidine (5-AzaDC) was used as a positive control (D) TOP/FOP assay demonstrated activated Wnt signaling by overexpression of piR-823 (E) Immunofluorescence staining assay indicating relocation of β-catenin from the cytoplasm to the nucleus in MCF-7 cells after overexpression of piR-823 (F) piR-823 promoted DNA methylation of gene APC promoter in MCF-7 cells DNA methylation-specific primers were applied for PCR analysis to distinguish methylated and unmethylated APC (G,H) Suppressed expression of APC at both mRNA (G) and protein (H) levels by piR-823 (I) Western blot analyses indicating the upregulation of APC and downregulation of DNMT 1 and DNMT 3B in the tumor samples of mice treated with anti-piR-823 (J) Schematic representation of the mechanism through which piR-823 promotes tumorigenesis in the luminal subtype of breast cancer by methylating the APC promoter and activating Wnt signaling thereby regulating breast cancer stem cells we propose that high expression of piR-823 contributes to tumorigenesis in luminal breast cancer Reduction of piR-823 may be considered as an indicator of patients’ therapeutic response to hormone treatment It has been demonstrated that CSCs are the main source of drug resistance Development of novel therapeutic strategies targeting CSCs will shed light on the campaign to conquer cancer we identified a small non-coding RNA piR-823 as a new regulator of CSCs in luminal breast cancer Targeted knockdown of piR-823 significantly inhibited cancer cell proliferation and tumor growth in vitro and in vivo Our finding not only demonstrates piR-823 as a novel target to treat the luminal subtype of breast cancer but also provides help to understand the role that piR-823 may play when hormone therapy is applied to prevent and treat breast cancer The original contributions presented in the study are included in the article/Supplementary Material further inquiries can be directed to the corresponding author/s The studies involving human participants were reviewed and approved by the Institutional Review Board (IRB) of Tongji University Shanghai East Hospital The patients/participants provided their written informed consent to participate in this study The animal study was reviewed and approved by the Institutional Animal Care and Use Committee of the Tongji University School of Medicine ZY and CL designed the project and wrote the manuscript RP involved in the revision and language editing All authors contributed to the article and approved the submitted version This work was supported by the grant from the National Key Research and Development Program of China Stem Cell and Translational Research (2016YFA0101202); grants 82002789 (JL) and 81972476 (ZY) from the National Natural Science Foundation of China; and grant 20JC1410400 from the Science and Technology Commission of Shanghai Municipality This work was supported in part by the NIH R01CA132115 (RP) and a DOD Breakthrough Breast Cancer Research Program grant award (# W81XWH1810605) (RP) RP holds ownership interests (value unknown) for several patents and submitted patent applications The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fcell.2021.641052/full#supplementary-material Supplementary Figure 1 | Knockdown of piR-823 suppressed cell proliferation in T-47D breast cancer cells (A) Validation of piR-823 knockdown in T-47D cells (B) CCK8 assay showing decreased cell proliferation by piR-823 knockdown in T-47D cells Supplementary Figure 2 | Overexpression of piR-823 promoted cell proliferation in T-47D breast cancer cells (A) Validation of piR-823 overexpression in T-47D cells (B,C) CCK8 assay showing increased cell proliferation in piR-823-overexpressing T-47D cells Supplementary Figure 3 | piR-823 promoted cancer cell stemness in T-47D breast cancer cells (A) piR-823 knockdown in T-47D cells decreased ALDH + CSC population (C) Mammosphere formation assays using T-47D cells with or without knockdown of piR-823 (D,E) Quantitative analysis of C showing decreased sphere number (D) and size (E) by piR-823 knockdown in T-47D cells and h-TERT were downregulated by anti-piR-823 (F) and upregulated by piR-823 overexpression (G) in T-47D cells Myeloid-derived suppres-sor cells endow stem-like qualities to multiple myeloma cells by inducing piRNA-823 expression and DNMT3B activation Prospective identification of tumorigenic breast cancer cells The impact of the RASSF1C and PIWIL1 on DNA methylation: the identification of GMIP as a tumor suppressor PubMed Abstract | CrossRef Full Text | Google Scholar APC promoter hypermethylation contributes to the loss of APC expres-sion in colorectal cancers with allelic loss on 5q Circular RNA: an emerging non-coding RNA as a regulator and biomarker in cancer Analysis of adenomatous polyposis coli promoter hypermethyla-tion in human cancer Google Scholar Breast and cervical cancer in 187 countries between 1980 and 2010: a systematic analysis ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome RNA sequencing identifies specific PIWI-interacting small non-coding RNA expression patterns in breast cancer Cyclin D1 promotes secretion of pro-oncogenic immuno-miRNAs and piRNAs Unique somatic and malignant expression patterns implicate PIWI-interacting RNAs in cancer-type specific biology Google Scholar Estrogen and androgen hormone levels modulate the expression of PIWI interacting RNA in prostate and breast cancer Promoter methylation analysis of WNT/β-catenin pathway regulators and its association with expression of DNMT1 enzyme in colorectal cancer The piRNA pathway in Drosophila ovarian germ and somatic cells Defining the ex-pression of piRNA and transposable elements in Drosophila ovarian germline stem cells and somatic support cells PIWI-interacting RNA-36712 restrains breast cancer progression and chemoresistance by interac-tion with SEPW1 pseudogene SEPW1P RNA Wnt signaling promoter hypermethylation distinguishes lung primary ade-nocarcinomas from colorectal metastasis to the lung Gene ex-pression profiling predicts clinical outcome of breast cancer Cyclin D1-mediated microRNA expression signature predicts breast cancer outcome Long noncoding RNAs in regulation of human breast cancer Long non-coding RNA CCAT2 promotes oncogenesis in triple-negative breast cancer by regulating stem-ness of cancer cells piRNA-823 contrib-utes to tumorigenesis by regulating de novo DNA methylation and angiogenesis in multiple myeloma piR-823 contributes to colorectal tumorigenesis by enhancing the transcriptional activity of HSF1 Small non-coding RNAs govern mammary gland tumorigenesis The expression of stem cell protein Piwil2 and piR-932 in breast cancer Liang C and Yu Z (2021) piRNA-823 Is Involved in Cancer Stem Cell Regulation Through Altering DNA Methylation in Association With Luminal Breast Cancer Copyright © 2021 Ding, Li, Lü, Zhao, Guo, Lu, Ma, Liu, Pestell, Liang and Yu. 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Volume 12 - 2024 | https://doi.org/10.3389/fcell.2024.1450227 DEAD-box RNA helicase Vasa is required for gonad development and fertility in multiple animals Vasa is implicated in many crucial aspects of Drosophila oogenesis and maintenance of germline stem cells (GSCs) data about Vasa functions in Drosophila spermatogenesis remain controversial Here we showed that loss-of-function vasa mutations led to failures of GSC maintenance in the testes and a cessation of male fertility over time Defects in GSC maintenance in vasa mutant testes were not associated with an increasing frequency of programmed cell death indicating that a premature loss of GSCs occurred via entering differentiation We found that Vasa is implicated in the positive regulation of rhino expression both in the testes and ovaries The introduction of a transgene copy of rhino encoding a nuclear component of piRNA pathway machinery in vasa mutant background allowed us to restore premeiotic stages of spermatogenesis including the maintenance of GSCs and the development of spermatogonia and spermatocytes piRNA-guided repression of Stellate genes in spermatocytes of vasa mutant testes with additional rhino copy was not restored Our study uncovered a novel mechanistic link involving Vasa and Rhino in a regulatory network that mediates GSC maintenance but is dispensable for the perfect biogenesis of Su(Ste) piRNAs in testes we have shown that Vasa functions in spermatogenesis are essential at two distinct developmental stages: in GSCs for their maintenance and in spermatocytes for piRNA-mediated silencing of Stellate genes DEAD-box helicases are known to contribute to all aspects of intracellular RNA metabolism the molecular functions of Vasa in oogenesis remain poorly understood Since Vasa contributes to various intracellular processes in the germline It remains unclear whether its functions in the piRNA pathway and in other developmental processes are mutually linked or relatively independent Vasa is required for male fertility and germ cell maintenance in the testes (A) A scheme of Drosophila spermatogenesis green) are located adjacent to the hub (violet) at the apical testis tip and are encapsulated by two somatic cyst stem cells (CySCs undergoes four mitotic divisions to create cysts of 16 spermatogonia overlapping two undivided somatic cyst cells the germ cells within the cyst switch to the spermatocyte program Spermatocytes undergo extensive cell growth and then enter synchronous meiosis with the appearance of 64 haploid round spermatids Spermatids elongate and move to the basal end of the testis Mature individual spermatozoa enter the seminal vesicle and are stored there as mature sperm (B) Western blot analysis of the testis lysates of vasa mutants Note that the testes of vasEP812/vasD1 heteroallelic combination expressed VIG but not Vasa whereas vasEP812/vasPH165 and vasEP812/vasEP812 mutations caused a loss of expression of both Vasa and VIG proteins Anti-Actin antibodies were used as a loading control (C) Fertility test of vasa mutant males with vasEP812/vasD1 heteroallelic combinations (violet lines) in comparison with their heterozygous siblings (green lines) The average offspring number of male per day with standard errors is presented for indicated time intervals after parent male eclosion (D) The bar diagram depicts the distribution of testis phenotypes of vasEP812/vasD1 mutants and control heterozygous males (3 days after eclosion): wild-type-like phenotype (green) early germ cells and a few spermatocytes (blue) The numbers of examined testes are indicated (E) A loss of germ cells in the testes of vasEP812/vasD1 heteroallelic mutants Left: testes of heterozygous control males (3 days after eclosion) exhibited a wild-type phenotype Right: testes of vasEP812/vasD1 heteroallelic mutants (3 days after eclosion) rapidly lost germline content Whole-mount fixed testes were immunostained with Vasa (green) and lamin (violet) antibodies Confocal slices with the apical tips of the testes oriented leftward are shown Fragments in the white boxes of the top images are present in higher magnifications in the corresponding bottom images with Vasa and DAPI staining Scale bars are 100 µm for the top images and 50 µm for the bottom ones Yellow arrows indicate seminal vesicles containing mature sperm Our study explored the molecular functions of Vasa in testes of D Here we found that loss-of-function vasa mutations led to a rapid loss of GSC number in testes and a decrease in male fertility with aging Defects in GSC maintenance in vasa mutant testes were not associated with an elevated frequency of premature cell death indicating that a loss of GSCs occurred through entering differentiation without a self-renewal two main functions of RNA helicase Vasa in the testes of Drosophila melanogaster are carried out in distinct cohorts of germ cells: the maintenance of GSCs in early spermatogenesis and piRNA silencing of Stellate genes Both of these functions are necessary for male fertility underscoring the essential role of Vasa in fly spermatogenesis these results demonstrate that male reproductive capacity is substantially lowered in the absence of Vasa expression indicating the indispensable role of Vasa in male fertility maintenance a rapid decline in germ cell number in the testes is accompanied by reduced fertility These data indicate that Vasa is required intrinsically for the maintenance of testis GSCs indicating that a loss of GSCs was not caused by unrepaired DNA breaks and subsequent cell death the obtained results indicate that defects in the maintenance of GSCs in the testes of vasa mutant males are generally not associated with premature cell death and rather support a loss of GSCs by entering differentiation without a self-renewal (A) Violin plots present GSC number per testis in the testes of rhi and aub mutants and heterozygous control The medians and quartiles of GSC number sets are marked The number of testes counted for each genotype and the age of the analyzed males are shown at the bottom * Significant differences are found by the indicated pairwise comparison of GSC numbers in the mutant and control males of the same age (Wilcoxon/Mann-Whitney (U) test) C) RT-qPCR analysis of transcription levels of rhi (B) and aub (C) in the testes and ovaries of vasa mutants The expression levels of mRNAs were normalized to rp49 transcripts Error bars represent standard errors of the mean Differences between the mutant and control heterozygous samples siblings were analyzed by Student’s t-test The asterisks indicate significant differences between conditions; *p < 0.05 (D) RIP-qPCR assay for Vasa target mRNA identification Left: Western blot analysis of immunoprecipitation of vasa-GFP ovarian lysate using antibodies to GFP and normal rabbit serum for control Middle and right: RT-qPCR analysis of experimental and control RNA-immunoprecipitates with transcript-specific primers for rhi and aub with normalization to rp49 transcripts * The asterisks indicate that significant differences between the experiment and the control (Student’s t-test p < 0.05) were found only for rhi transcripts It should be proposed that Vasa is able to facilitate translation of aub mRNA or provide stabilization of Aub protein in nuage granules middle) of rhi transcripts in complex with Vasa-GFP according to the results of three independent experiments our data in sum suggest that RNA helicase Vasa is a positive regulator of rhi expression in the gonads of D our results allow us to propose that Vasa contributes to the stability of Aub protein within nuage granules which indicates a similar filling of the testes with premeiotic germ cells βNACtes signal of similar intensity was observed only for yw (wild type control) and vasEP812;rhi-GFP males whereas for vasEP812 testes it was more than 10-fold weaker This indicates that the amount of germ cells is sharply reduced in the testes of 6-day-old vasEP812 males but remains at a level comparable to the wild type in the testes of vasEP812;rhi-GFP males Effects of additional rhi expression in the testes of vasa mutants B) Immunofluorescence analysis of vasEP812−/− (A) and vasEP812;rhi-GFP (B) testes Testes of 6-day-old males were stained with antibodies to Fasciclin III (FasIII Internal confocal slices of the immunostained whole-mount fixed testis preparations are shown The fusomes are indicated by yellow arrows (B) (C) Western blot analysis of testis lysates of 0- and 6-day-old yw and vasEP812;rhi-GFP males using antibodies to Vasa βNACtes is a marker of spermatocytes which in normal circumstances constitute the most significant part of the germinal content of testes (D) Violin plots present GSC number per testis in vasEP812+/− The number of testes counted for each genotype and the age of males are shown at the bottom * Significant differences are found by indicated pairwise comparisons (Wilcoxon/Mann-Whitney (U) test) F) Stellate expression in the vasEP812;rhi-GFP (E) and vasEP812−/− (F) testes Testes of 0-day-old males were stained with antibodies to Stellate (green) and lamin (violet) Images of whole testes are presented on the left Fragments in the white boxes of the left images are present in higher magnifications on the right the expression of the additional rhi dose in the background of vasa mutation leads to a significant restoration of GSC number and total content of premeiotic germ cells in the testes but does not restore piRNA silencing of Stellate genes This experimental model allowed us to clearly separate two distinct functions of Vasa protein in spermatogenesis of D To analyze a generation pattern of Su(Ste) piRNAs we prepared and sequenced libraries of 18–29 nt small RNAs isolated from the testes of vasa mutants and males with the transgenic construct rhi-GFP in the background of vasa mutations our data suggest that the presence of maternally inherited transposon-derived piRNAs for the initiation of Su(Ste) piRNA generation by the phasing process itself is not enough for perfect repression of Stellates Vasa-dependent amplification ping-pong process is strongly required for effective Su(Ste) piRNA biogenesis and Stellate silencing the exact mechanism and direct mRNA targets of translational regulation with the aid of Vasa remain obscure to date and nothing is known about Vasa targets in Drosophila spermatogenesis The genetic and functional regulatory relationship established between Vasa and Rhi supports the assumption that the piRNA pathway could be involved in gene regulatory process directed to GSC maintenance in the testes of D further studies are needed to explore the molecular mechanism of this process we propose that Vasa contributes to the stability of Aub protein by recruiting it into the nuage granules for assembling functional piRNA-Aub complexes and providing piRNA silencing of mRNA targets Here we showed that RNA helicase Vasa essentially contributes to male fertility in D melanogaster at least on two distinct stages: GSC maintenance in early spermatogenesis and piRNA-dependent Stellate silencing need for correct passage through meiosis in spermatocytes Our study uncovers a novel mechanistic link involving Vasa and Rhi in a regulatory network that mediates GSC maintenance The open questions include whether observed relationships indicate a possible role of piRNA silencing in this important developmental process carries a deletion of the bulk of the Su(Ste) locus on the Y chromosome TUNEL signal of whole cyst of germ cells was estimated as a single event p-values for pairwise comparison in control and experimental testes were calculated using Wilcoxon/Mann–Whitney (U) test The following antibodies were used for immunofluorescence staining: a mix of murine monoclonal anti-Lamin Dm0 ADL67.10 and ADL84.12 antibodies (Developmental Studies Hybridoma Bank, University of Iowa (DSHB)), 1:500; rabbit polyclonal anti-Lamin antibodies (Osouda et al., 2005) 1:500; rat monoclonal anti-Vasa antibody (DSHB) 1:100; murine monoclonal anti-α-spectrin 3A9 antibody (DSHB) 1:200; murine monoclonal anti-Fasciclin III 7G10 antibody (DSHB) 1:25; rabbit polyclonal anti-γH2Av pS137 antibodies (Rockland) Alexa Fluor-labeled secondary goat anti-rat IgG and goat anti-mouse IgG (Invitrogen) were used as secondary reagents at a dilution of 1:500 DAPI (4′,6-diamidino-2-phenylindole) (Sigma) was used for chromatin staining For RNA immunoprecipitation (RIP) experiments rabbit polyclonal anti-GFP antibody ab6556 (Abcam) was used Samples were resolved by SDS-PAGE and blotted onto PVDF membrane Immobilon-P (Sigma) Alkaline phosphatase-conjugated anti-mouse anti-rat and anti-goat antibodies (Sigma) were used as secondary reagents at a dilution of 1:20,000 Blots were developed using the Immun-Star AP detection system (Bio-Rad Laboratories) All experiments were performed at least in triplicate with independent preparations of testis or ovarian lysates Total RNA was isolated from sets of 50–100 pairs of dissected testes or 30–50 pairs of ovaries using TRIzol Reagent (Invitrogen) according to the manufacturer’s recommendations cDNA was synthesized using random hexamers and SuperScript II reverse transcriptase (Invitrogen) cDNA samples were analyzed by real-time quantitative PCR using the incorporation of SYTO-13 (Invitrogen) Thermal cycling consisted of 5 min at 95°C followed by 45 cycles of denaturation (94°C and a final extension of 5 min at 72°C All experiments were performed with at least three independent RNA samples; each sample was analyzed in duplicate The following primers were used for RT-qPCR and RIP-RT-qPCR: rp49 fw 5′-ATG​ACC​ATC​CGC​CCA​GCA​TAC-3′ rev 5′-GCT​TAG​CAT​ATC​GAT​CCG​ACT​GG-3′; aub fw 5′-CAT​GAG​TGA​ACA​TAC​CAG​GCT​GAA-3′ rew 5′-GCG​GAG​TCC​AGC​TCG​ATG​TT-3′; rhi fw 5′-CGG​TTT​TCC​GAA​CGA​GAA​C-3′ rew 5′-CGG​CCT​TCC​GAT​GCA-3′ The number of X-linked Stellate genes in the genome was assessed by quantitative PCR with genomic DNA using highly specific Stellate primers the efficiency of which was previously tested and compared with the efficiency of rp49 primers (normalization control) genomic DNA of both males and females of D melanogaster was used as an additional control; the obtained value of the relative number of Stellate genes for males was 2-fold lower compared to females Stellate primers: fw 5′-GAT​TGG​TTC​CTC​GGG​ATC​AA-3′ rev 5′-CCG​TAC​AAC​AAG​CCA​GAG​GAA​CT-3′ Sets of 25–30 experimental males and their heterozygous siblings as a control were analyzed for their fertility Individual adult male (0 days after eclosion) was placed with three virgin yw females 4- to 6-day-old for 5 days in separate vials at 25°C After that the parent flies were removed from the vial The males were translocated in other vials with new three virgin females for the next 5 days up to six times The adult progeny in each vial was counted within 13–18-day interval after mating The tests were carried out at 25°C The flies were subjected to a light–dark cycle of 12:12 h the offspring from one male were counted per female for each time period Ovaries of vas-GFP flies 3–5 days after eclosion were dissected in ice-cold phosphate-buffered saline (PBS) Freshly dissected gonads were cross-linked in PBS supplemented with 0.25% formaldehyde for 30 min were washed by three times for 5 min and were stored at −70°C until further use Total cellular lysates were obtained from 500 pair of cross-linked ovaries using pre-chilled Dounce homogenizer on ice in a cold lysis buffer (50 mM Tris-HCl pH 8.0; 100 mM KCl; 2 mM MgCl2; 1 mM DTT; 0.5% Nonidet P-40 (NP-40); in the presence of 1% protease and phosphatase inhibitors (Sigma) with the addition of 2% Ribolock (Thermo Fisher Scientific) by two series of 100 strokes of pestle A with a 5 min incubation between the series The lysates were transferred to 1.7-mL polypropylene microcentrifuge tubes and were precipitated at 5000 g for 10 min at 4°C The supernatant fractions (crude extracts) were cleared by subsequent centrifugation at 16,000 g for 20 min at 4°C The clarified lysates were transferred to new tubes and diluted to protein concentration of 7–10 mg/mL with lysis buffer We used 50 µL of 50%-slurry of Protein A beads (Invitrogen) for each tube for immunoprecipitation The beads were previously washed two times with 500 µL cold PBS supplied by Tween 20 (1 × PBS; 0.1% Tween 20 (PBST)) and incubated with anti GFP-antibodies (Abcam) or normal rabbit serum for negative control in 200 µL PBST 20 min at room temperature on the rotator The antibody-coated beads were washed and pre-equilibrated with lysis buffer Then the equal volumes of clarified lysates were added to the experimental and control tubes for immunoprecipitation After incubation 40 min at RT on the rotator washing was performed three times with 1 mL washing buffer NT2 (50 mM Tris-HCl pH 8.0; 150 mM NaCl; 0.5 mM DTT; 0.3% NP-40) three times with 1 mL washing buffer NT3-Urea (50 mM Tris-HCl pH 8.0; 150 mM NaCl; 1M urea; 0.5 mM DTT; 0.3% NP-40) and one time NT2 buffer Small portions of immunoprecipitated material from each tube were put aside for Western blot analysis For that the bound material was eluted from the beads by boiling in 30 μL 2×sample buffer containing 0.2 M DTT Samples were subsequently resolved by % SDS-PAGE and blotted onto PVDF membrane Immobilon-P (Sigma) The rest parts of the material were subjected to proteolysis to remove a bulk of proteins performed on-bead in solution of 4 mg/ml Proteinase K (Promega) in PK2 buffer (10 mM Tris-HCl pH 7.5; 50 mM NaCl; 5 mM EDTA; 0.5% SDS; 1 mM DTT; in the presence of 2% Ribolock) for 2 hours at 55°C with extensive agitation After that the tubes were subjected by incubation for 10 min at 65°C for removing of cross-linking between RNA and peptide material Soluble fractions were separated from beads and were processed with TRIzol LS reagent (Thermo Fisher Scientific) followed by RNA isolation RT-qPCR analysis was performed as mentioned above Total RNA for RNA-seq libraries were isolated from vasEP812−/− and vasEP812−/−; rhi-GFP D melanogaster gonads with ExtractRNA (Evrogen) rRNA was depleted with Dynabeads MyOne Streptavidin C1 (Thermo Scientific Fisher) conjugated with synthesized anti-rDNA oligos RNA-seq libraries were generated using the NEBNext Ultra™ II Directional RNA Library Prep Kit for Illumina (#E7760 NEB) according to the manufacturer’s instructions Rhibo-depleted RNA-seq libraries were sequenced in 100 bp single-end mode on NovaSeq 6000 platform Small RNAs of 18–29 nt in size from gonads of D mix of vasEP812/+ with vasD1/+ and vasEP812/vasD1; rhi-GFP were isolated with TRIzol Reagent (Invitrogen) Small RNA fraction (19–29 nt) was purified by 15% polyacrylamide gel and libraries were prepared using NEBNext Multiplex Small RNA Sample Prep Set for Illumina (E7300S) and sequenced on NovaSeq 6000 in 50 bp single-end mode The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://www.ncbi.nlm.nih.gov/geo/ This work was conducted under the Institute of Developmental Biology Russian Academy of Sciences Government basic research program in 2024 The funder had no role in the design of the study; in the collection or interpretation of data; in the writing of the manuscript; or in the decision to publish the results We thank the Developmental Studies Hybridoma Bank and the Bloomington Stock Center for providing fly strains and antibodies used in this study The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fcell.2024.1450227/full#supplementary-material Stellate genes and the piRNA pathway in speciation and reproductive isolation of Drosophila melanogaster PubMed Abstract | CrossRef Full Text | Google Scholar RNA helicase vasa as a multifunctional conservative regulator of gametogenesis in eukaryotes PubMed Abstract 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Essential functions of RNA helicase Vasa in maintaining germline stem cells and piRNA-guided Stellate silencing in Drosophila spermatogenesis Received: 17 June 2024; Accepted: 29 July 2024;Published: 09 August 2024 Copyright © 2024 Adashev, Kotov, Bazylev, Kombarov, Olenkina, Shatskikh and Olenina. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use *Correspondence: Ludmila V. Olenina, b2xlbmluYV9sdWRtaWxhQG1haWwucnU= †These authors have contributed equally to this work and share first authorship Please press and hold the button until it turns completely green If you believe this is an error, please contact our support team. 147.45.197.102 : 653fd822-360f-4884-85f9-cf1830f2 Volume 8 - 2021 | https://doi.org/10.3389/fvets.2021.635013 This article is part of the Research TopicThe Advances in Semen EvaluationView all 14 articles Cryopreservation induces sperm cryoinjuries including physiological and functional changes the molecular mechanisms of sperm cryoinjury and cryoresistance are still unknown Cryoresistance or the freeze tolerance of sperm varies across species and boar sperm is more susceptible to cold stress giant panda sperm appears to be strongly freeze-tolerant and is capable of surviving repeated cycles of freeze-thawing differentially expressed (DE) PIWI-interacting RNAs (piRNAs) of fresh and frozen-thawed sperm with different freeze tolerance capacity from giant panda and boar were evaluated The results showed that 1,160 (22 downregulated and 1,138 upregulated) and 384 (110 upregulated and 274 downregulated) DE piRNAs were identified in giant panda and boar sperm Gene ontology (GO) enrichment analysis revealed that the target DE messenger RNAs (mRNAs) of DE piRNAs were mainly enriched in biological regulation and metabolic processes in giant panda and boar sperm Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated that the target DE mRNAs of DE piRNAs were only distributed in DNA replication and the cyclic adenosine monophosphate (cAMP) signaling pathway in giant panda and mitogen-activated protein kinase (MAPK) signaling pathways in boar sperm were considered as part of the olfactory transduction pathway we speculated that the difference in the piRNA profiles and the DE piRNAs involved in the cAMP signaling pathway in boar and giant panda may have contributed to the different freeze tolerance capacities between giant panda and boar sperm which helps to elucidate the molecular mechanism behind sperm cryoinjury and cryoresistance Despite the extensive progress that has been achieved in optimizing the cryopreservation process through the selection of friendly cryoprotectants and the design of better freezing and thawing procedures to ameliorate cryodamage the underlying mechanisms of freeze tolerance or freezability involved in cryopreservation have not been completely elucidated yet These previous studies demonstrated that cryopreservation induces different transcriptomic modifications and may explain why sperm with different freeze tolerance or cryoresistance capacities are susceptible to cold stress we speculated that piRNAs may be involved in post-thawed sperm cryoinjury or cryoresistance we first evaluated the differences in the piRNA profiles of fresh and frozen-thawed boar and giant panda sperm which will help to uncover the underlying molecular mechanisms of sperm cryoresistance and freeze tolerance and improve post-thawed sperm quality and fertility Fresh ejaculates from five sexually mature giant pandas with normal physiological parameters were obtained by electrical stimulation from the Bifengxia Base of China Conservation and Research Center for the Giant Panda (Yaán, Sichuan, China) according to a previous protocol (11) giant pandas were anesthetized by an intramuscular injection of 10 mg/kg ketamine HCl and maintained with 0–5% isofluorane gas Electroejaculation was conducted by using an electroejaculator (Boring USA); the period of electrical stimuli (2–8 V repeated three times) was 2 s following an intermittent break of 2 s When penile erection occurs during stimulation semen was collected into a sterile glass container Fresh ejaculates from 11 boars were collected with the glove-handed technique All procedures were carried out while strictly following the Regulations of the Administration of Affairs Concerning Experimental Animals (Ministry of Science and Technology revised in June 2004) and were accredited by the Institutional Animal Care and Use Committee in the College of Animal Science and Technology All ejaculates from giant panda and boar were pooled separately and two equal groups were generated (fresh sperm and cryopreserved sperm). Direct RNA extraction was performed with fresh sperms, and the other group was cryopreserved according to a previously procedure (19) TES–Tris (TEST) egg yolk buffer was used to dilute the giant panda sperm (Irvine Scientific CA) to obtain 5% concentration of glycerol This diluted material was filled into 0.25-ml semen straws and gradually cooled to 4°C in 4 h then kept at 7.5 cm for 1 min over liquid nitrogen (LN) to obtain the cooling rate of −40°C/min and then at 2.5 cm for 1 min above LN (approximate cooling rate was −100°C/min) before plunging in LN until further processing Thawing was performed by immersing the semen straws for 30 s in a water bath with constant temperature of 37°C Semen was diluted with an equal volume of Ham's F10 (HF10) containing 5% fetal calf serum and 25 mM HEPES Boar sperm was cryopreserved according to the following procedure; firstly the sperm was centrifuged (for 5 min at 1,800 rpm and 17°C) and then diluted with a lactose–egg yolk (LEY) extender containing 10 ml hen's egg yolk and 40 ml 11% β-lactose the sperm and the extender mixture were cooled to 4°C (at 0.2°C/min) and further dilution with LEY was performed to obtain a final 3% concentration of glycerol and kept 3 cm above LN for 10 min before being submerged into it until future use Before RNA extraction, seminal plasma was removed from all the samples by washing with RNase-free water three times. Then, 0.5% Triton (X-100) was employed in accordance with a previous study (16) to minimize the somatic cell count as they hinder the spermatic RNA extraction process USA) was utilized to extract total RNA from all sperm samples according to the manufacturer's instructions The RNA samples were pooled together equally in their respective groups before constructing RNA libraries USA) equipment was used to determine the purity and concentration of the RNA and an Agilent 2100 Bioanalyzer (Agilent Technologies a NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB The small RNA libraries were built by using the NEB Next Ultra RNA Library Prep Kit for Illumina (NEB USA) and the NEBNext Multiplex Oligos for Illumina (NEB USA) according to the manufacturer's guidelines After confirming the quality using Qubit 2.0 and the Agilent Bioanalyzer 2100 system (Agilent Technologies) all libraries were sequenced with the Illumina Hiseq 2500 platform (Illumina and those with adjusted p < 0.01 and absolute value of log2 fold change (FC) >1 were classified as DE piRNAs hierarchical clustering analysis was performed by R heatmap.2 on the selected DE piRNAs; piRNAs with similar expressions were clustered based on the log10(TPM + 1) value All data were shown as the means ± SEM 20.0) with independent samples t test was used to determine statistical differences and p < 0.05 were considered as statistically significant The 24-nt (21.76%) and 31-nt (34.21%) piRNAs were the most abundant in fresh and frozen-thawed giant panda sperms the 30-nt (25%) and 32-nt (1.96%) piRNAs showed the highest and the lowest respective abundances Overview of piRNA sequencing of fresh and frozen-thawed sperm in giant panda and boar Volcano plot and clustering analysis of differentially expressed PIWI-interacting RNAs (DE piRNAs) in fresh and frozen-thawed giant panda and boar sperm (A) Volcano plot of DE piRNAs in fresh and frozen-thawed giant panda sperm and red dots represent the upregulated piRNAs (B) Heat maps of the cluster analysis of piRNAs Red indicates high expression while green means low expression of piRNAs no target DE mRNAs were found for these common DE piRNAs Comparative analysis of differentially expressed PIWI-interacting RNAs (DE piRNAs) in fresh and frozen-thawed giant panda and boar sperm (A) Comparison of DE piRNAs and target DE mRNAs Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of differentially expressed PIWI-interacting RNAs (DE piRNAs) in giant panda and boar sperm (A) GO analysis of the target DE messenger RNAs (mRNAs) of DE piRNAs (B) Top 10 KEGG pathways of the target DE mRNAs of DE piRNAs Notably, most of the target DE mRNAs of DE piRNAs were distributed in the cyclic adenosine monophosphate (cAMP) signaling pathway in giant panda sperm, except for DNA replication (Figure 3B) the target mRNAs of the DE piRNAs in boar sperm were mainly distributed in the peroxisome and spliceosome the cAMP pathway was found in both giant panda and boar sperm but was extremely enriched in giant panda sperm Further analysis indicated that DE piRNAs involved in the cAMP signaling pathway may regulate the post-thawed sperm function by targeting cyclic nucleotide-gated (CNG) ion channel-related genes According to our previous study, the DE miRNAs and target mRNAs of giant panda sperm were mainly enriched in olfactory transduction pathways, including the cAMP and cGMP signaling pathways (14) we found that the target DE mRNAs of DE piRNAs in giant panda sperm were mainly distributed in the cAMP signaling pathway and partially involved in DNA replication few targets of the DE piRNAs in boar sperm were also enriched in the cAMP signaling pathway but the ratio was much lower than that of giant panda sperm we speculated that the 1,132 specific piRNAs involved in the cAMP signaling pathway in giant panda sperm may be closely related to the freeze tolerance of sperm we speculated that cryopreservation can affect the expression levels of olfactory transduction pathway-related genes and is probably involved in the regulation of capacitation and even the freeze tolerance of post-thawed sperm the regulatory mechanism of the olfactory transduction signaling pathway on post-thawed sperm is still unknown the target mRNAs of the DE piRNAs in giant panda sperm are mainly enriched in the cAMP pathway which indicates that cAMP and calcium may be associated with frozen-thawed sperm quality of giant panda Differences in the cAMP pathway-related piRNAs and mRNAs between the giant panda and boar sperm may have contributed to sperm cryotolerance our study first revealed that piRNAs might be regulating the cAMP signaling pathway to regulate post-thawed sperm quality which provides new insights into the cryoinjury or the freeze tolerance mechanisms of sperm varying across species Future exploration should focus on the biological roles of these DE piRNAs in sperm freeze tolerance or cryoresistance and their association with post-thawed sperm quality which may provide some insights regarding the molecular mechanisms of cryoinjury we first conducted a comparative analysis of the piRNAs and target mRNAs between giant panda sperm and boar sperm during cryopreservation The differentially expressed piRNAs and their target DE mRNAs are mainly involved in the cAMP signaling pathway and DNA replication which indicated that these piRNAs play a critical role in sperm cryoresistance and cryoinjury during cryopreservation Our study provides new insights into the cryoinjury or freeze tolerance mechanisms of sperm varying across species The datasets presented in this study can be found in online repositories The names of the repository/repositories and accession number(s) can be found below: Gene Expression Omnibus The animal study was reviewed and approved by the Regulations of the Administration of Affairs Concerning Experimental Animals (Ministry of Science and Technology revised in June 2004) the Institutional Animal Care and Use Committee in the College of Animal Science and Technology Written informed consent was obtained from the owners for the participation of their animals in this study and YZha revised the manuscript critically and given final approval to be published given final approval version of the manuscript to be published All authors reviewed and approved the final manuscript This research was supported by National Natural Science Foundation of China (No: 31872356 and No: 31570533) We offer special thanks to the Sichuan TIEQILISHI group for semen collection The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fvets.2021.635013/full#supplementary-material All DE piRNAs in giant panda and boar sperm DE piRNAs and their target DE mRNAs in giant panda and boar sperm The common DE piRNAs in giant panda and boar sperm Artificial insemination with frozen-thawed boar sperm CrossRef Full Text | Google Scholar Freezing-thawing induces alterations in histone H1-DNA binding and the breaking of protein-DNA disulfide bonds in boar sperm Recent advances in boar sperm cryopreservation: State of the art and current perspectives PubMed Abstract | CrossRef Full Text | Google Scholar Changes in sperm membrane and ROS following cryopreservation of liquid boar semen stored at 15°c Sperm cryopreservation: a review on current molecular cryobiology and advanced approaches Good and bad freezability boar ejaculates differ in the integrity of nucleoprotein structure after freeze-thawing but not in ROS levels Freeze-thawing induces alterations in the protamine-1/DNA overall structure in boar sperm Differences in sperm protein abundance and carbonylation level in bull ejaculates of low and high quality susceptibility to peroxidation and fatty acid composition of boar semen during liquid storage Cryopreservation of boar semen and its future importance to the industry Acrosomal integrity and capacitation are not influenced by 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using cryopreserved spermatozoa in the giant panda (Ailuropoda melanoleuca) proTRAC–a software for probabilistic piRNA cluster detection High-throughput sequencing of Arabidopsis microRNAs: evidence for frequent birth and death of MIRNA genes IDEG6: a web tool for detection of differentially expressed genes in multiple tag sampling experiments The KEGG resource for deciphering the genome A comprehensive evolutionary classification of proteins encoded in complete eukaryotic genomes Automated genome annotation and pathway identification using the KEGG Orthology (KO) as a controlled vocabulary piRNA cluster database: a web resource for pirna producing loci PubMed Abstract | CrossRef Full Text | Google Scholar Spermatozoa cryopreservation: state of art and future in small ruminants Dietary fatty acids affect semen quality: a review Sperm cryopreservation in three species of endangered gazelles (Gazella cuvieri Effect of method of collection on seminal characteristics of the domestic cat 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bovines The activation of the chymotrypsin-like activity of the proteasome is regulated by soluble adenyl cyclase/cAMP/protein kinase A pathway and required for human sperm capacitation Semi-automatized segmentation method using image-based flow cytometry to study sperm physiology: the case of capacitation-induced tyrosine phosphorylation Tyrosine phosphorylation signaling regulates Ca2+ entry by affecting intracellular pH during human sperm capacitation Sperm capacitation and acrosome reaction in mammalian sperm Central role of soluble adenylyl cyclase and cAMP in sperm physiology cAMP and 1,2-diacylglycerol in cryopreserved buffalo (Bubalus bubalis) spermatozoa on supplementation of taurine and trehalose in the extender Catsper channels are regulated by protein kinase A Shedding light on the role of cAMP in mammalian sperm physiology Current knowledge on the acute regulation of steroidogenesis Steroid hormone receptors and direct effects of steroid hormones on ram spermatozoa Role of the cAMP pathway in glucose and lipid metabolism Luteinizing hormone (LH) acts through PKA and PKC to modulate T-type calcium currents and intracellular calcium transients in mice leydig cells Effects of cryopreservation on cAMP-dependent protein kinase and AMP-activated protein kinase in Atlantic salmon (salmo salar) spermatozoa: relation with post-thaw motility Zhou G and Zeng C (2021) Comparative Analysis of piRNA Profiles Helps to Elucidate Cryoinjury Between Giant Panda and Boar Sperm During Cryopreservation Received: 29 November 2020; Accepted: 17 February 2021; Published: 22 April 2021 Copyright © 2021 Wang, Zhou, Ali, Zhang, Wang, Huang, Luo, Zhang, Qin, Zhang, Zhang, Zhou and Zeng. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) *Correspondence: Changjun Zeng, emVuZ2NoakBzaWNhdS5lZHUuY24= Metrics details The PIWI-interacting RNA (piRNA) pathway is an adaptive defense system wherein piRNAs guide PIWI family Argonaute proteins to recognize and silence ever-evolving selfish genetic elements and ensure genome integrity Driven by this intensive host–pathogen arms race the piRNA pathway and its targeted transposons have coevolved rapidly in a species-specific manner but how the piRNA pathway adapts specifically to target silencing in mammals remains elusive we show that mouse MILI and human HILI piRNA-induced silencing complexes (piRISCs) bind and cleave targets more efficiently than their invertebrate counterparts from the sponge Ephydatia fluviatilis The inherent functional differences comport with structural features identified by cryo-EM studies of piRISCs MILI and HILI piRISCs adopt a wider nucleic-acid-binding channel and display an extended prearranged piRNA seed as compared with EfPiwi piRISC consistent with their ability to capture targets more efficiently than EfPiwi piRISC the seed gate—which enforces seed–target fidelity in microRNA RISC—adopts a relaxed state in mammalian piRISC revealing how MILI and HILI tolerate seed–target mismatches to broaden the target spectrum A vertebrate-specific lysine distorts the piRNA seed shifting the trajectory of the piRNA–target duplex out of the central cleft and toward the PAZ lobe Functional analyses reveal that this lysine promotes target binding and cleavage Our study therefore provides a molecular basis for the piRNA targeting mechanism in mice and humans and suggests that mammalian piRNA machinery can achieve broad target silencing using a limited supply of piRNA species Ten things you should know about transposable elements Adaptation to P element transposon invasion in Drosophila melanogaster The Piwi-piRNA pathway provides an adaptive defense in the transposon arms race Transposable element domestication as an adaptation to evolutionary conflicts Dynamic interactions between transposable elements and their hosts Molecular domestication of transposable elements: from detrimental parasites to useful host genes Regulation of transposable elements by DNA modifications Initial sequencing and analysis of the human genome piRNA clusters need a minimum size to control transposable element invasions Initial sequencing and comparative analysis of the mouse genome Discrete small RNA-generating loci as master regulators of transposon activity in The piRNA response to retroviral invasion of the koala genome piRNAclusterDB 2.0: update and expansion of the piRNA cluster database Crystal structure of silkworm PIWI-clade Argonaute Siwi bound to piRNA Rapid and specific purification of Argonaute-small RNA complexes from crude cell lysates indicates that thousands of human genes are microRNA targets Structure of an argonaute silencing complex with a seed-containing guide DNA and target RNA duplex propagation and cleavage of target RNAs in Ago silencing complexes Argonaute divides its RNA guide into domains with distinct functions and RNA-binding properties Relaxed targeting rules help PIWI proteins silence transposons The initial uridine of primary piRNAs does not create the tenth adenine that is the hallmark of secondary piRNAs Slicing and binding by Ago3 or Aub trigger Piwi-bound piRNA production by distinct mechanisms Helix-7 in Argonaute2 shapes the microRNA seed region for rapid target recognition elegans VASA homologs control Argonaute pathway specificity and promote transgenerational silencing In vivo PIWI slicing in mouse testes deviates from rules established in vitro The evolutionarily conserved piRNA-producing locus pi6 is required for male mouse fertility Sperm acrosome overgrowth and infertility in mice lacking chromosome 18 pachytene piRNA Ubiquitination-deficient mutations in human Piwi cause male infertility by impairing histone-to-protamine exchange during spermiogenesis Automated acquisition of cryo-electron micrographs for single particle reconstruction on an FEI Tecnai electron microscope Coot: model-building tools for molecular graphics PHENIX: a comprehensive Python-based system for macromolecular structure solution Schrödinger, L., & DeLano, W. PyMOL http://www.pymol.org/pymol (2020) UCSF ChimeraX: meeting modern challenges in visualization and analysis A logarithmic approximation to initial rates of enzyme reactions Zeng, L. GuidewithTarget (Degradome Sequencing Bioinformatics Pipeline). GitHub https://github.com/CMACH508/GuidewithTarget (2023) Piuco, R. & Galante, P. A. piRNAdb: a piwi-interacting RNA database. Preprint at BioRxiv https://doi.org/10.1101/2021.09.21.461238 (2021) fastp: an ultra-fast all-in-one FASTQ preprocessor Download references Yu (Westlake University) for critical proofreading of the manuscript; I Zamore for guidance in analyzing Degradome Sequencing data members of Shen and Wu laboratories for discussions; the Cryo-EM Facility of Westlake University for providing support on cryo-EM data collection; the Key Laboratory of Zhejiang Province for Aptamers and Theranostics for their technical support in the SPR assay and the Mass Spectrometry and Metabolomics Core Facility of Westlake University for protein analysis This work was supported by Westlake Education Foundation Zhejiang Provincial Foundation of China (2021R01013) and National Natural Science Foundation of China (NSFC32070628) to E.-Z.S. National Natural Science Foundation of China (32271261) Zhejiang Provincial Natural Science Foundation of China (LR22C050003) Westlake University (1011103860222B1) and Institutional Startup Grant from the Westlake Education Foundation (101486021901) to J.W and the National Natural Science Foundation of China (NO 92068103) and Westlake Laboratory of Life Science to C.-Q.S These authors contributed equally: Zhiqing Li Key Laboratory of Growth Regulation and Translational Research of Zhejiang Province Westlake Laboratory of Life Sciences and Biomedicine Key Laboratory of Structural Biology of Zhejiang Province Department of Computer Science and Engineering Center for Cognitive Machines and Computational Health (CMaCH) Key Laboratory of Reproductive Dysfunction Management of Zhejiang Province Zhejiang Provincial Clinical Research Center for Obstetrics and Gynecology Key Laboratory of Insect Developmental and Evolutionary Biology Shanghai Institutes for Biological SciencesChinese Academy of Sciences Department of Pharmacology and Cancer Biology reviewed and edited the manuscript.C.-Q.S. Nature Structural & Molecular Biology thanks Claus Kuhn and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available Sara Osman was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team Ratio of retrotransposon (a) and piRNA cluster (b) of invertebrate and mammalian genomes Phylogram of the PIWI proteins shows that MILI and EfPiwi proteins belong to the same branch Size-exclusion chromatography profile of the purified MILI-piRNA complex (arrow) (b) and HILI-piRNA complex (arrow) (c) using a modified oligo capture approach SDS-PAGE analysis of the purified MILI-piRNA complex (d) and HILI-piRNA complex (e) Gels were stained with Coomassie blue (representative of protein purifications performed at least ten times independently) Guide RNA-mediated cleavage assay with MILI (f) or HILI (g) MILI or HILI loaded with or without synthetic guide RNAs were incubated with 5′-FAM-labeled fully matched (FM) target RNAs or non-complementary (NC) target RNAs for 1 h at 37 °C Products were resolved on a denaturing urea-PAGE gel alongside synthetic 22-nt marker and 1-nt RNA ladder Similar results are seen in three experiments The fluorescein amidite (FAM) modification in the target strand is highlighted in green and the cleavage site is indicated with a red arrow The cleaved 5′ fragment with FAM label is 24 nucleotides Source data Representative PAGE of the protein-guide RNA complexes binding to another fully matched (FM) target RNA Non-complementary (NC) target is a negative control Similar results are seen in two experiments The guide and target RNAs used in this experiment Equilibrium dissociation constant (KD) of g2-g8 complementary target for protein-guide RNA complexes SPR traces showing binding kinetics of g2-g8 complementary target for Ago2-miRNA binary complex SPR sensorgrams of non-complementary target RNA binding to Ago2-miRNA complex Data processing workflow of cryo-EM analysis of MILI-piRNA (a) and MILI-piRNA-target (15 nt) (b) Representative motion-corrected dose-weighted micrographs were selected out of 4,602 and 8,107 micrographs for the two datasets gold-standard FSC curves of the final maps (resolution cut-off at FSC = 0.143) map curves of the final maps against the final models (resolution cut-off at FSC = 0.5) were presented Please note that an open state was also observed during 2D and 3D classifications in the MILI-piRNA-target (15 nt) dataset Data processing workflow for HILI-piRNA cryo-EM analysis Representative motion-corrected dose-weighted micrograph was selected out of 2,136 micrographs map curves of the final map against the final model (resolution cut-off at FSC = 0.5) were presented The density of the bound piRNA in the selected 3D class (with a dotted box) was indicated by a red arrow a, Superposition of the individual domains of MILI (pink), HILI (orange), EfPiwi (PDB: 7KX7) (aquamarine), and Siwi (PDB: 5GUH) (chartreuse) The density map (grey mesh) for the entire bound piRNA in MILI–piRISC is shown in the left panel A close-up view of the EM density map (grey mesh) for the piRNA 5′ segment is shown in the right panel Modeled guide RNAs are shown as red sticks The density map (grey mesh) for the entire bound piRNA in HILI-piRISC is shown in the left panel Two close-up views of the EM density map (grey mesh) for the piRNA 5′ segment and 3′ half are shown in the middle and right panels Non-seed interactions between piRNA-target RNA duplex and EfPiwi protein (d) and MILI protein (e) The relative ratio here is mismatched target binding % over fully matched target binding % Source data Coomassie blue-stained SDS-PAGE of purified recombinant Flag-tagged GTSF1 proteins from different species (representative of protein purifications performed three times independently) piRISC target cleavage assays in the presence of increasing amounts of GTSF1 Note: The Ephydatia fluviatilis genome sequence is unavailable GTSF1 from the related sponge Ephydatia muelleri was therefore incubated with EfPiwi Representative native PAGE analysis of protein-guide RNA complexes binding to fully matched (FM) target RNA non-complementary (NC) target negative control Representative in vitro cleavage reactions in the presence of equimolar substrate (target RNA) and enzyme (Piwi-piRNA complexes) Guide-target-pairing schematic for targets with 3-nt mismatch regions to guide RNA used in (f Representative urea-PAGE showing the ability of EfPiwi and HILI to cleave targets with three consecutive mismatches between g2 and g22 Quantification of the in vitro cleavage assays (right) showing changes in cleavage activity for three consecutive mismatches between g2 and g22 Source data The secondary structure of MmMILI is indicated above the sequences, color coded by domains as indicated in Fig. 2 Data processing workflow of cryo-EM analysis of EfPiwiN959K-piRNA (a) and EfPiwiN959K-piRNA-target (16 nt) (b) Representative motion-corrected dose-weighted micrographs were selected out of 2,279 and 3,983 micrographs for the two datasets piRNA queries (Ago3_IP.fa/Ago3_bg.fa) were aligned to candidate targets whose cleaved 5′ ends are complementary to g2–g10 of a piRNA Targets with extended pairing were identified from g2–g25 the fraction of cleaved targets was calculated as the fraction of cleaved targets explained by Ago3-associated piRNAs minus the fraction explained by Ago3-non-associated piRNAs Plot of the fraction of cleaved targets in fly ovaries for contiguous pairing from nucleotide g2–gX Ago3-non-associated piRNA were sampled 10 times Source data Download citation DOI: https://doi.org/10.1038/s41594-024-01287-6 Metrics details An Author Correction to this article was published on 14 August 2024 This article has been updated exposing a molecular link between immune responses in mammals and deeply conserved RNA-based mechanisms that control transposable elements The magnified view shows the active site of SLFN13 Label-free proteomic quantification of TOFU-2–HA and wild-type immunoprecipitates from young adult extracts The x axis shows the median fold enrichment of individual proteins P values were calculated using Welch two-sided t-tests The dashed lines represent enrichment thresholds at P = 0.05 and fold change > 2 curvature of enrichment threshold c = 0.05 The dots represent enriched (blue/red) or quantified (grey) proteins Wide-field fluorescence microscopy analysis of adult hermaphrodites carrying the piRNA sensor in the following three genetic backgrounds: tofu-2(E216A) (top) prg-1(n4357) (middle) and wild type (bottom) Germlines are outlined by white dashed lines A representative image from a series of ten is shown Total mature piRNA levels (type 1) in wild-type and tofu-2(E216A)-mutant young adult hermaphrodites P values were calculated using two-tailed unpaired t-tests The relative abundance of type 1 piRNA precursors from individual loci in tofu-2(E216A)-mutant versus wild-type young adult hermaphrodites reads per million non-structural small RNA reads Source data Although PETISCO has been implicated in precursor stabilization and is required for piRNA production the nuclease that mediates 5′ precursor processing and how it interacts with PETISCO remain unclear We conclude that a TOFU-1–TOFU-2 complex could be the nuclease that processes piRNA precursors Source data We conclude that SLFL-3 and SLFL-4 function redundantly in piRNA processing TOFU-2 and either SLFL-3 or SLFL-4 constitutes the enzyme that processes the 5′-end of piRNA precursors in C We conclude that PUCH is a type of cap- and sequence-specific ribonuclease Source data We conclude that PUCH can cleave piRNA precursors Source data whereas PUCH is brought to mitochondria through a C-terminal TM helix on SLFL-3/4 Deletion of the TM helix results in a strong reduction in mature piRNAs without precursor accumulation which is different to the tofu-2 and slfl-3/4 phenotypes This may indicate that precursor processing per se is not affected the loading of processing intermediates into PRG-1 critically depends on mitochondrial tethering We did not detect PRG-1 in any of our experiments this interaction is too transient to be detected through immunoprecipitations including the role that we identify in piRNA biogenesis point to a deeply conserved role for SLFN-like domains in immunity- and stress-related mechanisms Our results show that SLFN domains can form multimeric complexes and that multimerization can unveil highly specific nucleolytic activities It is conceivable that combinations of proteins with SLFN-related folds may generate highly specific enzymes that help organisms to fight off infectious nucleic acids Blinding or randomization of strains and samples was not applied in this work young adult hermaphrodite animals were used Sample size calculations were not performed or required Images of piRNA-sensor-carrying strains were obtained using the Leica DM6000B system Young adults and adult worms were washed in a drop of M9 (22 mM KH2PO4 1 mM MgSO4) and immobilized with 30 mM sodium azide in M9 buffer Imaging of Bm4 cells was performed using the Leica TCS SP5 system with the LAS AF 2.7.3.9723 software Images were processed using ImageJ and Adobe Illustrator All IP–MS experiments were performed in quadruplicates grown until the gravid adult stage and bleached again The embryos were left to hatch in M9 buffer (22 mM KH2PO4 L1-stage worms were seeded on standard OP50 plates and collected at the young adult stage The worms were washed three times with M9 buffer and one time with cold sterile water Worm aliquots (200 µl) were pelleted and frozen in liquid nitrogen and stored at −80 °C A total of 200 µl of synchronized young adult worms was thawed on ice and resuspended in 250 µl of 2× lysis buffer (50 mM Tris HCl pH 7.5 The Bioruptor Plus (Diagenode) sonicator was used to lyse the worms (10 cycles 30/30 s the supernatant was accurately removed without the lipid phase the protein concentration of the lysate was determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific 550 µl of worm lysate containing 0.75 mg protein was resuspended in a final volume of 550 µl of 1× lysis buffer Anti-HA immunoprecipitation was performed using 2 µg of custom-made anti-HA antibodies (mouse The lysate was incubated with the antibodies for 2 h at 4 °C 30 µl of protein G magnetic beads (Dynabeads Invitrogen) was washed three times in washing buffer (25 mM Tris HCl pH 7.5 equilibrated beads were added to the lysis and incubated for an additional hour at 4 °C by end-over-end rotation beads were washed 6 times with wash buffer resuspended in 2× NuPAGE LDS sample buffer (containing 200 mM DTT) and boiled for 15 min at 95 °C elegans database (August 2014; 27,814 entries) carbamidomethylation at cysteine was set as a fixed modification whereas methionine oxidation and protein N-acetylation were considered variable modifications The match-between-run option was activated protein groups based on one unique peptide and known contaminants were removed the label-free quantification values were log2-transformed and the median across the replicates was calculated This enrichment was plotted against the −log10-transformed P value (Welch’s t-test) using the ggplot2 package in the R environment 666 mM NaOH) and were left to hatch overnight in M9 buffer L1-stage worms were seeded onto OP50 plates and collected as young adults 500 µl of TRIzol LS (Thermo Fisher Scientific 10296-028) was added to the 50 µl worm aliquot and five cycles of freezing in liquid nitrogen and thawing in a 37 °C water bath were performed The samples were centrifuged for 5 min at 21,000g at room temperature An equal volume of 100% ethanol was added to the supernatant before proceeding with RNA extraction using the Direct-zol RNA MicroPrep (Zymo Research) kit RNA was eluted into 13 µl of nuclease-free water (Ambion Invitrogen) and each sample was divided into two aliquots for piRNA-precursor and mature piRNA library preparation CIP treatment of 1.5 µg of isolated RNA was performed in rCutSmart Buffer (B6004S) using 3 µl of Quick CIP (M0525L) in a 40 µl reaction The reaction was incubated at 37 °C for 20 min followed by heat-inactivation for 2 min at 80 °C The CIP-treated RNA was subjected to another round of purification using the Direct-zol RNA MicroPrep (Zymo Research) kit RppH (NEB) treatment was performed with a starting amount of 500 ng Next-generation sequencing library preparation was performed using the NEXTflex Small RNA-Seq Kit V3 following step A to step G of Bioo Scientific’s standard protocol Amplified libraries were purified by running an 8% TBE gel and size-selected for 15–40 nucleotides Libraries were profiled using a High Sensitivity DNA Chip on the 2100 Bioanalyzer (Agilent Technologies) quantified using the Qubit dsDNA HS Assay Kit in the Qubit 2.0 Fluorometer (Life Technologies) and sequenced on the Illumina NextSeq 500/550 system 3′-end labelling of substrate RNA (the sequence is shown in Supplementary Table 3) was performed in a 25 µl reaction containing 2.5 µl DMSO 1 µl of synthetic RNA precursor (5 pmol µl−1) The reaction was mixed and 2.5 µl of [5′-32P]pCp (SCP-111 Hartmann analytic) was added before overnight incubation at 16 °C the labelled RNA was purified using G25 columns (Cytiva) according to the manufacturer’s protocol The 3′-end-labelled synthetic RNA precursor was used for in vitro cleavage assays and in EMSAs A total of 5 pmol synthetic RNA oligonucleotide was labelled with ATP, [γ-32P] (PerkinElmer) using T4 PNK(NEB), according to the manufacturer’s protocol. The sequences of the RNA substrates are provided in Supplementary Table 3 It was not authenticated and was not tested for mycoplasma Approximately 4 × 106 BmN4 cells were collected from each 10 cm dish (see above) washed once in 5 ml ice-cold PBS and once more in 1 ml ice-cold PBS cells were pelleted by centrifugation for 5 min at 500g at 4 °C and frozen at −80 °C BmN4 cell pellets were thawed on ice and lysed in 1 ml IP-150 lysis buffer (30 mM HEPES (pH 7.4) 2 mM Mg(OAc)2 and 0.1% IGEPAL freshly supplemented with EDTA-free protease inhibitor cocktail and 5 mM DTT) for 1 h by end-over-end rotation at 4 °C Cells were further lysed by passing the lysate ten times through a 20-gauge syringe needle followed by five passes through a 30-gauge needle Cell debris was pelleted by centrifugation at 17,000g for 20 min at 4 °C The supernatant fractions were collected and processed for GFP immunoprecipitation using GFP-Trap beads (Chromotek) The GFP-Trap beads (15 µl bead suspension per reaction) were washed three times in 1 ml of IP-150 lysis buffer Equilibrated beads were subsequently incubated with the BmN4 cell lysate and incubated overnight by end-over-end rotation at 4 °C immunoprecipitated complexes were washed five times using 1 ml of IP-150 lysis buffer and were subsequently used for in vitro cleavage assays or for immunodetection using western blot analysis Samples were prepared in 1× Novex NuPage LDS sample buffer (Invitrogen) supplemented with 100 mM DTT and were heated at 95 °C for 10 min before resolving on a 4–12% Bis-Tris NuPage NOVEX gradient gel (Invitrogen) in 1× Novex NuPAGE MOPS SDS running buffer (Invitrogen) at 140 V Separated proteins were transferred to a nitrocellulose membrane (Amersham) overnight at 20 V using 1× NuPAGE transfer buffer (Invitrogen) supplemented with 10% methanol the membrane was incubated for 1 h in 1× PBS-Tween-20 (0.05%) supplemented with 5% skimmed milk and incubated for 1 h with primary antibodies diluted in PBS-Tween-20 (1:1,000 monoclonal anti-Flag M2 Sigma-Aldrich; 1:1,000 monoclonal anti-GFP antibodies (B-2) in house); 1:1,000 anti-actin (A5060) rabbit monoclonal antibodies the membrane was washed three times for 5 min in PBS-Tween-20 before incubation with secondary antibodies using 1:10,000 IRDye 800CW goat anti-mouse and IRDye 680LT donkey anti-rabbit IgG (LI-COR) and imaged on the Odyssey CLx imaging system (LI-COR) The blots were scanned using Image Lab (v.6.0.1) Strains RFK 1269 and RFK1280 were grown and lysed as described in the ‘MS analysis’ section A total of 15 µg of protein was mixed with 2× gel loading buffer (2× Novex NuPage LDS sample buffer (Invitrogen) supplemented with 200 mM DTT) and heated at 95 °C for 10 min before resolving on a 4–12% Bis-Tris NuPage NOVEX gradient gel (Invitrogen) in 1× Novex NuPAGE MOPS SDS Running Buffer (Invitrogen) at 150 V Separated proteins were transferred to nitrocellulose membrane (Amersham) 1 h at 120 V using 1× NuPAGE transfer buffer (Invitrogen) supplemented with 10% methanol The membrane was incubated for 30 min in 1× PBS-Tween-20 (0.05%) supplemented with 5% skimmed milk cleaved and incubated overnight with primary antibodies diluted in PBS-Tween-20 (1:1,000 monoclonal anti-HA (12CA5 Sigma-Aldrich) rabbit polyclonal antibodies) the membrane was washed five times for 5 min in PBS-Tween-20 before incubation with secondary antibodies using 1:10,000 horse anti-mouse HRP-linked antibody (7076 Cell Signaling) and goat anti-rabbit HRP-linked antibodies (7074 Cell Signaling) and imaged using the SuperSignal West Pico Plus (Thermo Fisher Scientific) kit 50 young adult worms were picked into 13 µl of M9 buffer 5 µl of 4× Novex NuPage LDS sample buffer (Invitrogen) and 2 µl of 1 M DTT boiled for 30 min at 95 °C and loaded onto the 4–12% Bis-Tris NuPage NOVEX gradient gel (Invitrogen) staining and imaging were performed as described above; anti-MYC (1:1,000 Blots were scanned using Image Lab (v.6.0.1) The PUCH complex used for in vitro cleavage assays was obtained using two different methods The full-length PUCH complex was obtained from GFP immunoprecipitates using BmN4 cell lysates (see above) whereas the minimal catalytic complex (mini-PUCH) was purified from E For the in vitro cleavage assays performed with immunoprecipitated material from BmN4 cells beads were washed in the cleavage buffer (CB) containing 40 mM Tris-HCl The beads were subsequently resuspended in 10 µl of CB and incubated with 0.2 pmol of the labelled RNA substrate for 1 h at room temperature For cleavage assays with mini-PUCH purified from E 0.2 pmol of labelled RNA substrate was incubated in 10 µl CB with 27 nM mini-PUCH protein complex (final concentration) at 20 °C for 30 min The cleavage reaction was terminated by adding 1 µl of 20 mg ml−1 proteinase K One volume of the 2× RNA gel loading dye (Thermo Fisher Scientific R0641) was added and the RNA was resolved on a 15% TBE-UREA gel (Novex) for 90 min at 180 V with 1× TBE as the running buffer Capped RNA oligonucleotides were labelled at the 3′ end and 0.2 pmol (1 µl) of RNA per sample was used in the cleavage reaction For the reaction with immunoprecipitated material to obtain 5′P-containing piRNA precursor oligonucleotide 5′OH-piRNA precursor had been labelled at the 3′ end as described above M0201S) according to the NEB T4 PNK protocol For the reaction with mini-PUCH 5′OH-piRNA precursor the oligonucleotide had been labelled at the 5′ end as described above A total of 18 µl of 0.2 pmol µl−1 RNA substrate (AAU or CAU) was added to the 162 µl of CB containing recombinant mini-PUCH at a final concentration 27 nM The samples were transferred to 20 °C and the samples for each timepoint were taken The reaction was stopped by adding proteinase K A total of 10 µl of CB with 27 nM mini-PUCH was added to a 2 µl mix of 0.2 pmol labelled AAU substrate and 0.4 pmol cold RNA of choice Cleavage reactions were incubated at 20 °C for 15 min and were stopped by adding protease K BmN4 cells were lysed in EDTA + lysis buffer (30 mM HEPES (pH 7.4) 1 mM EDTA and 0.1% IGEPAL freshly supplemented with EDTA-free protease inhibitor cocktail and 5 mM DTT) after the immunoprecipitation beads were washed five times with EDTA + lysis buffer followed by one wash in CB containing 1 mM EDTA the cleavage reactions were performed in CB containing MgCl2 MnCl2 or CaCl2 at the indicated concentrations (1 A total of 2 pmol of labelled RNA was incubated in 35 µl of CB containing mini-PUCH (or mutated mini-PUCH) at a final concentration of 40 nM and was incubated at 20 °C for 1 h 3 volumes of TRIzol LS reagent (Thermo Fisher Scientific and RNA was purified using Direct-zol RNA MicroPrep (Zymo Research) according to the manufacturer’s protocol the RNA was ligated to 10 pmol of 5′OH-rGrUrCrUrGrUrUrUrArA-OH3′ oligonucleotide using T4 RNA ligase according to the manufacturer’s protocol the reaction was terminated by proteinase K and RNA was resolved on a 15% TBE-UREA gel (Novex) for 90 min at 180 V with 1× TBE as the running buffer A total of 16 µl of 3′-end-labelled piRNA precursor (0.2 pmol µl−1) was incubated with five times molar excess of PETISCO protein complex on ice for 1 h in 160 µl of CB PUCH-immunoprecipitate-containing beads were added The same procedure was performed in parallel for RNA incubated without PETISCO Reactions were incubated at 20 °C with mild shaking and 10 µl samples were taken for each timepoint The same experiment was performed with recombinant mini-PUCH at the concentrations described for cleavage reactions Gels were scanned using the Typhoon FLA 9500 system (software version V.0 build 1.0.0.185) in 10 μl of binding buffer (20 mM HEPES pH 7.5 150 mM NaCl) for 1 h at the room temperature each sample was mixed with 15% Ficoll with bromophenol blue Native 6% TBE gel was pre-run for 30 min at 180 V at room temperature in 1× TBE PETISCO and its subunits (IFE-3, TOFU-6, PID-3, ERH-2) were purified and reconstituted as described previously28 TOFU-2 and SLFL-3 were cloned into modified pET vectors All proteins were produced as an N-terminal His-Tagged fusion protein with varying fusion tags that can be removed by the addition of 3C protease Proteins or protein complexes were produced in the E coli BL21(DE3) derivate strains in terrific broth medium and when the culture reached an optical density at 600 nm (OD600) of 2–3 0.2 mM IPTG was added to induce protein production for 12–16 h overnight For interaction studies using the co-expression co-purification strategy two plasmids containing the gene of interest and different antibiotic-resistance markers were co-transformed into BL21(DE3) derivative strains to allow co-expression A total of 50 ml of cells was grown in TB medium under shaking at 37 °C and Protein production was induced after 2 h at 18 °C through the addition of 0.2 mM IPTG for 12–16 h overnight Cells were collected by centrifugation and the cell pellets were resuspended in 2 ml of lysis buffer (50 mM sodium phosphate 5 mM 2-mercaptoethanol pH 8.0) per gram of wet cell mass Cells were lysed by ultrasonic disintegration and insoluble material was removed by centrifugation at 21,000g for 10 min at 4 °C 500 µl of supernatant was applied to 20 Strep-Tactin XT resin (IBA Lifesciences); for MBP pull-downs 500 µl supernatant was applied to 20 µl amylose resin (New England Biolabs) and incubated for 2 h at 4 °C the resin was washed three times with 500 µl of lysis buffer The proteins were eluted in 50 µl of lysis buffer supplemented with 10 mM maltose or 50 mM biotin in the case of amylose beads or Strep-Tactin XT beads Input material and eluates were analysed by SDS–PAGE and Coomassie brilliant blue staining To analyse protein interactions with purified proteins appropriate protein mixtures (bait 10–20 µM prey in 1.2-fold molar excess) were incubated in binding buffer containing 20 mM Tris/HCl (pH 7.5) the indicated beads were added to the protein mixtures were then incubated with the indicated beads for 2 h on ice: Glutathione Sepharose beads (Cube Biotech) Amylose Sepharose beads (New England Biolabs) and Strep-Tactin XT beads (IBA) the beads were washed three times with 200 μl binding buffer and the retained material was eluted with 0.05 ml incubation buffer supplemented with 20 mM of reduced glutathione Input material and eluates were analysed using SDS–PAGE and Coomassie brilliant blue staining TOFU-2 (residues 200–433) and SLFL-3 (residues 1–345) were co-expressed in BL21(DE3) cells an inactive TOFU-2 mutant (residues 200–433 TOFU-1 carried an N-terminal His10-MBP tag TOFU-2 an N-terminal His10-MBP and a C-terminal Strep II tag and SLFL-3 an N-terminal His6-GST tag All of the purification steps were performed on ice or at 4 °C Cells were lysed by sonication in lysis buffer (50 mM sodium phosphate 10% (v/v) glycerol and 5 mM 2-mercaptoethanol at pH 8.0) PUCH was purified by immobilized metal affinity chromatography (IMAC) using a 5 ml Ni2+-chelating HisTrap FF column (Cytiva) Proteins were eluted with lysis buffer supplemented with 500 mM imidazole and dialysed overnight against 20 mM Tris/HCl 10% (v/v) glycerol and 5 mM 2-mercaptoethanol at pH 7.5 PUCH was subjected to heparin affinity chromatography on a 5 ml HiTrap Heparin HP (Cytiva) followed by size-exclusion chromatography on the HiLoad Superdex 200 16/600 (Cytiva) column in 20 mM Tris/HCl pH 7.5 The thermal stability of mini-PUCH WT and the E216 mutant versions was determined using differential scanning fluorimetry 0.1 mg ml−1 mini-PUCH was mixed with a final concentration 5× SYPRO Orange (Thermo Fisher Scientific) in a buffer containing 20 mM Tris/HCl pH 7.5 Unfolding transitions were measured using the CFX96 Touch real-time PCR machine (Bio-Rad) by increasing the temperature from 15 °C to 95 °C in 0.5 °C increments (10 s hold time) Data analysis was performed using the CFX Manager software (Bio-Rad) included with the real-time PCR machine Purified proteins were incubated alone or in different mixtures at concentrations between 20 µM and 40 µM (total volume of 50 µl) in size-exclusion buffer (20 mM Tris/HCl pH 7.5 2 mM DTT) as indicated in the figure legends The samples were incubated for 1 h on ice to allow complex formation Complex formation was assayed by comparing the elution volumes in size-exclusion chromatography on the Superdex 200 Increase 3.2/300 (Cytiva) column The size-exclusion chromatography peak fractions were analysed using SDS–PAGE and visualized by Coomassie brilliant blue staining Unicorn7 software was used for data acquisition Isothermal titration calorimetry (ITC) experiments to quantitatively analyse the interaction between TOFU-1 peptide (residues 82–113) and the TOFU-6 eTUDOR domain (residues 119–314) interaction were performed using the PEAQ-ITC Isothermal titration calorimeter (Malvern) The TOFU-182–113 peptide does not contain tyrosine or tryptophane residues To be able to determine the concentration precisely we engineered a TOFU-1 peptide (TOFU-1W-82–113) that contains a Tryptophan residue at the N terminus Data processing and analysis was performed using the PEAQ-ITC software (Malvern) the samples were dialysed overnight simultaneously against 1 l of ITC buffer (20 mM Tris TOFU-1W-82–113 (the reactant) samples were concentrated to 45–48 µM and TOFU-6eTUDOR (the injectant) to 400–450 µM Titrations were carried out at 25 °C with 2 µl of the injectant per injection added to 200 µl of reactant cell solution The reported Kd and stoichiometry are the average of three experiments and the reported experimental error is the s.d The MicroCal PEAQ-ITC Control Software v.1.41 was used for data acquisition Purified TOFU-6eTUDOR and TOFU-1W-82–113 were mixed with TOFU-1W-82–113 being in 1.5-fold molar excess and subjected to size-exclusion chromatography on the HiLoad Superdex S75 16/600 (Cytiva) column equilibrated in 20 mM Tris/HCl The complex-containing fractions were concentrated to 10 mg ml−1 by ultrafiltration Crystallization trials were performed at 4 °C and 22 °C at 8–10 mg ml−1 using a vapour-diffusion set-up Drops were set up using the mosquito Crystallization Robot (SPT Labtech) on 96-Well 2-Drop MRC Crystallization Plates (Swissci) by mixing the protein complex and crystallization solution at 200 nl:200 nl and 400 nl:200 nl ratios Crystals were soaked with a mother liquor supplemented with 20% (v/v) glycerol for cryoprotection and then frozen in liquid nitrogen Data were collected at the ESRF (Grenoble, France) beamline ID30A-3 on 26 September 2021 (https://doi.org/10.15151/ESRF-DC-1033968485) Initial structural homology was detected using HHPRED71 Predicted complexes contained either SLFL-3 or SLFL-4 As SLFL-3 and SLFL-4 are paralogues that are 90% identical and 93% similar at the protein-sequence level we focused on predictions of the trimeric PUCH containing TOFU-1 the following residue boundaries were used: TOFU-1 residues 156–373 encompassing the SLFN domain with an N-terminal extension; TOFU-2 residues 200–433 encompassing the SLFN domain and two C-terminal alpha helices; SLFL-3 residues 103–300 Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article All computational tools are public and details on their use are provided in the Methods A Correction to this paper has been published: https://doi.org/10.1038/s41586-024-07938-4 A field guide to eukaryotic transposable elements piRNA-guided genome defense: from biogenesis to silencing PETISCO is a novel protein complex required for 21U RNA biogenesis and embryonic viability and functional insights into Schlafen proteins Defining the functions of PIWI-interacting RNAs PIWI-interacting RNAs: from generation to transgenerational epigenetics Crystal structure of the primary piRNA biogenesis factor Zucchini reveals similarity to the bacterial PLD endonuclease Nuc The structural biochemistry of Zucchini implicates it as a nuclease in piRNA biogenesis Structure and function of Zucchini endoribonuclease in piRNA biogenesis piRNA-guided slicing specifies transcripts for Zucchini-dependent piRNA-guided transposon cleavage initiates Zucchini-dependent The RNase PARN-1 trims piRNA 3′ ends to promote transcriptome surveillance in C PIWI associated siRNAs and piRNAs specifically require the Caenorhabditis elegans HEN1 ortholog henn-1 methylates and stabilizes select subclasses of germline small RNAs Differential impact of the HEN1 homolog HENN-1 on 21U and 26G RNAs in the germline of Caenorhabditis elegans PRG-1 and 21U-RNAs interact to form the piRNA complex required for fertility in C Piwi and piRNAs act upstream of an endogenous siRNA pathway to suppress Tc3 transposon mobility in the Caenorhabditis elegans germline Large-scale sequencing reveals 21U-RNAs and additional microRNAs and endogenous siRNAs in C Blumenthal, T. Trans-splicing and operons in C. elegans. WormBook https://doi.org/10.1895/wormbook.1.5.2 (2012) Structural basis of PETISCO complex assembly during piRNA biogenesis in C Molecular basis for PICS-mediated piRNA biogenesis and cell division Functional proteomics identifies a PICS complex required for piRNA maturation and chromosome segregation Structure of Schlafen13 reveals a new class of tRNA/rRNA-targeting RNase engaged in translational control Structural and biochemical characterization of human Schlafen 5 Structure of PDE3A-SLFN12 complex reveals requirements for activation of SLFN12 RNase Extremely stable Piwi-induced gene silencing in Caenorhabditis elegans PID-1 is a novel factor that operates during 21U-RNA biogenesis in Caenorhabditis elegans Deciphering arginine methylation: Tudor tells the tale The paternal gene of the DDK syndrome maps to the Schlafen gene cluster on mouse chromosome 11 The retroelement Lx9 puts a brake on the immune response to virus infection Codon-usage-based inhibition of HIV protein synthesis by human schlafen 11 Structures of diverse poxin cGAMP nucleases reveal a widespread role for cGAS-STING evasion in host-pathogen conflict Camelpox virus encodes a schlafen-like protein that affects orthopoxvirus virulence CRISPR/Cas9-mediated genome-edited mice reveal 10 testis-enriched genes are dispensable for male fecundity NONU-1 encodes a conserved endonuclease required for mRNA translation surveillance Schweinsberg, P. J. & Grant, B. D. C. elegans gene transformation by microparticle bombardment. WormBook https://doi.org/10.1895/WORMBOOK.1.166.1 (2013) WormBase: a modern model organism information resource Efficient marker-free recovery of custom genetic modifications with CRISPR/Cas9 in Caenorhabditis elegans Precision genome editing using CRISPR-Cas9 and linear repair templates in C pre-fractionation and storage of peptides for proteomics using StageTips high-resolution proteomics for data-driven systems biology MaxQuant enables high peptide identification rates individualized p.p.b.-range mass accuracies and proteome-wide protein quantification The MORPHEUS protein crystallization screen Towards rationalization of crystallization screening for small- to medium-sized academic laboratories: the PACT/JCSG+ strategy Data processing and analysis with the autoPROC toolbox How good are my data and what is the resolution Macromolecular structure determination using X-rays neutrons and electrons: recent developments in Phenix The Buccaneer software for automated model building Towards automated crystallographic structure refinement with phenix.refine MolProbity: more and better reference data for improved all-atom structure validation UCSF ChimeraX: structure visualization for researchers A completely reimplemented MPI bioinformatics toolkit with a new HHpred server at its core AlphaFold protein structure database: massively expanding the structural coverage of protein-sequence space with high-accuracy models Evans, R. et al. Protein complex prediction with AlphaFold-Multimer. Preprint at bioRxiv https://doi.org/10.1101/2021.10.04.463034 (2022) The PRIDE database resources in 2022: a hub for mass spectrometry-based proteomics evidences Download references We thank all of the members of the Ketting and Falk laboratories for discussions This work was funded by the Deutsche Forschungsgemeinschaft (DFG German Research Foundation) project IDs 439669440-TRR319 252386272 and 504320275 (to R.F.K.) and the Austrian Science Fund (FWF) I6110-B (to S.F.) was supported by the Peter und Traudl Engelhorn Foundation Some strains were provided by the Caenorhabditis Genetics Center (CGC) funded by NIH Office of Research Infrastructure Programs (P40 OD010440) We acknowledge support by the members of the IMB Genomics Core Facilities and the use of the NextSeq 500 system (funded by the DFG Möckel from the IMB Protein Production Core Facilities Miksch for their contribution to the early stages of this project; and the beamline scientists from the European Synchrotron Radiation Facility (ESRF) beamline ID30A-3 (Grenoble Present address: Institute of Molecular Virology and Cell Biology These authors contributed equally: Nadezda Podvalnaya International PhD Programme on Gene Regulation Department of Structural and Computational Biology Institute of Developmental Biology and Neurobiology performed and analysed the genetic experiments as well as the RNA binding and cleavage assays set up the experiments to produce the PUCH complex from BmN4 cells co-designed the cleavage assays and assisted in data analysis and interpretation purified proteins for biochemical experiments and crystallization trials and performed protein interaction experiments purified proteins and designed and performed protein interaction experiments performed computational analysis of the small RNA datasets performed and interpreted AlphaFold predictions ITC experiments and all crystallography-related work assisted in data analysis and interpretation The study was conceived and designed by S.F contributed to writing the manuscript and making the figures Nature thanks Zissimos Mourelatos and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available Source data The predicted C-terminal transmembrane helix is highlighted with a box AlphaFold2 predicted structures of SLFL-3/4 shown as cartoon and coloured by pLDDT score Widefield fluorescent microscopy of adult hermaphrodites carrying the mCherry::H2B-piRNA sensor in two genetic backgrounds: slfl-3(xf248) on top and wild type at the bottom The germlines are outlined by a white dashed line Representative image from a series of 10 is shown Total piRNA levels (type 2) in young adult hermaphrodites of the indicated genotypes (n = 3 biological replicates) Red lines depict group means and P-values were calculated using one-way ANOVA test followed by a Tukey’s HSD test Scatter plots depicting the relative abundance of type 1 piRNA precursors from individual loci in slfl4(-/-) slfl-3(ΔTM);slfl-4(-/-) and slfl-3(-/-) mutants versus wild type young adult hermaphrodites (n = 3 biological replicates) RPM: Reads per million non-structural sRNA reads Prediction of transmembrane helices in SLFL-3 and SLFL-4 using TMHMM - 2.0 Source data Predicted alignment error (PAE) plots for the five models predicted by Alphafold for full-length TOFU-1 The zoom-in highlights the predicted interaction between the SLFN domains of TOFU-2 and SLFL-3 suggesting that the TOFU-2 SPRY domain is no involved in complex formation The expected position error in angstroms (Å) is colour coded where blue colour indicates low PAE (high confidence) and red colour indicates high PAE (low confidence) Predicted alignment error (PAE) plots for the five models predicted by Alphafold for core regions of TOFU-1 Schematic summary of the interaction results presented in a and b AlphaFold predicts a trimeric complex consisting of TOFU-1 TOFU-2 residues 200-433 and SLFL-3 residues 103-300 were used for the prediction The predicted alignment error (PAE) plots are shown in (a) the five superposed models are shown as cartoon in (b) The settings used for the prediction are shown on the top The best of the five predicted models is coloured per chain (c) or per pLDDT score (d) yellow low and orange very low model confidence Source data Source data All data from this figure were obtained at least in duplicate Source data All data from this Figure was obtained at least in duplicate Source data The eTUDOR domains are shown in different shades of blue the TOFU-1pep in yellow and the PIWIpep containing the dimethyl-arginine residue in grey The zoom-in view shows the region of the degenerated aromatic cage of TOFU-6eTUDOR (b) and the canonical aromatic cage of PAPIeTUDOR (c) including annotations and information on controls Peptide count and peptide intensity data corresponding to the MS experiment displayed in Fig Peptide count and peptide intensity data corresponding to the MS experiment displayed in Extended Data Fig RNA substrate sequences and primers used for cloning and genotyping Reprints and permissions Download citation DOI: https://doi.org/10.1038/s41586-023-06588-2 Metrics details The intermitochondrial cement (IMC) and chromatoid body (CB) are posited as central sites for piRNA activity in mice with MIWI initially assembling in the IMC for piRNA processing before translocating to the CB for functional deployment The regulatory mechanism underpinning MIWI translocation We unveil that piRNA loading is the trigger for MIWI translocation from the IMC to CB piRNA loading facilitates MIWI release from the IMC by weakening its ties with the mitochondria-anchored TDRKH augmenting its binding affinity for TDRD6 and ensuring its integration within the CB loss of piRNA-loading ability causes MIWI entrapment in the IMC and its destabilization in male germ cells leading to defective spermatogenesis and male infertility in mice our findings establish the critical role of piRNA loading in MIWI translocation during spermatogenesis offering new insights into piRNA biology in mammals the mechanistic underpinnings of how mature piRNAs produced in the IMC transition into functional entities within piRNA machinery in the CB remain elusive the mechanism underlying the sequential translocation of MIWI from the piRNA-producing IMC to the piRNA-functioning CB during male germ cell development has remained an enigmatic aspect of piRNA biology we elucidate the pivotal role of piRNA loading in mediating the translocation of MIWI from the IMC to CB Our results indicate that piRNA loading instigates the disengagement of MIWI from TDRKH this detachment primes the MIWI protein for arginine methylation which subsequently fosters its association with TDRD6 via its methylated arginine residues thereby culminating in its integration into the CB By generating piRNA loading-deficient Miwi mutant mice we demonstrate that loss of piRNA-loading ability in MIWI impedes its translocation from the IMC to CB during spermatogenesis This obstruction critically undermines MIWI stability in developing male germ cells and leads to defective spermatogenesis and male infertility in mice our study sheds light on the molecular dynamics underpinning the sequential translocation of MIWI in developing male germ cells and establishes a mechanistic bridge between piRNA processing and functionality during male germ cell differentiation in mice a A schematic model showing the domain composition of MIWI and trajectory of the 5′ and 3′ ends of piRNA anchored with MID and PAZ-domain The Y569/K573 and Y346/Y347 conserved in PIWI proteins are required for the 5′ end or 3′ end piRNA loading capacity of MIWI b RNA co-IP assay of MIWI-associated-piRNAs (top) in wild-type (lane 1) with anti-MIWI IB as a loading reference (bottom) red) on testis sections from adult wildtype Left: representative staining images of indicated mouse testis sections The developmental stages of spermatocytes and spermatids were distinguished according to γH2AX (green) and DAPI (greyscale) staining White arrows indicated MIWI (c) or TDRKH (d) aggregations e Co-immunostaining of MIWI (red) and TDRKH (green) on testis sections from adult wildtype MiwiYY/YY and MiwiYK/YK mice using confocal microscopy with nuclei counterstained by DAPI (greyscale) White arrowheads indicated colocalization sites; yellow and white open arrowheads respectively indicated the unique localization of MIWI and TDRKH at non-colocalization sites The results shown are representative of three independent experiments suggesting that the piRNA-loading-deficient MIWI mutants may exert a dominant-negative effect on mitochondrial clustering These results together indicate that the loss of piRNA-loading ability leads to the retention of MIWI in the IMC reinforcing the notion that piRNA loading is indispensable for the translocation of MIWI into the CB during the differentiation of male germ cells in mice This supports that the fundamental architecture of the IMC and CB is preserved in both MiwiYY/YY and MiwiYK/YK male germ cells these results indicate that piRNA loading-deficient mutations lead to a failure in MIWI translocation from the IMC to CB suggesting that piRNA loading is a prerequisite for MIWI to be released from the IMC and subsequently integrated into the CB b Co-IP assay of the association of MIWI and TDRKH in mouse testes from 18 dpp wildtype Quantification of blot intensity of TDRKH protein in anti-MIWI pellets is shown in parentheses [the one from wildtype control mouse (lane 4) is set as 1.0 after normalization with MIWI blotting] c RNA co-IP assay of MIWI-interacting piRNAs in anti-MIWI (lane 1) and anti-TDRKH IP pellets (lane 2) from adult wildtype mouse testes d Anti-MIWIunloaded preferably pulled down piRNA-unloaded MIWI (left) and TDRKH (right) in adult mouse testicular lysate RNA co-IP assays using anti-MIWIunloaded and control anti-MIWI antibodies in adult wildtype mouse testicular lysate co-IP assay of the association of MIWI and TDRKH using anti-MIWIunloaded (lane 3) and control anti-MIWI antibodies (lane 4) in adult wildtype mouse testicular lysate with testicular lysate (lane 1) and IgG IP (lane 2) serving as positive and negative controls Quantification of blot intensity of TDRKH is shown in parentheses [the one anti-MIWI IP (lane 4) is set as 1.0 after normalization with MIWI blotting] e RNase A treatment enhanced the MIWI-TDRKH interaction in wild-type mouse testes Quantification of blot intensity of TDRKH protein in anti-MIWI pellets is shown in parentheses [the one from RNase A-untreated (lane 4) is set as 1.0 after normalization with MIWI blotting] f Transfection of piRNA attenuated the MIWI-TDRKH interaction in Flag-tagged MIWI-stable-expressed GC-2spd (ts) cells Quantification of blot intensity of TDRKH is shown in parentheses [the one with RNase A-untreated and piRNA-free condition in Flag-tagged MIWI-stable-expressed GC-2spd (ts) cell lysates (lane 2) is set as 1.0 after normalization with MIWI blotting] g Schematic diagram showing that piRNA loading facilitates the dissociation of MIWI from TDRKH Quantification of western blot analysis are represented as mean ± SD thereby facilitating the translocation of MIWI from the IMC to CB during male germ cell development a Co-immunostaining of TDRKH (red) and TDRD6 (green) on testis sections from adult wildtype mice using confocal microscopy b Co-immunostaining of MIWI (red) and TDRKH (green) on testis sections from adult wildtype and Tdrd6−/− mice using confocal microscopy d Co-IP assay of the association of MIWI with TDRD6 and TDRKH in mouse testes from adult wildtype (c and d lanes 4 and 6) were immunoblotted by the indicated antibodies lanes 3 and 5) serving as positive and negative controls Quantification of blot intensity of indicated proteins in anti-MIWI IP pellets is shown in parentheses [the one from wildtype control mouse (lane 4) is set as 1.0 after normalization with MIWI blotting] e Co-immunostaining of MIWI (red) and TDRD6 (green) on testis sections from adult wildtype White arrowheads indicated colocalization sites These results indicate that piRNA loading is essential for MIWI protein to efficiently interact with TDRD6 in mouse male germ cells a RNase A treatment barely altered MIWI methylation and MIWI-TDRD6 interaction in the adult testicular lysate Quantification is shown in parentheses [the one from RNase A-untreated (lane 2) is set as 1.0 after normalization with MIWI blotting] b Co-IP assay of the association of MIWI (lane 2) or arginine methylation-deficient MIWIR-K mutant (lane 3) with TDRD6 in co-transfected HEK293T cells Quantification is shown in parentheses [the one with wildtype MIWI (lane 2) is set as 1.0 after normalization with MIWI blotting] c Co-IP assay of the effect of methyltransferase inhibitor methylthioadenosine (MTA D5011) on the MIWI-TDRD6 interaction in co-transfected HEK293T cells Quantification is shown in parentheses [the one with DMSO treatment (lane 1) is set as 1.0 after normalization with MIWI blotting] e piRNA loading-deficient mutations impaired arginine methylation of MIWI in mouse testes Quantification is shown in parentheses [the one from the wildtype control mouse (lane 2) is set as 1.0 after normalization with MIWI blotting] f Anti-MIWIunloaded antibody pulled down less methylated MIWI and TDRD6 in adult wildtype mouse testicular lysate (lane 3) compared with control anti-MIWI antibody (lane 4) Quantification is shown in parentheses [the one anti-MIWI IP (lane 4) is set as 1.0 after normalization with MIWI blotting] g Sequential co-IP showing that TDRKH is mainly associated with unmethylated MIWI Quantification is shown in parentheses [the first anti-MIWI IP (lane 2) is set as 1.0 after normalization with MIWI blotting] h TDRKH reduced MIWI methylation in co-transfected HEK293T cells Quantification is shown in parentheses [the one without TDRKH transfection (lane 1) is set as 1.0 after normalization with MIWI blotting] i Schematic diagram showing that piRNA loading promotes MIWI dissociation from TDRKH leading to the exposure of the N-terminal of MIWI for arginine methylation by PRMT5 to enhance the MIWI-TDRD6 interaction further showing a weak interaction of unmethylated MIWI with TDRD6 in mouse testes These findings together indicate that piRNA loading augments the arginine methylation of MIWI which in turn promotes its interaction with TDRD6 in mouse male germ cells a All tested MiwiYY/YY and MiwiYK/YK males were infertile b Testes from adult MiwiYY/YY and MiwiYK/YK mice were moderately reduced compared with wildtype control a representative image of testes from indicated mice; right the average weight of testes from wildtype MiwiYY/YY (p = 0.033) and MiwiYK/YK (p = 0.014) mice (n = 6 P values were calculated using two-tailed Student’s t-test c PAS staining of the testis (top) and H&E staining of the epididymis (bottom) sections from adult wildtype Developmental stages of the seminiferous tubules were distinguished according to γH2AX (green) and DAPI (grayscale) staining e TUNEL assays (red) of testis sections from adult wildtype Results shown in c–e are representative of three independent experiments a Detection of piRNA expression in adult wildtype b The length distribution of small RNAs from adult wildtype Data were normalized by miRNA reads (21–23 nt) c Nucleotide distributions at the first position in the piRNAs from adult wildtype d Genomic annotation of the piRNAs from adult wildtype e Scatter plot of total piRNA reads mapped to individual piRNA clusters from adult wildtype f Western blotting of MIWI and MILI expression in testes from wildtype Quantification of blot intensity of MIWI is shown in parentheses (the one in wildtype testis is set as 1.0 after normalization with β-actin) Results shown in a and f are representative of three independent experiments and small RNA-seq experiments shown in b–e with two replicates suggesting that piRNA-loading-deficient MIWI proteins are subjected to degradation via the ubiquitin-proteasome pathway in mouse testes these results suggest that loss of piRNA-loading ability compromises not only the correct subcellular localization of MIWI but also significantly impairs its protein stability and piRNA production in mouse testes Upon its expression in mid-pachytene spermatocytes MIWI protein is recruited to the IMC for piRNA processing via interacting with TDRKH through its unmethylated N-terminus while piRNA loading induces a conformational change of MIWI and the disassociation of MIWI with TDRKH simultaneously results in the arginine residues in its N-terminus exposed for methylation by PRMT5 thereby enhancing TDRD6 binding to prime its localization in the CB for piRNA function but the physiological significance of this particular characteristic of TDRKH remains unknown Our results suggest that TDRKH acts as an initial receptor interacting with unmethylated MIWI and simultaneously masking its methylation sites and that the subsequent dissociation between them facilitates the arginine methylation of MIWI which in turn strengthens its interaction with TDRD6 and promotes its subsequent translocation in the CB the arginine methylation-modulated differential interaction of MIWI with the two Tudor family proteins TDRKH and TDRD6 dictates the localization of MIWI in specific germ granules during male germ cell differentiation which underscores the multifaceted roles of Tudor family proteins in the piRNA pathway suggesting a role for TDRD9 in MIWI2 translocation akin to TDRD6 in MIWI translocation suggests that mouse PIWI proteins employ distinct molecular mechanisms governing their translocation from the piRNA processing sites to functional deployment sites but how MILI and MIWI2 translocation is precisely controlled requires further investigation in future studies our current study demonstrates that piRNA loading facilitates the translocation of MIWI from the IMC to CB during the progression of male germ cell differentiation in mice This piRNA loading-regulated process only allows piRNA-loaded MIWI complexes transported to the CB while “leftover” piRNA-unloaded MIWI proteins are degraded This serves as a quality control mechanism ensuring the delivery of competent MIWI/piRNA complexes to their functional site After fulfilling their functions in the late stage of spermatid development piRNA loading also triggers MIWI ubiquitination and degradation and promotes the coordinated elimination of MIWI and piRNAs which is crucial for the histone-to-protamine exchange to produce functional sperm All experimental animal procedures were approved by the Institutional Animal Care and Research Advisory Committee at SIBCB All experiments with mice were performed ethically according to the Guide for the Care and Use of Laboratory Animals and institutional guidelines Female Human Embryonic Kidney 293T (HEK293T) cells (ATCC CRL-3216) and male mouse spermatocyte-derived GC-2spd (ts) cells (ATCC CRL-2196) were cultured in DMEM with 10% FBS according to the manufacturer’s instructions A GC-2spd (ts) cell line stably expressing MIWI was generated using the pLVX-Puro system (Clontech) the coding sequence of MIWI was subcloned from pCMV-3×Flag-MIWI into the lentiviral vector pLVX-Puro The lentiviral vectors were then co-transfected into HEK293T cells along with two packaging plasmids using Lipofectamine and used to infect GC-2spd (ts) cells for 24 h The infected GC-2spd (ts) cells were subsequently selected with 2 μg/mL puromycin All cell lines were recently authenticated and tested for mycoplasma contamination Transfection was performed with Lipofectamine 2000 (Thermo Fisher 11668019) for HEK293T cells or Lipofectamine 3000 (Thermo Fisher L3000015) for GC-2spd (ts) cells according to the manufacturer’s instructions All DNA plasmids were made free of endotoxins which we found specifically recognizes piRNA-unloaded MIWI protein was generated using a synthetic 100-amino acid (aa) peptide corresponding to position 700–800 of human PIWIL1 (Q96J94) Complementary DNA corresponding to TDRD6 1911–2135 aa (TDRD6 NP_001154838.1) was cloned into pET-28a (His-tag) vectors His-tagged recombinant protein was used as the antigen to generate rabbit anti-TDRD6 polyclonal antisera The antisera were affinity-purified with His-tagged TDRD6 antigen using an AminoLink Plus immobilization kit (Thermo Scientific) Immunoprecipitation (IP) and immunoblotting (IB) assays were performed using standard IP and IB protocols mouse testes or cells were homogenized in lysis buffer A [50 mM Tris-HCl (pH 7.4) 4693132001)] or lysis buffer B [20 mM Tris-HCl (pH 7.0) Primary antibody-coupled Protein A/G beads (Thermo Fisher 88803) were added to the precleared tissue or cell lysates and incubated for 6 h at 4 °C After washing with washing buffer [50 mM Tris-HCl (pH 7.4) IP pellets or tissue/cell extracts were diluted in SDS-loading buffer and then analyzed with standard SDS-PAGE and IB procedures the first IP pellets were eluted with 5 volumes of 0.1 M glycine-HCl (pH 3.0) and rotated at room temperature for 5 min Tris buffer (pH 8.0) was added into the supernatant to adjust pH = 7.0 and the supernatant was incubated with the second antibody-coupled beads for 4 h at 4 °C the second IP pellets were then analyzed with standard SDS-PAGE and IB procedures Western blotting images were obtained by the Tanon-5200 Chemiluminescent Imaging System (Tanon) For immunoprecipitation with RNase A Treatment adult mouse testes or GC-2spd (ts) cells were homogenized in lysis buffer A [50 mM Tris-HCl (pH 7.4) proteinase inhibitor cocktail (Roche)] and treated with 250 μg/mL RNase A (Thermo Fisher Additional 125 μg/mL RNase A (Thermo Fisher) and primary antibody-coupled Protein A/G beads were added to the precleared tissue or cell lysates and incubated for 5 h at 4 °C to fully degrade RNAs Testes were fixed overnight at 4 °C in PBS containing 4% PFA and then embedded in paraffin Tissue sections were cut to a thickness of 5 μm Antigen retrieval was achieved by microwaving the sections in 0.01 M Tris-EDTA buffer (pH 9.0) for 2 min the sections were blocked with 5% normal goat serum (NGS) for 30 min The sections were subsequently incubated with primary antibodies diluted in 5% NGS at 37 °C for 1 h The antibodies used included: anti-ACRV1 (1:50; 14040-1-AP or FITC-conjugated anti-γH2AX (1:500; 16–202 A L32460) conjugated with Alexa Fluor Cy3 were used for acrosome staining the sections were incubated with Alexa Fluor 555 anti-rabbit IgG (1:500; A31572 Thermo Fisher) and mounted using an antifade mounting medium containing DAPI (Beyotime Fluorescence microscopy was carried out using a CKX53 fluorescence microscope (Olympus Japan) or a Ti2-E confocal microscope (Nikon Image J software (National Institutes of Health USA) was employed for grayscale conversion of DAPI signals tissue sections were first incubated with either anti-MIWI (1:100; 2079 Cell Signaling Technology) or anti-TDRKH (1:200; 13528-1-AP followed by Alexa Fluor 555 anti-rabbit IgG The sections were then re-blocked with 5% NGS and incubated with either anti-TDRKH or anti-TDRD6 which were pre-labeled with the Zenon Alexa Fluor 488 Rabbit IgG Labeling Kit (Thermo Fisher Z25302) according to the manufacturer’s instructions In vitro piRNA loading assay was performed as we recently described63 HEK293T cells were transfected with the Flag-tagged MIWI expression vector using Lipofectamine 2000 (Invitrogen) cells were harvested and lysed using lysis buffer [20 mM HEPES–KOH (pH 7.0) 1% triton X-100 and 1×Complete EDTA-free protease inhibitor tablets (Roche)] precleared lysates were incubated with Cy5-labeled synthetic piRNA for 1 h at 37 °C lysates were incubated with Protein A/G beads (Invitrogen) coupled with anti-MIWI or anti-MIWIunloaded antibodies half of the anti-MIWI or anti-MIWIunloaded IPed beads were used for immunoblotting of MIWI while the remaining half were used to determine the fluorescence intensities of Cy5 G1281) staining or Hematoxylin and Eosin (H&E) staining testicular or epididymal tissues were fixed in Bouin’s buffer The paraffin-embedded sections were then sequentially deparaffinized and stained with either Hematoxylin and Eosin or Periodic Acid Schiff Apoptosis assays were performed using the In Situ Cell Death Detection Kit Small RNA libraries from immunoprecipitated RNAs or total RNA were prepared using the NEBNext® Multiplex Small RNA Library Prep Kit (NEB Libraries with different barcodes were then pooled together and sequenced using the Illumina NovaSeq 6000 (Novogene Co. RNA reads were normalized by miRNA counts (21–23 nt) RNA reads were normalized by the total reads in each library USA) was employed for the quantification of western blot analysis The signal from each band was converted into intensity values These values were subsequently normalized and utilized to calculate fold changes providing a basis for comparing protein expression across various samples The samples in the same panel were derived from the same experiment and the gels/blots were processed in parallel Results are presented as mean ± standard deviation (SD) of three separate experiments The numbers (n) of biological replicates or animals used are indicated in the individual figure legends and no statistical methods were employed to predetermine the sample size We utilized Student’s t-test to compare the differences between treated groups and their respective paired controls Results are presented as the mean ± standard deviation (SD) P values are indicated either in the text or on the figures with values <0.05 (denoted by asterisks) considered significant (***P < 0.001 Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article Germ plasm and the differentiation of the germ cell line Next generation organelles: structure and role of germ granules in the germline LLPS of FXR1 drives spermiogenesis by activating translation of stored mRNAs Germ granule-mediated RNA regulation in male germ cells Ultrastructural characterization of spermatogenesis and its evolutionary conservation in the germline: germinal granules in mammals Observations on the fine structure and relationships of the chromatoid body in mammalian spermatogenesis Chromatoid body and small RNAs in male germ cells Tdrkh is essential for spermatogenesis and participates in primary piRNA biogenesis in the germline PIWI-interacting small RNAs: the vanguard of genome defence Identification of piRNA binding sites reveals the argonaute regulatory landscape of the C The piRNA targeting rules and the resistance to piRNA silencing in endogenous genes MIWI and piRNA-mediated cleavage of messenger RNAs in mouse testes Mitochondrial membrane-based initial separation of MIWI and MILI functions during pachytene piRNA biogenesis Aub and Ago3 are recruited to Nuage through two mechanisms to form a ping-pong complex assembled by Krimper Tudor domain containing 7 (Tdrd7) is essential for dynamic ribonucleoprotein (RNP) remodeling of chromatoid bodies during spermatogenesis TDRD5 is required for retrotransposon silencing Blockade of pachytene piRNA biogenesis reveals a novel requirement for maintaining post-meiotic germline genome integrity Mouse Piwi interactome identifies binding mechanism of Tdrkh Tudor domain to arginine methylated Miwi Proteomic analysis of murine Piwi proteins reveals a role for arginine methylation in specifying interaction with Tudor family members Structural basis for arginine methylation-independent recognition of PIWIL1 by TDRD2 Identification and functional analysis of the Pre-piRNA 3’ trimmer in silkworms is required for meiotic and post-meiotic male germ cell development The RNase PARN-1 trims piRNA 3’ ends to promote transcriptome surveillance in C modifies germline piRNAs and single-stranded siRNAs in RISC Mouse Piwi-interacting RNAs are 2’-O-methylated at their 3’ termini The 3’ termini of mouse Piwi-interacting RNAs are 2’-O-methylated mediates 2’-O-methylation of Piwi- interacting RNAs at their 3’ ends Deciphering arginine methylation: tudor tells the tale TDRD5 binds piRNA precursors and selectively enhances pachytene piRNA processing in mice SirT1 is required in the male germ cell for differentiation and fecundity in mice piRNA 3’ uridylation facilitates the assembly of MIWI/piRNA complex for efficient target regulation in mouse male germ cells Download references We thank members of the Mo-Fang Liu’ lab and Deqiang Ding’ lab for their helpful comments This work was supported by grants from the National Key R&D Program of China (No National Natural Science Foundation of China (No Science and Technology Commission of Shanghai Municipality (No the Young Elite Scientist Sponsorship Program of the China Association for Science and Technology (No Zhejiang Provincial Natural Science Foundation of China (No the Research Funds of Hangzhou Institute for Advanced Study the Fundamental Research Funds for the Central Universities (No the Foundation of Key Laboratory of Gene Engineering of the Ministry of Education and the New Cornerstone Science Foundation (No Chen Chen was supported by NIH grants (R01HD084494 and R01GM132490) These authors contributed equally: Huan Wei Key Laboratory of Systems Health Science of Zhejiang Province Hangzhou 310024; University of Chinese Academy of Sciences Shanghai Key Laboratory of Maternal Fetal Medicine Clinical and Translational Research Center Shanghai First Maternity and Infant Hospital Frontier Science Center for Stem Cell Research Shanghai Key Laboratory of Molecular Andrology Shanghai Institute of Biochemistry and Cell Biology Center for Excellence in Molecular Cell Science Chinese Academy of Sciences; University of Chinese Academy of Sciences was responsible for bioinformatic analyses All authors discussed the results and commented on the manuscript Nature Communications thanks Godfried van der Heijden and the other Download citation DOI: https://doi.org/10.1038/s41467-024-46664-3 Please enable JS and disable any ad blocker Metrics details PIWI proteins use guide piRNAs to repress selfish genomic elements protecting the genomic integrity of gametes and ensuring the fertility of animal species Efficient transposon repression depends on amplification of piRNA guides in the ping-pong cycle which in Drosophila entails tight cooperation between two PIWI proteins Here we show that post-translational modification Methylation is triggered by loading of a piRNA guide into Aub which exposes its unstructured N-terminal region to the PRMT5 methylosome complex sDMA modification is a signal that Aub is loaded with piRNA guide Amplification of piRNA in the ping-pong cycle requires assembly of a tertiary complex scaffolded by Krimper which simultaneously binds the N-terminal regions of Aub and Ago3 Krimper uses its two Tudor domains to bind Aub and Ago3 in opposite modification and piRNA-loading states Our results reveal that post-translational modifications in unstructured regions of PIWI proteins and their binding by Tudor domains that are capable of discriminating between modification states is essential for piRNA biogenesis and silencing PIWI proteins recognize transposon targets with help of the associated small (23–30 nt) non-coding RNA guides Loss of Prmt5 (encoded by the Csul and Vls genes) in Drosophila leads to reduced piRNA level and accumulation of transposon transcripts in germ cells suggesting that sDMA modification of PIWIs plays an important role in the piRNA pathway the architecture of the ping-pong piRNA processing (4 P) complex and the extent to which Krimper regulates ping-pong remained unresolved sDMA modification of PIWIs provides a binding platform for interactions with Tudor-domain proteins its biological function and regulation are not known Despite the essential role of ping-pong in transposon repression we similarly have little understanding of the molecular mechanisms that control this process Here we revealed the biological function of Aub and Ago3 sDMA modifications and show that it plays an essential role in orchestrating assembly of the 4 P complex in the ping-pong cycle The modification signals whether PIWI proteins are loaded with guide piRNA and this information is used to assemble a ping-pong complex that is receptive for directional transfer of RNA to an unloaded PIWI protein a Scheme depicting two-step sequential immunoprecipitation and GFP-tagged truncated Krimper lacking its N terminal dimerization domain were co-expressed in the S2 cell FLAG-immunoprecipitation was followed by elution and GFP IP b Krimper monomer interacts with Aub and Ago3 simultaneously and GFP-deltaN-Krimper in input and after the first FLAG- and second GFP-IP Experiment reproduced twice with similar results c Top: protein disorder prediction of Krimper Bottom panel: domain architecture of Krimper and constructs used for ITC and structural analyses Top: disorder prediction of Ago3 (1-155aa) Middle: conservation score of each amino acid within Ago3(1-155aa) Bottom: peptides used for ITC and structural analysis Middle: conservation score of each amino acid within Aub(1-145aa) The conserved RG-repeat region is enlarged Bottom: sequence difference between wtAub and mdAub Five arginines within the N terminus were replaced by lysines Aub-1 peptide used for ITC and structural analysis f eTud1 preferentially binds unmethylated Ago3 ITC analysis of Krimper eTud1 with different Ago3 or Aub peptides raising the question of how these two proteins interact the two Tudor domains of Krimper have different binding preferences ensuring that Aub and Ago3 can bind Krimper simultaneously without direct competition for a binding site These results demonstrate that eTud1 does not bind Aub while they also show that the presence of the self-interacting N-terminal region is indispensable for co-immunoprecipitation of Aub with the N + eTud1 fragment The most parsimonious explanation for these results is that formation of the complex between Aub and N + eTud1 observed in co-immunoprecipitation experiments is mediated by the full-length Krimper protein expressed in S2 cells that binds the N + eTud1 fragment through its self-interacting N-region our results indicate that binding of both Aub and Ago3 to Krimper is mediated by the RG/RA-motifs embedded in their unstructured N-terminal extended regions the two proteins interact with Krimper in opposite modification states: Aub must be sDMA-modified The distinct specificities of the two Tudor domains explain how Krimper can simultaneously bind Aub and Ago3 to bring them into physical proximity for ping-pong a Krimper contains the canonical eTud2 domain and the non-canonical eTud1 domain Alignments of Krimper Tudor domains with Tudor1 domain 11 are shown Residues forming the me-Arg binding pocket (red) and corresponding residues in tud1 (green) are shown Additional conserved residues marked yellow b Overall topology demonstration of Krimper eTud2 domain The Tudor and SN-like subdomain are indicated c Overall structure of the Krimper eTud2-Aub-R15me2 complex with the bound Aub-R15me2 peptide shown in yellow d Electrostatic surface of eTud2 with bound Aub-R15me2 peptide shown in yellow e Enlarged view of the eTud2 aromatic cage with R15me2 shown in yellow f Overall topology demonstration of Krimper eTud1 domain g The overall structure of the Krimper eTud1-Ago3-2 complex with bound Ago3-2 peptide shown in orange h Electrostatic surface of eTud1 with bound Ago3-2 peptide shown in orange i Enlarged the view of the eTud1 binding pocket with inserted Ago3 R70 residue indicated in orange the peptide-binding interface of eTud1 is much narrower compared with the extended and negatively charged interface of TDRD2 eTud1 specifically recognizes unmodified R(A/G) motif in Ago3 using a unique hydrophilic concave and a hydrophobic concave region in the interface between the Tudor and SN-like subdomains the N-terminal of the Aub sequence with an additional RG motif appears to be more hydrophilic and may not readily fit into the narrow hydrophobic concave cleft of eTud1 while the peptide of Ago3-1 with only one RG motif appears to be unable to form strong interactions with eTud1 our results reveal that the two Tudor domains of Krimper have distinct architectures that are responsible for the differential binding of two PIWI proteins with N-terminal (G/A)R motifs that are subject to sDMA modification in vivo while Aub interacts with the eTud2 domain in a methylated state through sDMA binding to a conserved aromatic cage Ago3 binds the eTud1 domain in an unmethylated state employing a binding pocket that is distinct from other Tudor domain interactions eTud1 possesses a ‘latch helix’ that must move out of the cleft to allow Ago3 binding providing a potential regulatory mechanism These results highlight the crucial role that sDMA modifications of PIWI proteins play in regulating the formation of the tertiary ping-pong complex Experiments showed similar fractions of mobile proteins respectively Right: microscopic quantification of the ratio of GFP signal originating from nuage versus cytoplasm is shown Experiments showed a similar ratio respectively All transgenes are expressed under the control of the endogenous Aub promoter d Arginine methylation of Aub is required for fertility and mutant females rescued with wt- or mdAub transgenes are indicated as % of eggs laid e Arginine methylation of Aub is required for TE suppression Fold changes of different TE transcripts in ovaries of flies with the indicated genotypes compared to the heterozygous control as measured by RT-qPCR n = 3 biologically independent experiments with similar results The inability of mdAub to rescue sterility and transposon activation suggests that sDMA modification is essential for Aub function in vivo a Length distributions of reads annotated as miRNAs and repeats in ovarian small RNA libraries from indicated flies The ratio of repeat-derived piRNAs to miRNAs in each genotype is indicated above the graphs b Arginine methylation of Aub is required for piRNA expression and ping-pong processing of transposon piRNA The heatmap shows the fold change compared to control (aub heterozygotes) for the top 20 most abundant TE families present in the control ovary The last two heatmaps show changes in 10 A bias for all sense piRNAs and non-1U sense piRNAs c Aub sDMA modification is required for the generation of piRNAs from piRNA clusters but not mdAub rescues piRNA expression from genomic regions affected by aub mutation Genomic windows with more than 5 RPM in control and more than 80% reduction in aub mutant are shown; uni-strand clusters flam and 20 A that are not affected in aub mutants are shown for comparison d Detailed analysis of piRNA generation from major piRNA clusters Reads uniquely mapping to four clusters were determined and plotted as the fold change compared to the het control The dot represents 1 kb windows with uniquely mapping piRNAs within the clusters e The fraction of piRNAs that have the signature of the ping-pong processing (piRNAs that map to TEs in sense orientation and have 10 A but not 1U bias) is reduced in aub mutants and only partially rescued by mdAub expression The graphs show the fraction of sense non-1U piRNAs that have 10 A in libraries from indicated ovaries f Aub Arginine methylation is not required for piRNA binding GFP-tagged mdAub and wtAub were immunoprecipitated from ovaries where the transgene was expressed in the wild-type background Relative piRNA abundance normalized to IP-ed protein is estimated based on band intensity g Arginine methylation of Aub does not greatly affect the TE and antisense fractions of Aub-bound piRNAs The bar chart shows normalized read counts (RPM) mapped to the 20 TE families with post abundant piRNAs Aub transgenes were expressed on the wild-type background Heatmap shows the % of antisense reads mapping to each TE family we did not find any abnormalities in the amount and composition of piRNA loaded into mdAub when it was expressed in the wild-type Aub background the methylation status of Aub plays no direct role in piRNA loading our results indicate that the sDMA modification of Aub has a crucial role in piRNA biogenesis and specifically in the ping-pong amplification cycle a RNA binding is required for Aub arginine methylation in vivo Transgenes were expressed on the wildtype background Total protein level and methylation was detected in Western blot The experiment reproduced three times with similar results b RNA binding is required for Aub interaction with Tudor in vivo GFP tagged Aub was immunoprecipitated from ovarian lysate followed by Western blot detection of the tagged transgenes c Arginine methylation is required for the localization of Aub to the pole plasm GFP tagged wtAub and mdAub were separately expressed in ovaries on the wild-type Aub background Ten independent ovaries were used for imaging with similar results d RNA binding is required for Aub localization into the pole plasm Upper panel: GFP-wtAub and mKate2-wtAub were co-expressed in ovaries in the wild-type Aub background Bottom panel: GFP-pdAub and mKate2-wtAub were co-expressed in ovaries in the wild-type Aub background e Zucchini KD leads to decreased Aub arginine methylation FLAG-tagged wtAub was immunoprecipitated from control and Zuc KD ovaries f Scheme of in vitro oligo-binding experiments Lysate of S2 cells expressing FLAG-Aub was incubated with or without 32P labeled 26 nt RNA oligos followed by FLAG immunoprecipitation and detection of methylation by Western blot and oligo binding on urea gel g RNA oligo binding promotes Aub sDMA modification 30 nt (30 M) ssRNA was spiked into each IP-ed sample to normalize for total IP-ed RNA amount h Aub methylation correlates with synthetic ssRNA concentration added to lysates of S2 cells expressing FLAG-Aub Methylation was quantified based on band intensity in FLAG and SYM11 Western blot and normalized to the no oligo control i Binding of the RNA 3′ end by Aub’s PAZ domain is required for sDMA modification Wild-type Aub and AubPAZmut were expressed in S2 cells sDMA modification was detected by western blot using SYM11 antibody suggesting that the modification status of wild-type Aub depends on its piRNA loading status loading of Aub with synthetic RNA triggers sDMA modification in cell lysate indicating a direct link between RNA binding and modification indicating that complete binding of the guide—both 5′ and 3′ ends—is required to induce modification a N-terminal region within Aub protein is not easily accessible to methylation FLAG-tagged full length and N-terminal truncated (1-105aa) Aub were expressed in S2 cells and immunoprecipitated Total protein and methylation level were assessed by Western blot Arrowheads indicate correct size for full-length and N-terminal fragment b Top: scheme showing architectures of different Aub constructs expressed and IP-ed from S2 cells EGFP inserted between the N-terminal and PAZ domain (pdAub-EXT) artificially exposes N-terminus in absence of piRNA binding Bottom: western blot analysis of methylation states Relative methylation level as estimated by the ratio of SYM11/FLAG band intensities normalized to wildtype is listed c Aub interacts with Csul and Vls in S2 cells Asterisk indicates band corresponding to GFP-Csul in the INPUT d Aub binding to Csul depends on RNA binding but not on Arg methylation FLAG-Csul and HA-Aub transgenes were expressed in S2 cells and coIP followed by Western detection e RNA loading of Aub leads to increased binding to Csul and increased methylation of Aub FLAG-Csul and HA-Aub were expressed in S2 cells lysates were incubated in the presence or absence of ssRNA oligo prior to coIP followed by Western detection Next, we tested if we can alter the accessibility of the N-terminal region to the methylation machinery by inserting an extra sequence between this region and the rest of the protein (named pdAub-EXT). While Aub deficient in piRNA binding was methylated only on a very weak level, insertion of a GFP sequence after AA105 caused robust methylation (Fig. 6b) insertion of an extended sequence between the N-terminus and the rest of the protein converts the N-terminal (GA)R motif into a good substrate for modification even if the protein is not loaded with RNA these experiments suggest that the N-terminal region is not readily accessible if Aub is not bound to RNA it can be readily methylated upon solvent exposure This indicated that the methylosome interacts with the N-terminal region of Aub harboring (G/A)R motif while other regions are dispensable for binding we determined that increased levels of sDMA modifications upon RNA binding appear to be a direct result of the stronger binding of RNA-loaded Aub to the methylosome complex We found that a post-translational modification specific to members of the PIWI clade of the Argonaute family encodes information about guide RNA loading status and regulates interactions Ping-pong employs the intrinsic RNA binding and processing capabilities of Ago proteins it creates new functionality through the cooperation between two PIWI proteins Our results indicate that the ping-pong cycle and sDMA-modification are tightly linked and that the modification status of PIWI proteins regulates the assembly of the ping-pong processing complex these results suggest that sDMA modification of Aub acts as a signal of its piRNA-bound state a Model sDMA dependent assembly of the ping processing complex The N-terminus of unloaded Aub is inaccessible Binding to piRNA guide leads to a conformational change of Aub exposing its N terminus and enabling methylation of its N-terminal arginines Methylation thus serves as a signal of loading-state and enables Aub binding to the Tud2 domain of Krimper which specifically recognizes methylated Arginines The Tud1 domain of Krimper binds unmethylated enabling Ago3 loading with the newly processed piRNA The N-terminal unstructured region of Krimper allows Krimper multimerization resulting in a Krimper scaffold that might assist in nuage assembly and ensuring high local concentration of Aub and Ago3 b The Ping-Pong cycle consists of two distinct stages Aub with its piRNA guide targets piRNA precursors or TE transcripts piRNA-loaded Ago3 targets antisense piRNA precursors The ping and pong processing could be accomplished by different complexes in nuage Krimper-bound Ago3 is both unloaded and unmethylated indicating that piRNA binding and modification are correlated for Ago3 as well as for Aub The poor similarity between N-terminal sequences of different Agos might endow them with distinct functions It might be worth exploring whether signaling of the guide-loading state through exposure of the N-terminal region is also conserved in Ago-clade proteins and whether it regulates their function information about the piRNA-loading state of PIWI proteins signaled by their sDMA modifications is used to assemble a complex that enables the transfer of the processed RNA from Aub to Ago3 The architecture of the tertiary complex assembled by Krimper permits Aub-dependent generation and loading of RNA into Ago3 the ping-pong cycle also includes the opposite step Ago3-dependent generation of Aub piRNA (henceforth we termed these steps “ping” and “pong”) Our results suggest that the ping and pong steps require the assembly of two distinct complexes discriminated by the modification status of Aub and Ago3 The high local concentration of proteins and RNA involved in the piRNA pathway in nuage might enhance the efficiency of ping-pong as well as the recognition of RNA targets by Aub and Ago3 Short hairpin RNA (shRNA) lines used for knockdown including sh-White (BDSC #33623) and sh-Zuc (BDSC #35227) maternal alpha-Tubulin 67C-Gal4 drivers on chromosome two (BDSC #7062) or chromosome three (BDSC #7063) in addition to the Aub mutant stocks aubHN2 cn1 bw1/CyO (BDSC #8517) and aubQC42 cn1 bw1/CyO (BDSC # 4968) were obtained from the Bloomington Drosophila Stock Center Flies were kept on yeast for 2 days and ovaries dissected 5 days after hatching Transgenic constructs for injection were generated using the Gateway cloning system (Life Technologies) cDNAs were obtained by RT-PCR from ovarian or testes RNA of adult Drosophila melanogaster and AubPAZmut were generated by overlap PCR and inserted into the pENTR-D-TOPO directional cloning vector (Life Technologies) Transgenes were cloned into the pUASP-Gateway-phiC31 fly injection vector derived from the pCasPeR5-phiC31 vector containing GFP or Strep-FLAG tags using the Gateway cloning system (Life Technologies) The expression of each transgene was controlled using the yeast upstream activation sequence promoter (UASp) stably crossed with a maternal a-Tubulin67c-Gal4-VP16 (MaG4) driver Transgenes were generated in flies by PhiC31-mediated transformation (BestGene) using PhiC31 landing pads on either chromosome two (BDSC #9736) or chromosome three (BDSC #9750) The GFP-wtAub and GFP-mdAub BAC line was generated by cloning the aub genomic locus from the BAC clone BACN04M10 into the pCasPeR4 vector using restriction sites XhoI and SpeI Bacterial recombineering (Gene Bridges Counter Selection kit) was used to insert an in-frame GFP tag in the start site of Aub GFP-wtAub and GFP-mdAub rescue lines were generated by crossing transgenic construct into the aub[HN]/ aub[QC] background Schneider S2 cells were cultured in a complete Schneider medium (10% heat-inactivated FBS; 100U penicillin [Life technologies]; 100 μg streptomycin [Life technologies]) Plasmids were generated using Gateway cloning (Life technologies) using the Drosophila gateway vector collection (DGVC) destination vectors Cells were transfected using TransIT-LT1 transfection reagent (Mirus biosciences) according to the manufacturer’s recommendation using 3 μg of total plasmid S2 cells were lysed in S2 lysis buffer (20 mM Tris at pH7.4 EDTA-free Complete Protease Inhibitor Cocktail [Roche] Supernatant was cleared by centrifugation at 4000×g for 20 min at 4 °C Input sample was collected from the supernatant at concentrations of 1–3 μg/μL and anti-GFP antibody (Covance) conjugated to Dynabeads (Thermo Fisher) were blocked in 5 mg/ml BSA for 10 min at 4 °C Beads were added to the supernatant and rotated at 4 °C for 4 h washed three times in lysis buffer and eluted by boiling in reducing SDS loading buffer FLAG beads were eluted using 3X FLAG peptide (Sigma- Aldrich) dissected ovaries were lysed in NT2 buffer (50 mM Tris at pH 7.4 EDTA-free Complete Protease Inhibitor Cocktail) while for IP experiments RIPA buffer (25 mM Tris at pH 7.4 EDTA-free Complete Protease Inhibitor Cocktail) was used for lysis Lysate was incubated in the presence or absence of 100 µg/mL RNase A and cleared by centrifugation lysates were incubated with anti-FLAG M2 beads (Sigma Aldrich) or with anti-GFP antibody (Covance) conjugated to Dynabeads (Thermo Fisher) at 4 °C for 4 h lysates were incubated with GFP nanobody (Chromo Tek) followed by washing and elution in reducing SDS buffer Western blot was probed with rabbit anti-GFP (Covance) (1:3 K) rabbit SYM11 antibody (Sigma Aldrich) (1:1 K) Small RNA libraries were cloned from the total ovarian lysate of Aub heterozygous aub[HN]/ aub[QC] mutant and aub[HN]/ aub[QC] and total RNA was isolated with Ribozol (Amresco Small RNAs within a 19- to 29-nt window were isolated from 15% polyacrylamide gels from 4 µg of ovarian total RNA 150 mM boric acid) and 200 mM sodium periodate were added to the size-selected small RNA and the samples were incubated for 30 min at 25 °C The NaIO4-treated small RNA was then ethanol-precipitated before proceeding to library construction The small RNA libraries were constructed using the NEBNext small RNA library preparation set for Illumina (no using NEBNext multiplex oligos for Illumina (no Libraries were sequenced on the Illumina HiSeq 2500 (SE 50-bp reads) platform Ovaries (∼100 per immunoprecipitation) from flies expressing GFP-wtAub and GFP-mdAub under the control of endogenous promoter were dissected and lysed on ice in 250 µL of lysis buffer [30 mM Hepes-KOH at pH7.4 Lysate was dounced and clarified by centrifugation at maximum speed at 4 °C The supernatant was incubated with rabbit polyclonal anti-GFP (Covance) conjugated to Dynabeads (Thermo Fisher) for 4 h at 4 °C 10% of immunoprecipitate was used for western blotting Five 5 pmol of 42 nt RNA oligonucleotide (42 M) was added to the beads to assess purification efficiency followed by proteinase K digestion and phenol extraction of RNA A fifth of the RNA was CIP-treated (New England Biolabs M0290S) in NEB buffer #3 (New England Biolabs B7003S) for 30 min at 37 °C and then ethanol-precipitated after phenol:chloroform and chloroform extraction The CIP-treated RNA was then PNK-treated with 1 µL of 10× T4 polynucleotide kinase buffer (New England Biolabs and 1 µL of T4 polynucleotide kinase (New England Biolabs The CIP- and PNK-treated RNA was added back to the remainder of the RNA isolated from the immunoprecipitation and analysis were performed as described in small RNA-seq except that fragments were gel-extracted based on labeled immunoprecipitation material while miRNAs were defined based on their mapping to annotated miRNA genes To analyze piRNA generation throughout the genome the genome was split into 5 kb intervals and windows that produce at least 5 reads per million of miRNAs in control (aub heterozygous) and showed more than 80% reduction of piRNAs in the aub mutants were further analyzed To compare piRNA abundance between the libraries piRNA reads were normalized to total miRNA reads For a detailed comparison of piRNA generation from 42AB To analyze piRNAs to individual TE families piRNA reads were mapped to TE consensus sequences (http://www.fruitfly.org/p_disrupt/TE.html) allowing for up to 2 mismatches To compare TE piRNA abundance between the libraries piRNA reads were normalized to total miRNA reads The fold-change in read count for each TE family was calculated by obtaining the base 2 logarithm of ratio of normalized reads in experimental libraries and heterozygous control libraries To obtain the fraction of ping-pong piRNA pairs for each TE family we measure the likelihood that a piRNA has a “partner” with 10 nt overlap We first matched all piRNA sequences to the Repbase transposon sequences allowing for plus and minus strand matches with up to 2 mismatches we computed a histogram of distances/offsets to “partner” piRNAs that match to the opposite strand for which each partner piRNA contributes its cloning/sequencing count normalized to 100% overall partner piRNAs up to a distance of 29 nt (note that each piRNA is a “partner” piRNA for all other piRNAs and also a “target” piRNA itself with all other piRNAs as “partners”) We then combined these histograms for each distance/offset by summing the values for that distance over all histograms but weighting each histogram’s contribution by the relative cloning/sequencing of the respective target piRNA for which the histogram was computed To compare the abundance of TE piRNA associated with wtAub- and mdAub reads mapping to each TE family were normalized to total library reads (RPM) after rRNA reads were discarded Ovaries were dissected in PBS and fixed in 4% PFA in PBS for 20 min permeabilized in 1% Triton-X100 in PBS for 10 min Ovaries were washed in PBS and mounted in Vectashield medium (Vector Labs) S2 cells were allowed to settle on coverslips treated with Poly-L-Lysine (Sigma-Aldrich) cells were fixed in 0.5% PFA in PBS for 20 min followed by staining with DAPI (Sigma-Aldrich) and mounted in Vectashield medium (VectorLabs) Images were captured using an AxioImager microscope; an Apotome structured illumination system was used for optical sections (Carl Zeiss) The in vitro assay for Aub slicer activity was adapted from a method described previously5 FLAG-tagged wild-type and mdAub proteins were expressed and immunopurified from S2 cells Proteins were eluted using 3X Flag peptide (Thermo Fisher) 50 K MWCO protein concentrator (Thermo Fisher) was used to remove the 3X Flag peptide 100 nM purified proteins were incubated with 100 nM of single-strand 26 nt guide RNA in cleavage buffer (25 mM Hepes-KOH and then 32P-5′-labeled 29 nt complementary RNA (PerkinElmer BLU502A250UC) was added and incubated for another 90 min The cleaved products were analyzed on urea-containing PAGE 10 ng/µl chymotrypsin stock solution was prepared in chymotrypsin reaction buffer (10 mM Tris-HCl [pH 8.0] Lysates were divided into two equal fractions one was incubated with 26 nt ssRNA oligo for 1 h at RT followed by immunoprecipitation using anti-FLAG M2 beads Beads were washed three times with lysis buffer and then incubated with a 1:2 K and 1:8 K serial dilution of the thermolysin protease for 30 min at 37 °C Samples were analyzed by western blot using an anti-FLAF M2 antibody Three-day-old mated adult female flies fed on yeast paste were transferred to fresh grape agar plates and allowed to lay eggs for 12 h and the hatching rate was determined over the following 36 h Counting was repeated on ten consecutive days To account for background and photo-bleaching effects during acquisition the mean intensity values from the bleach zone (BL) and the reference signal zone (REF) were used to calculate the corrected BL(BLcorr) for each acquisition frame using the equation: BLcorr values were normalized to the mean of five pre-bleach values which were used to estimate 100% fluorescence intensity the normalized post-bleach data were fit to an exponential recovery model: The mobile fraction was obtained by the sum of coefficients a and c which describes the maximal extent of recovery for each experiment The occupancy of GFP-tagged protein in either nuage versus cytoplasm was calculated by obtaining the ratio of mean signal intensities of a perinuclear nuage area (N); an adjacent cytoplasmic area (C) of the equal area; and a background area (G) using the following equation: All images were acquired from the fixed specimen using the Apotome structured illumination system and a 40× oil-immersion objective S2 cells were transfected with 10 μg of a plasmid expressing N terminally Flag tagged wtAub driven by the Actin5C promoter cells were lysed in S2 lysis buffer (20 mM Tris at pH7.4 Lysates were divided into four equal fractions 26 nt synthetic RNA oligo (IDT) labeled with [γ-P32] ATP (PerkinElmer BLU502A250UC) as described above was added into lysate fractions to a final concentration 1 μM Lysates were incubated at RT for 1 h followed by IP with FLAG M2 beads at 4 °C for 4 h Half of the beads were subject to RNA isolation In the oligo binding concentration gradient experiment lysates were divided into four equal fractions non-radioactivity labeled 5′-end phosphorylated ssRNA oligo (IDT) was added into each fraction to a final concentration of 0 Lysates were incubated at RT for 1 h followed by anti-FLAG IP at 4 °C for 4 h Protein and methylation levels were detected by Western blot and methylation level was estimated as the ratio of the background-subtracted methylation signal intensity and the background-subtracted protein signal intensity Relative methylation level was measured by normalizing to the methylation level of the no oligo control Krimper eTud1 (residues 272-512) was cloned into a self-modified pSumo vector with 10×His tag followed by a yeast sumo sequence coli strain BL21(DE3) Rosseta and cultured at 37 °C in LB medium The protein expression was induced by adding IPTG to a final concentration of 0.2 mM when the OD600 reached 0.7 The recombinant expressed protein was purified using a HisTrap column (GE Healthcare) The hexahistidine plus yeast sumo tag was removed by ulp1 protease digestion followed by a second step HisTrap column (GE Healthcare) The target protein was further purified using MonoQ and Superdex G75 columns (GE Healthcare) Krimper eTud2 (residues 562-746) was cloned into a self-modified His-MBP vector Protein production procedure was to with Krimper eTud1 The hexahistidine plus MBP tag was removed by TEV protease digestion followed by a second step HisTrap column (GE Healthcare) Protein was further purified using Q and Superdex G75 columns (GE Healthcare) Krimper eTud1 (272-512) was concentrated to 20 mg/ml and screened in sitting drop at 4 °C Crystals were grown for 5 days in 0.1 M HEPES pH 7.5 20 mg/ml Krimper eTud1 was mixed with Ago3-2 peptide with a molar ratio of 1:4 and incubated for 1 h before setting up sitting drop screening at 4 °C 10 mg/ml Krimper eTud2 was mixed with AubR15me2 peptide with a molar ratio of 1:4 and incubated for 1 h before sitting drop Crystals appeared in 2 days in 16% PEG3350 and 0.1 M NH4Ac All ITC was performed using the Malvern PEAQ ITC instrument (Malvern) in a buffer of 50 mM NaCl Data analysis was performed using the Malvern data analysis software and Origin 7.0 Further information on experimental design is available in the Nature Research Reporting Summary linked to this paper Argonaute protein PIWI controls mobilization of retrotransposons in the Drosophila male germline Collapse of germline piRNAs in the absence of argonaute3 reveals somatic piRNAs in flies Double-stranded RNA-mediated silencing of genomic tandem repeats and transposable elements in the D-melanogaster germline A slicer-mediated mechanism for repeat-associated siRNA 5′ end formation in Drosophila Gene silencing mechanisms mediated by aubergine-piRNA complexes in Drosophila male gonad The Argonaute family: tentacles that reach into RNAi piwi encodes a nucleoplasmic factor whose activity modulates the number and division rate of germline stem cells Structural evolution and functional diversification analyses of argonaute protein Crystal structure of Argonaute and its implications for RISC slicer activity The structure of human argonaute-2 in complex with miR-20a Crystal structure of the MID-PIWI lobe of a eukaryotic Argonaute protein Structural basis for 5′-nucleotide base-specific recognition of guide RNA by human AGO2 Structural basis for overhang-specific small interfering RNA recognition by the PAZ domain The crystal structure of the Argonaute2 PAZ domain reveals an RNA binding motif in RNAi effector complexes Structure and conserved RNA binding of the PAZ domain Crystal structure of a PIWI protein suggests mechanisms for siRNA recognition and slicer activity RISC is a 5′ phosphomonoester-producing RNA endonuclease piRNA biogenesis in Drosophila melanogaster The making of a slicer: activation of human Argonaute-1 Structure of yeast Argonaute with guide RNA Multidomain convergence of argonaute during RISC assembly correlates with the formation of internal water clusters Arginine methylation as a molecular signature of the Piwi small RNA pathway Analysis of sDMA modifications of PIWI proteins Arginine methylation of Piwi proteins catalysed by dPRMT5 is required for Ago3 and Aub stability Loss of the mili-interacting Tudor domain-containing protein-1 activates transposons and alters the Mili-associated small RNA profile Functional involvement of Tudor and dPRMT5 in the piRNA processing pathway in Drosophila germlines Structural basis for dimethylarginine recognition by the Tudor domains of human SMN and SPF30 proteins Structural basis for methylarginine-dependent recognition of Aubergine by Tudor Structural basis for recognition of arginine methylated Piwi proteins by the extended Tudor domain How does the royal family of Tudor rule the PIWI-interacting RNA pathway A systematic analysis of Drosophila TUDOR domain-containing proteins identifies Vreteno and the Tdrd12 family as essential primary piRNA pathway factors Tudor-domain containing proteins act to make the piRNA pathways more robust in Drosophila Arginine methylation of Aubergine mediates Tudor binding and germ plasm localization Crystal structure of silkworm PIWI-clade argonaute siwi bound to piRNA Krimper enforces an antisense bias on piRNA pools by binding AGO3 in the Drosophila germline The Drosophila SDE3 homolog armitage is required for oskar mRNA silencing and embryonic axis specification The role of Tudor domains in germline development and polar granule architecture Tudor and its domains: germ cell formation from a Tudor perspective Germ cell specification and migration in Drosophila and beyond is involved in assembly of these structures and binds to Tudor and the methyltransferase Capsuleen Structural basis for piRNA 2’-O-methylated 3′-end recognition by Piwi PAZ (Piwi/Argonaute/Zwille) domains Recognition of 2′-O-methylated 3′-end of piRNA by the PAZ domain of a Piwi protein Identification and functional analysis of the pre-piRNA 3′ trimmer in silkworms Armitage determines Piwi-piRISC processing from precursor formation and quality control to inter-organelle translocation Piwi nuclear localization and its regulatory mechanism in Drosophila ovarian somatic cells produces dimethylarginine-modified Sm proteins Methylation of Sm proteins by a complex containing PRMT5 and the putative U snRNP assembly factor pICln A novel WD repeat protein component of the methylosome binds Sm proteins Arginine methyltransferase Capsuleen is essential for methylation of spliceosomal Sm proteins and germ cell formation in Drosophila is essential for germ-cell specification and maintenance HSP90 protein stabilizes unloaded argonaute complexes and microscopic P-bodies in human cells Phosphorylation of Argonaute 2 at serine-387 facilitates its localization to processing bodies Prolyl 4-hydroxylation regulates Argonaute 2 stability Structure of Aquifex aeolicus Argonaute highlights conformational flexibility of the PAZ domain as a potential regulator of RNA-induced silencing complex function Structural and mechanistic insights into an archaeal DNA-guided Argonaute protein Argonaute of the archaeon Pyrococcus furiosus is a DNA-guided nuclease that targets cognate DNA Structure of the guide-strand-containing argonaute silencing complex Structural basis for the recognition of guide RNA and target DNA heteroduplex by Argonaute Structural insights into mRNA recognition from a PIWI domain-siRNA guide complex Eukaryote-specific insertion elements control human ARGONAUTE slicer activity Human Argonaute2 mediates RNA cleavage targeted by miRNAs and siRNAs The expanded universe of prokaryotic argonaute proteins Structure-based cleavage mechanism of Thermus thermophilus Argonaute DNA guide strand-mediated DNA target cleavage The N domain of Argonaute drives duplex unwinding during RISC assembly a protein with both E3 ligase and tudor domains PrDOS: prediction of disordered protein regions from amino acid sequence Protein sequence alignments: a strategy for the hierarchical analysis of residue conservation HKL-3000: the integration of data reduction and structure solution–from diffraction images to an initial model in minutes Download references We thank members of the Aravin lab for their discussion and comments We thank the BL19U1 beamlines staff at the Shanghai Synchrotron Radiation Facility for assistance during data collection We thank Igor Antoshechkin (Caltech) for help with sequencing This work was supported by grants from the National Institutes of Health (R01 GM097363) and by the HHMI Faculty Scholar Award to A.A.A and the National Natural Science Foundation of China (31870755) and the Guangdong Innovation Research Team Fund (2016ZT06S172) to S.L Max Planck Institute for Biophysical Chemistry These authors contributed equally: Xiawei Huang Division of Biology and Biological Engineering Pasadena California Institute of Technology National Key Laboratory of Plant Molecular Genetics and Shanghai Center for Plant Stress Biology Center for Excellence in Molecular Plant Sciences Southern University of Science and Technology of China Department of Biochemistry and Molecular Biology conceived and supervised ITC and structural work; A.A.A conceived and supervised all other experiments developed tools for the analysis of small RNA libraries Peer review information nature communications thanks Jinrong Min and other reviewers for their contributions to the peer review of this work Download citation DOI: https://doi.org/10.1038/s41467-021-24351-x Nature Reviews Molecular Cell Biology (2023) Metrics details Piwi-interacting RNAs (piRNAs) are predominantly expressed in germ cells and function in gametogenesis in various species Piwi-deficient female mice are fertile and mouse oocytes express a panel of small RNAs that do not appear to be widely representative of mammals the function of piRNAs in mammalian oogenesis remains largely unclear we generated Piwil1- and Mov10l1-deficient golden hamsters and found that all female and male mutants were sterile with severe defects in embryogenesis and spermatogenesis the oocytes and embryos displayed aberrant transposon accumulation and extensive transcriptomic dysregulation and the embryos were arrested at the two-cell stage with impaired zygotic genome activation PIWIL1-piRNAs exert a non-redundant function in silencing endogenous retroviruses in the oocytes and embryos our findings demonstrate that piRNAs are indispensable for generating functional germ cells in golden hamsters and show the value of this model species for piRNA studies in gametogenesis especially those related to female infertility Piwi proteins and piRNAs in mammalian oocytes and early embryos Identification and characterization of two novel classes of small RNAs in the mouse germline: retrotransposon-derived siRNAs in oocytes and germline small RNAs in testes Single-cell CAS-seq reveals a class of short PIWI-interacting RNAs in human oocytes Zili is required for germ cell differentiation and meiosis in zebrafish A role for Piwi and piRNAs in germ cell maintenance and transposon silencing in zebrafish piRNA-associated germline nuage formation and spermatogenesis require mitoPLD profusogenic mitochondrial-surface lipid signaling A retrotransposon-driven Dicer isoform directs endogenous small interfering RNA production in mouse oocytes Endogenous siRNAs from naturally formed dsRNAs regulate transcripts in mouse oocytes Pseudogene-derived small interfering RNAs regulate gene expression in mouse oocytes The golden (Syrian) hamster as a model for the study of reproductive biology: past Hamster PIWI proteins bind to piRNAs with stage-specific size variations during oocyte maturation Efficient gene targeting in golden Syrian hamsters by the CRISPR/Cas9 system Acrosin is essential for sperm penetration through the zona pellucida in hamsters The control of DNA repair by the cell cycle Efficient generation of targeted large insertions by microinjection into two-cell-stage mouse embryos A microtubule-organizing center directing intracellular transport in the early mouse embryo Nimble and ready to mingle: transposon outbursts of early development Mechanisms regulating zygotic genome activation Mitochondria in early development: linking the microenvironment Restricted and non-essential redundancy of RNAi and piRNA pathways in mouse oocytes Global profiling of RNA-binding protein target sites by LACE-seq Loubalova, Z. et al. Formation of spermatogonia and fertile oocytes in golden hamsters requires piRNAs. Nat. Cell Biol. https://doi.org/10.1038/s41556-021-00746-2 (2021) Hasuwa, H. et al. Production of functional oocytes requires maternally expressed PIWI genes and piRNAs in golden hamsters. Nat. Cell Biol. https://doi.org/10.1038/s41556-021-00745-3 (2021) Transcriptome landscape of human folliculogenesis reveals oocyte and granulosa cell interactions Enzymatic isolation of human primordial and primary ovarian follicles with Liberase DH: protocol for application in a clinical setting Multiplex genome engineering using CRISPR/Cas systems Distribution of crossing over on mouse synaptonemal complexes using immunofluorescent localization of MLH1 protein Chromosome spread analyses of meiotic sex chromosome inactivation Smart-seq2 for sensitive full-length transcriptome profiling in single cells Tn5 transposase and tagmentation procedures for massively scaled sequencing projects piClust: a density based piRNA clustering algorithm Comprehensive integration of single-cell data The dynamics and regulators of cell fate decisions are revealed by pseudotemporal ordering of single cells RepeatModeler2 for automated genomic discovery of transposable element families Gapped BLAST and PSI-BLAST: a new generation of protein database search programs Identifying repeat domains in large genomes GtRNAdb: a database of transfer RNA genes detected in genomic sequence Download references Li’s laboratories for their discussion and comments on this project; M Shum for their comments and suggestions; Z Xu for his assistance with high-performance computing; staff at the HPC storage and network service platform of SIBCB for supplying the computing resources; H Japan) for sharing unpublished data; and Y This work was supported by the following funding: the National Natural Science Foundation of Jiangsu Province (BE2019730) to J.L.; the National Key R&D Program of China (2017YFA0504401) the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB19040102) and the National Natural Science Foundation of China (31970607 and 31470781) to L.W.; the National Basic Research (973) Program of China (2009CB941700) Natural Science Foundation (SKLRM-2021B5) of State Key Lab of Reproductive Medicine and the National Natural Science Foundation of China (31171443) to J.L.; the National Key Research and Development Program of China (2018YFC1003800) the National Natural Science Foundation of China (31871507 the National Basic Research (973) Program of China (2014CB943200 and 2013CB945500) and the National Natural Science Foundation of Jiangsu Province (BK20140061) to Y.-Q.S These authors contributed equally: Hongdao Zhang State Key Laboratory of Reproductive Medicine Jiangsu Animal Experimental Center of Medicine and Pharmacy Jiangsu Key Laboratory of Xenotransplantation Collaborative Innovation Center for Cardiovascular Disease Translational Medicine The First Affiliated Hospital with Nanjing Medical University conceived and developed the methodology for genome editing of golden hamsters generated the Piwil1 and Mov10l1-deficient golden hamsters with the help of J.Z analysed the phenotype of Piwil1 mutants and Z.Z. conducted small-RNA- and RNA-sequencing analysis performed bioinformatics analyses with the help of Y.X interpreted the data of the experiments and wrote the manuscript Peer review information Nature Cell Biology thanks René Ketting Jeremy Wang and William Theurkauf for their contribution to the peer review of this work (a) Strategy for the generation of albino (Tyr mutants) golden hamsters Two-cell embryos were injected with CRISPR/Cas9 and sgRNAs and transferred to naturally pregnant recipients (b) Diagram of the golden hamster Tyr gene and the resulting Tyr mutants (c) Production of albino golden hamster lines Albino golden hamster lines were established by 7 mutant founders (d) The live birth rate after transfer of injected 1-cell or 2-cell embryos n = 3 or n = 5 biologically independent experiments for PN injection or 2 C injection Statistical analysis was performed using unpaired two-tailed t-test Statistical data are provided in the source data (e) Strategy for the generation of Piwil1 mutant golden hamsters by CRISPR/Cas9 Both mutant1 and mutant2 are frameshift variants (g-h) Immunostaining shows loss of PIWIL1 expression in MII oocytes (g) and 2-cell embryos (h) of Piwil1m1/m1 The experiments were independently repeated twice with similar results (i) Structure of golden hamster the Mov10l1 gene and generation of Mov10l1 mutants (Mov10l1ins1/ins1) The Mov10l1ins1/ins1 contained one thymine (T) insertion in exon 2 (j) Diagram of wild-type and MOV10L1 mutant protein Mov10l1ins1/ins1 caused a frameshift that generated a premature stop codon in Mov10l11 mRNAs Source data (a) PAS staining of wild type and Piwil1m1/m1 ovarian sections (b) The average numbers of ovulated oocytes collected from wild-type and Piwil1m1/m1 females p-value = 0.2 by unpaired two-tailed t-test (n = 10 superovulated golden hamsters per group) (c) Immunofluorescence staining of the MII oocytes and early embryos using Actin- and Tubulin-specific antibodies The results show normal morphology of spindles in Piwil1-deficient MII oocytes and the absence of the microtubule bridge (white arrow) in maternal Piwil1-deficient embryos Piwil1m1/m1 indicates Piwil1-deficient oocytes or maternal Piwil1-deficient embryos (d) Representative images of in vitro cultured embryos obtained from wild-type and Piwil1m1/m1 oocytes fertilized in vivo with wild-type sperm at 9 (e) Representative images of embryos collected from the oviducts of wild-type and Piwil1m2/m2 females at 52 h.p.e.a The assays in a and c-d were performed twice Source data (a) Comparison of the testes from 8-week-old wild-type Piwil1m2/m2 and Mov10l1ins1/ins1 golden hamsters Testes were collected from 8-week-old wild-type Piwil1m1/m1 and Mov10l1ins1/ins1 golden hamsters n = 4 Piwil1 mutants or n = 5 Mov10l1 mutants (c-d) Periodic acid-Schiff (PAS) staining of adult caput epididymis (c) (e) H&E staining of WT and Mov10l1ins1/ins1 testes at 8 d and e were independently repeated twice and showed similar results Source data (a) The piRNAs identified in oocytes and embryos collected from wild-type females showed three main peaks in their size distribution: 18–20-nucleotide (19-nucleotide piRNA) and 28–30-nucleotide (29-nucleotide piRNA) All three piRNA populations preferentially carried a 5′ uracil (U) Oocytes and embryos collected from Piwil1m1/m1 females only expressed 19-nucleotide piRNAs which showed a strong preference for a 5′ U Data shown were average value of biological replicates at each time point: n = 3 (PF) 4 (44 h.p.e.a.) or 3 (52 h.p.e.a.) for WT; n = 2 (PF) 4 (44 h.p.e.a) or 4 (52 h.p.e.a) for Piwil1 mutants (b) Sequence analysis of piRNAs bound to PIWIL1 and PIWIL3 in MII oocytes (c) The 5’-5’ overlap between sense- and antisense strands of TE-derived piRNAs bound to PIWIL1 or PIWIL3 were analyzed for the presence of Ping-Pong signatures The number of pairs of piRNA reads at each position is plotted Significance of 10-nucleotide overlap (‘Ping-Pong’) was determined based on Z score; a Z score > 1.96 corresponds to p-value < 0.05 (a) Heat maps show the expression level (RPKM) of top piRNA clusters which generated >90% of the unique mapped piRNAs in each oocyte and embryo at different developmental stages (b) The relative abundances of 23-nucleotide 29-nucleotide and 19-nucleotide piRNAs among total piRNAs (a) in each cluster are shown Almost all of the piRNA clusters that produce PIWIL1 23-nucleotide and 29-nucleotide piRNAs are identical while several piRNA clusters uniquely produced PIWIL3 19-nucleotide piRNAs (c) The ratio of piRNAs with identical 5’ ends (up) and 3’ ends (bottom) among PIWIL1 23-nucleotide PIWIL1 29-nucleotide and PIWIL3 19-nucleotide piRNAs Only uniquely mapped piRNAs are calculated in (a) and (b) Data shown in a-c were average value of biological replicates at each time point: n = 3 (PF) 4 (44 h.p.e.a.) or 4 (52 h.p.e.a.) for Piwil1 mutants (d) Differential analysis of miRNA expression during oogenesis and early embryo development miRDeep2 was used for de novo miRNA identification Piwil1-deficient oocytes or maternal Piwil1-deficient embryos; w (e) Sequence analysis of piRNAs expressed in testes The 28–31-nucleotide piRNAs found in wild-type testes and the remaining 26–28-nucleotide piRNAs in Piwil1m1/m1 testes both showed a strong preference for 5′ U (f) Composition of small RNAs in wild-type and Piwil1m1/m1 testes with or without NaIO4 oxidation treatment The small RNA counts were normalized by exogenous spike-in (g) Size distribution of PIWIL1-bound piRNAs in wild-type and Piwil1m1/m1 testes immunoprecipitated with PIWIL1-specific antibody Rabbit non-specific immunoglobulin G (IgG) served as a negative control (h) Sequence analysis of PIWIL1-piRNAs expressed in testis (a) Genomic annotation of piRNA counts of different sizes identified in oocytes at the primary follicle stage through embryos at 52 h.p.e.a in wild-type and Piwil1m1/m1 golden hamsters (b) Bar graphs show the distribution of piRNAs generated from different genomic regions and PIWIL3 19-nucleotide piRNAs were designated based on their location on the genome (piRNA cluster) and length Histograms left of the vertical dashed line show different families of repeat elements; histograms right of the vertical dashed line show the gene-related regions Data shown a-b were average value of biological replicates at each time point: n = 3 (PF) 4 (44 h.p.e.a.) or 4 (52 h.p.e.a) for Piwil1 mutants (a) Volcano plots show the differentially expressed TEs between two adjacent stages during oogenesis and embryo development in wild-type golden hamsters The highly significant up- or down-regulated TEs (≥ 2 folds; Welch two sample t-test p-value < 0.05) are indicated in red or blue (b) Heat map of up-regulated TE expression levels in oocytes and embryos derived from WT and Piwil1-deficient females The highly significant up-regulated TEs (≥ 2 folds) in Piwil1-deficient oocytes and maternal Piwil1-deficient embryos compared to wild-type oocytes and embryos at PF (a-b) The bar graph shows the expression level (RPKM) of piRNAs mapped to the sense (gray) or anti-sense (red) sequences of each TE family in GV oocytes (a) and 1-cell embryos at 9 h.p.e.a Different TE family-derived piRNAs are designated based on their location on the genome (piRNA cluster) and length The significantly up-regulated TE families are listed with log2 fold change in expression level between Piwil1 mutant versus wild-type GV oocytes or maternal Piwil1-deficient and WT zygotes The fold change level is indicated by differences in orange hue in the heat map Data shown in a are average value from 3 biological replicates for WT and 2 biological replicates for mutants Data shown in b are average values from 3 biological replicates for WT or Piwil1 mutants (c) Examples of PIWIL1-piRNA and PIWIL3-piRNA distribution in ERV families that were up-regulated in Piwil1m1/m1 MII oocytes The expression levels (RPKM) represent the normalized number of all mapped piRNAs with the same 5’ end at each position The dotted boxes indicate the Ping-Pong signal between the adjacent sense and antisense piRNAs (d) The 5’-5’ overlap between piRNA sense- and antisense strands were analyzed for the presence of Ping-Pong signatures These PIWIL1-piRNAs (23-nucleotide and 29-nucleotide piRNAs) and PIWIL3-piRNAs (19-nucleotide piRNAs) in panel c and d are designated based on their location on the genome (piRNA cluster) and length Data shown in c-d are average value from 3 biological replicates for WT and 2 biological replicates for Piwil1 mutants Top-ranking GO terms (biological processes) of differential expressed genes in maternal Piwil1-deficient embryos at 9 (a-b) Heat maps of gene expressions in oocytes and embryos derived from WT and Piwil1-deficient females Only the genes which were up-regulated in wild-type embryos from 9 h.p.e.a The up-regulation of gene expression in early embryogenesis of wild-type embryos was barely detectable in maternal Piwil1-deficient embryos (c) Degradation of some maternal mRNAs is inhibited during the development of the Piwil1-deficient MII oocytes to maternal Piwil1-deficient 1-cell embryos at 9 h.p.e.a and the maternal Piwil1-deficient 1-cell embryos at 9 h.p.e.a X- and Y-axes represent the log2 fold change of gene expression between adjacent developmental stages in WT (X-axis) or (maternal) Piwil1-deficient oocytes (embryos) (Y-axis) Supplementary Table 1: list of oligonucleotide sequences Supplementary Table 2: sequencing data summary of small- and long-RNAs Supplementary Table 3: the top piRNA clusters that generated >90% of the unique mapped piRNAs in each oocyte and embryo at different developmental stages are shown The expression level of unique mapped piRNAs generated by these top clusters in different samples were calculated Supplementary Table 4: predicted miRNAs information and expression levels (RPM) in oocytes and early embryos derived from WT and Piwil1-deficient golden hamsters Supplementary Table 5: consensus-TE expression levels in oocytes and early embryos derived from WT and Piwil1-deficient golden hamsters Supplementary Table 6: gene expression levels in oocytes and early embryos derived from wild-type and Piwil1-deficient golden hamsters Download citation DOI: https://doi.org/10.1038/s41556-021-00750-6 Journal of Assisted Reproduction and Genetics (2024) Metrics details In the male germ cells of placental mammals 26–30-nt-long PIWI-interacting RNAs (piRNAs) emerge when spermatocytes enter the pachytene phase of meiosis pachytene piRNAs derive from ~100 discrete autosomal loci that produce canonical RNA polymerase II transcripts These piRNA clusters bear 5′ caps and 3′ poly(A) tails and often contain introns that are removed before nuclear export and processing into piRNAs What marks pachytene piRNA clusters to produce piRNAs and what confines their expression to the germline We report that an unusually long first exon (≥ 10 kb) or a long unspliced transcript correlates with germline-specific transcription and piRNA production and epigenome datasets across multiple species reveals that a long first exon is an evolutionarily conserved feature of pachytene piRNA clusters often containing a low or intermediate level of CG dinucleotides correlates with germline expression and somatic silencing of pachytene piRNA clusters Pachytene piRNA precursor transcripts bind THOC1 and THOC2 THO complex subunits known to promote transcriptional elongation and mRNA nuclear export these features may explain why the major sources of pachytene piRNA clusters specifically generate these unique small RNAs in the male germline of placental mammals 46 are divergently transcribed from a central promoter including 18 pairs that produce piRNA precursors from both arms and 3 that generate a lncRNA from the other arm What genic or epigenetic features direct the transcripts of pachytene piRNA clusters into the piRNA pathway what determines which arm of these divergently transcribed loci makes piRNAs and what prevents the expression of pachytene piRNAs outside the testis remain unknown comparison of testis germ cells with a variety of somatic tissues and cell types suggests that a highly methylated promoter and pre-pachytene) and sorted by tissue-specificity score (“Methods”) within each group Each piRNA gene is further annotated by its function as an mRNA (black/grey) length of first exon or length of intronless gene (≥10 or <10 kb; in red and pink) and transcript expression level (RPKM) in the testis piRNA abundance and transcript expression levels are log2 and log10 transformed with a pseudo-count of 1 and share the same color scale b Each panel indicates the levels of RNA pol II binding or a histone mark; tissues are color coded Pachytene piRNA clusters exhibit high levels of activating histone marks (H3K4me3 low levels of the repressive histone mark H3K27me3 and high levels of RNA pol II binding specifically in adult testis ChIP-seq signals are shown in a ±2 kb window around the TSS in 10 bp bins H3K4me2 ChIP-seq data were publicly available only for testis We performed two-sided Wilcoxon signed-rank tests to compare epigenetic signals at pachytene piRNA clusters in the testis versus somatic tissues 2.3 × 10−16 < p < 6.9 × 10−16; H3K4me2 1.0 × 10−11 < p < 3.0 × 10−10; H3K4me3 2.3 × 10−11 < p < 7.9 × 10−6; H3K36me3 Sixty nine of the 100 annotated pachytene piRNA clusters but only one of the 17 piRNA pathway genes were exclusively expressed in the testis (<0.1 RPKM in all somatic tissues examined; Chi-square test p value = 3.9 × 10−5) most pachytene piRNA clusters are expressed exclusively or specifically in the mouse testis and generally show greater tissue specificity than genes encoding piRNA pathway proteins a Bar plot reports the Spearman correlation coefficient between piRNA abundance and first exon length and promoter O/E CG chromatin accessibility measured by ATAC-seq and RNA pol II binding at the promoters (TSS ± 2 kb) of pachytene piRNA clusters in pachytene spermatocytes Significant correlations (Benjamin-adjusted p values < 0.05; n = 100) are marked in black while nonsignificant correlations are marked in grey b Histograms of first exon length of intronless pachytene piRNA clusters and the first exons of intron-containing pachytene piRNA clusters Eight groups are shown: pachytene piRNA clusters Boxplots and meta-gene plots show histone modifications and RNA pol II levels at 100 pachytene piRNA clusters and 164 testis-specific lncRNAs in pachytene spermatocytes The x-axis for the box and the y-axis for the meta-gene plots report log2 ChIP signal or ATAC-seq read coverage relative to input Asterisks indicate statistical significance (two-sided Wilcoxon rank-sum test p value < 0.001) for pairwise comparisons between 100 pachytene piRNA clusters and each of the other gene types Red (blue) indicates that the epigenetic level is significantly higher (lower) in pachytene piRNA clusters boxes represent the first and third quartiles none of these long-first-exon isoforms is expressed in the testis No hybrid and only three pre-pachytene piRNA clusters have long-first-exon isoforms (i.e. supported by RNA-seq reads; Chi-square test between pachytene and other piRNA clusters p value = 6.8 × 10−14) for two of these pre-pachytene piRNA clusters the long-first-exon isoforms produced more piRNAs than the short-first-exon isoforms (piRNA density was 15.8 vs 0.1 RPKM for pi-Phf20.1 in postnatal day 10.5 testes) and it makes abundant piRNAs (242.6 RPKM in postnatal day 10.5 testes) our data indicate that a long first exon (or a long unspliced transcript) is a specific feature of pachytene piRNA clusters which may distinguish them from protein-coding and lncRNA genes right panels; we note that the apparent enrichment of signals upstream of pachytene piRNA cluster is due to the large number of bidirectionally transcribed piRNA clusters and this apparent enrichment is not observed for the RIP signal described below that is specific to the transcribed strand) other active genes showed a narrow peak of histone acylation at the transcriptional start site These data suggest that such broad domains of acylated histones enable testis-specific transcription of pachytene piRNA clusters despite their high levels of promoter DNA methylation a Heatmap reports the enrichment of the pan-lysine acetylation ChIP signal relative to input in pachytene spermatocytes for 100 pachytene piRNA clusters in the −10 kb to +80 kb window flanking the TSS b A meta-gene plot shows average lysine acetylation signal in pachytene spermatocytes for long-first-exon (or long intronless) pachytene piRNA clusters and other groups of genes in the −10 kb to +80 kb window flanking the TSS c UCSC genome browser view of 4-qC5-17839.1 intronless pachytene piRNA-producing gene with a low-CG promoter All signal tracks are from pachytene spermatocytes except for BTBD18 d UCSC genome browser view of 6-qF3-8009.1 piRNA-producing gene with a low-CG promoter Vertical dashed line marks the end of the first exon e Scatter plot compares the change in transcript and piRNA abundance for Btbd18 mutant compared to Btbd18 heterozygous testis Green: BTBD18-dependent pachytene piRNA clusters; yellow: BTBD18-independent pachytene piRNA clusters The transcripts of pachytene piRNA clusters are further classified as long and intronless (n = 39); short and intronless (n = 13); spliced with long first exons (n = 5); spliced with short first exons (n = 40); intronless transcripts isoforms are long and spliced transcript isoforms have long first exons (n = 2); intronless transcript isoforms are long and spliced transcript isoforms have short first exons (n = 1) This locus failed to qualify as BTBD18-dependent because in Btbd18Null homozygous testis exonic reads increased 12% while intronic reads decreased 33% Yet piRNAs mapping to the locus decrease 16-fold in Btbd18Null mutant testis 85% of piRNAs from the locus map to introns suggesting that the piRNAs derive from the long intronic piRNAs decreased 17-fold in Btbd18Null testis Just 4.6% of pi-1700016M24Rik.1 piRNAs mapped to sequences unique to the two short-first-exon transcript isoforms and even these piRNAs decreased only 4.3-fold in Btbd18Null testes these data suggest that BTBD18 is essential for transcription of long unspliced and long-first-exon pachytene piRNA clusters 10-qB4-6488.1 makes three piRNA-producing transcript isoforms: a long unspliced RNA and two long-first-exon RNAs Ddx50 has a short first exon and makes no piRNAs their distinct transcriptional regulation and the contrasting fates of their transcripts likely reflects their different first-exon lengths and not BTBD18 binding per se THO complex components specifically bind piRNA precursor transcripts and loss of THO complex subunits disrupts transposon silencing Our data suggest that in adult mouse testis the THO complex also binds pachytene piRNA precursor transcripts a RIP-seq was used to measure the abundance of RNA bound to THOC1 (n = 5) or THOC2 (n = 3) compared to IgG control (n = 4) in adult testis Each dot represents the mean abundance normalized for transcript length (RPKM) b Heatmaps report the enrichment of THOC1 or THOC2 binding relative to IgG control for pachytene piRNA clusters in adult testis Each row corresponds to pachytene piRNA gene in first-exon (spliced) or transcript (unspliced) length order the black bar denotes the first exon–intron boundary; the blue bar indicates the 3′ end of the transcript a piRNA and transcript abundance for pachytene piRNA clusters in pachytene spermatocytes Promoter O/E CG is indicate by color: Blue Two-sided Wilcoxon rank-sum test was used to determine p value; n.s and RNA pol II for pachytene piRNA clusters in pachytene spermatocytes Asterisks indicate two-sided Wilcoxon rank-sum test p values < 0.05 between 49 low-CG and 33 intermediate- or 18 high-CG promoters Red (blue) indicates that the epigenetic level is significantly higher (lower) in low-CG pachytene piRNA clusters Asterisks indicate two-sided Wilcoxon rank-sum test p values < 0.05 between 49 pachytene piRNA clusters and 397 testis-specific mRNAs and all 2050 lncRNAs with low-CG promoters DNA methylation levels for pachytene piRNA clusters and other groups of genes DNA methylation levels of pachytene piRNA clusters grouped as in a Vertical line indicates the 50% DNA methylation level e DNA methylation levels of pachytene piRNA clusters between forebrain and pachytene spermatocytes Colors indicate promoter CG level; genes with forebrain expression level higher than 0.1 RPKM are denoted as triangles Only the 23 pachytene piRNA clusters in the bottom-left quadrant defined in e are included and further classified by their expression levels in the forebrain: RPKM ≥ 0.1 (n = 12) or RPKM < 0.1 (n = 11) g First exon length of the same 23 pachytene piRNA clusters as in f We conclude that despite their hypermethylation pachytene piRNA clusters with low-CG promoters can be expressed to high levels in the testis because of compensating 5hmC DNA modification What prevents expression of pachytene piRNA clusters in somatic cells which generally lack the machinery to process precursor transcripts into piRNAs We found that two genic features associated with pachytene piRNA clusters—low-CG promoters and long first exons—correlated with their somatic silencing we identified two epigenomic signals—DNA methylation and the repressive histone mark H3K27me3—likely underpin this silencing the median methylation level for the testis-specific protein-coding genes with intermediate-CG promoters was 17% in pachytene spermatocytes vs 71–81% in the soma (p values < 2.2 × 10−16) These results suggest that DNA methylation may be an essential mechanism for silencing these intermediate-CG genes in the soma DNA methylation likely silences a majority of pachytene piRNA clusters in the soma especially those with low- or intermediate-CG promoters high H3K27me3 may be another mechanism for silencing pachytene piRNA clusters in the soma especially those with high-CG and intermediate-CG promoters that are not DNA methylated in the soma likely repress these genes in the soma: among 1171 testis-specific protein-coding genes 452 and 157 were expressed in the forebrain at >0.1 and >1 RPKM testis-specific protein-coding genes and pachytene piRNA-producing genes may be silenced in the soma by similar mechanisms unspliced and long-first-exon mouse pachytene piRNA clusters are more likely to be syntenic in other species than those with short first exons We did not detect significant conservation of low-CG promoters between the species examined The silhouettes have not been altered in any way Two-sided Wilcoxon rank-sum tests were used to calculate p values are expressed almost exclusively in the male germline 47 are long and unspliced or have long first exons a lack of splicing or a low O/E CG promoter is anticipated to prevent somatic expression genes with these features are silenced in the soma Yet pachytene piRNA clusters with these same features are highly expressed in the germline and produce most of the pachytene piRNAs in the adult mouse and other eutherian mammals We suggest that the high 5-hydroxymethylation of the low-CG promoters of pachytene piRNA clusters allows them to maintain high lysine acylation and active transcription in the male germline Three additional features likely act to repress pachytene piRNA clusters in the soma both low-CG and intermediate-CG promoters show high levels of DNA methylation in the soma the subset of pachytene piRNA clusters with high- or immediate-CG promoters show a high density of the repressive histone mark H3K27me3 pachytene piRNA clusters tend to have long first exons or long unspliced transcripts Pachytene piRNA clusters with intermediate-CG promoters may particularly rely on DNA methylation and H3K27me3 for their somatic silencing: just one-third (11/33) of intermediate-CG pachytene piRNA clusters are unspliced with long transcripts or spliced with long first exons; nearly two-thirds (30/49) of low-CG pachytene piRNA clusters display these features Clearly a major question for future study is how BTBD18 or other proteins identify pachytene piRNA precursor transcripts Such a mechanism would provide an initial defense against these pathogens until the establishment of an antisense piRNA response that can directly target the spliced protein-coding transcripts of retroviruses and transposons All mice were maintained and used according to the guidelines of the Institutional Animal Care and Use Committee of the University of Massachusetts Medical School C57BL/6 J mice (RRID: IMSR_JAX:000664) were used as wild-type controls ChIP-seq libraries were sequenced as 79-nt paired-end reads using NextSeq500 (Illumina) Gene annotations and ribosomal RNA (rRNA) sequences were from Ensembl (Release 82) To determine whether Ensembl Release 100 might yield more lncRNAs with long first exons we reanalyzed the data using the latest Ensembl gene annotation Ensembl Release 100 contains 924 more lncRNAs than Release 82 (5,586 vs but just one of the 924 lncRNAs (AC160336.1) has long first exon RNA-seq data indicate that this lncRNA is not expressed in adult mouse testis our conclusions remain unchanged using Ensembl Release 100 For each protein-coding or lncRNA gene (Ensembl Release 82) and piRNA-producing locus we then calculated and normalized the number of uniquely mapped reads as Reads Per Million mapped reads (RPM) and the number of uniquely mapped reads normalized by the total transcript length of each gene as RPKM The H3K27ac ChIP-seq data in primary spermatocytes and round spermatids (accession GSE107398) were not provided with input so we calculated read density normalized by sequencing depth we normalized read density by sequencing depth for all ATAC-seq data To calculate the level of each epigenetic mark for the various groups of genes (piRNA clusters we averaged the enrichment or read density at the promoter (TSS ± 2 kb) or gene body (TSS + 2 kb till the 3′-end) as indicated We used a control gene set to normalize ChIP-seq signal levels across different datasets We first identified 1230 genes with similar expression levels (0.66 < change < 1.5 and maximal expression >0.1 RPKM) in three cell types (spermatogonia We then used the median levels of each histone mark for these 1230 control genes in three cell types to normalize the levels of all histone marks and RNA pol II binding in the corresponding cell types We used the same strategy to normalize the H3K27me3 levels in testis and somatic tissues We identified a set of control genes whose expression levels differed by <1.5-fold between testis and each somatic tissue: 1132 and 817 control genes for comparing testis with forebrain This normalization method reduced the bias caused by the difference in antibody specificity across different batches or different tissue or cell types Only C in CG positions was used for computing methylation levels Methylation level was computed as mCG/CG averaged over all detected CG sites in a region and we calculated the methylation level for each region in each replicate and then averaged the levels across the four replicates piRNA abundance was reported normalized to the total number of reads mapping uniquely to one genomic location As most of the mapped reads are uniquely mapped to the mouse genome (90.6% in adult testis normalizing to uniquely mapping or uniquely mapping plus multiply mapping reads result in similar piRNA abundance and the same conclusions 5hMe-DIP (5hmC) data were mapped to the mouse genome (mm10) using Bowtie2 with the parameter–very-sensitive The number of aligned reads were processed into enrichment relative to input in the bedGraph format The 5hmC level for each gene was calculated by averaging the enrichment in the TSS ± 500 bp window where the expected number = [(Fraction of C + Fraction of G)/2]2 Promoters were classified into three groups: low-CG Testis-specific genes corresponded to those genes with expression levels in testis >4-fold higher than the maximal expression in any somatic tissues that we examined We also calculated a tissue-specific score (ts-score) where n denotes the total number of tissues examined, Expi denotes the expression levels in a particular tissue, and Expts denotes the expression level in testis (we set the ts-score to 0 if any Expi > Expts). We used ts-score to rank piRNAs in Fig. 1a (2) We removed the intron in 5-qF-14224.1 which is 59 nt long with a noncanonical splice site motif CC-CT and supported by few reads (3) We changed the TSS of 17-qC-59.1 from chr17: 50,237,659 to chr17:50,239,160 based on A-MYB ChIP-seq (4) We changed the main TSS of 4-qB3-639.1 from chr4: 62,230,936 to chr4: 62,228,511 based on A-MYB ChIP-seq (5) We added a long and intronless isoform (chr2: 92,529,805–92,540,950) for Gm13817 which is divergently transcribed from 2-qE1-35981.1 Gm13817 is an unannotated pachytene piRNA-producing gene and produces >100 piRNAs per million unique mapped reads (6) We removed the long-first-exon isoform of pi-Zfp652.1 (a hybrid piRNA clusters) which is not supported by RNA-seq or small RNA-seq reads (7) We changed the main TSS of 10-qC-875.1 from chr10: 86,617,011 to chr10: 86,591,510 based on A-MYB and H3K4me3 ChIP-seq We also separated 10-qC-875.1 from a spliced lncRNA Gm48485 which is primarily expressed in round spermatid We defined BTBD18-dependent pachytene piRNA clusters as those whose expression level was ≥2-fold lower in pachytene spermatocytes of Btbd18 mutant mice than Btbd18 heterozygous mice and whose piRNA abundance was also ≥2-fold lower in the 18-day postpartum testis tissue of Btbd18 mutant mice than that of Btbd18 heterozygous mice We considered 24–32 nt small RNA reads that could map to each mammalian genome piRNA abundance was then computed for 20 kb sliding windows (with a 1 kb step) in the genome and windows with >100 piRNAs per million uniquely mapped piRNAs were deemed potential piRNA clusters To remove false positives due to unannotated miRNA which mostly produce reads with the same sequences we also filtered out the 20-kb genomic windows with fewer than 200 distinct reads We then calculated the first-nucleotide composition for each 20-kb window and discarded those windows with fewer than 50% of its piRNAs having a 1 U or 10 A (with the 10 A possibly resulting from ping-pong amplification) The remaining contiguous 20-kb windows were deemed putative piRNA clusters To obtain the precise promoter position of each piRNA gene we performed trimming from the 5′ and 3′ ends by examining adjacent 100-bp windows The first and last two nearby windows (closer than 1000 bp) each with more than two piRNAs per million uniquely mapped piRNAs were considered as the 5′ and 3′ ends of the piRNA gene We used the RNA-seq reads after rRNA removal for annotating introns of piRNA clusters we performed manual curation for each piRNA gene using piRNA profile and exon–exon junctions detected using RNA-seq reads The transcriptional direction of a piRNA gene was indicated by the direction of the main long RNA transcript The final piRNA clusters were classified according to their genomic location and those with >50% base pairs overlapping protein-coding genes were defined as genic and the rest as intergenic We treated intergenic piRNA clusters as pachytene piRNA clusters in rhesus We defined 17 piRNA pathway genes in rhesus 593 A-MYB-regulated protein-coding genes using one-to-one orthology between mouse and rhesus The coordinates of the syntenic region in the other species which overlapped a mouse piRNA-producing gene on the same genomic strand were adjusted to be the piRNA gene coordinates we annotated in that species To be inclusive of piRNAs that map to the boundaries of the syntenic regions in that other species that did not overlap piRNA clusters on the same genomic strand we extended these syntenic regions by 10 kb in both ends and then calculated the piRNA abundance in the regions using small RNA-seq data in that other species eutherian-conserved pachytene piRNA clusters were defined as those for which three or more eutherian mammals (among human or cow) could produce similar amounts of piRNAs (change < 5) to mouse in the syntenic regions The remaining pachytene piRNA clusters were defined as murine-conserved when the syntenic region in rat produced similar amount of piRNAs to mouse In order to quantify how much the signals of histone modification or factor binding extend into the gene body we first cut the genome into 200-bp bins and then computed the average enrichment of each type of signal over input or average read count per one million reads over these 200-bp bins Bins far downstream pachytene piRNA clusters (TSS + 80 kb to TSS + 100 kb) were considered background bins and bins with signal higher than 95% quantile of the background bins were regarded as signal-enriched bins Transposon copies reside in pachytene piRNA clusters can hinder the signal identification and lead to signal gaps we identified the furthest continually enriched bins for each pachytene piRNA clusters allowing 3800-bp gaps (19 bins) The extension index was defined as the distance from the TSS to the furthest continually enriched bin The relative extension index was defined as the extension index divided by the relative first-exon length Further information on research design is available in the Nature Research Reporting Summary linked to this article The data supporting the findings of this study are available from the corresponding authors upon reasonable request piRNAs from pig testis provide evidence for a conserved role of the piwi pathway in post-transcriptional gene regulation in mammals A-MYB (MYBL1) transcription factor is a master regulator of male meiosis Chromatin and transcription transitions of mammalian adult germline stem cells and spermatogenesis BTBD18 regulates a subset of piRNA-generating loci through transcription elongation in mice interacts directly with Mex67p and is required for mRNA export an evolutionary conserved family of hnRNP-like proteins interacts with TAP/Mex67p and participates in mRNA nuclear export Genes with internal repeats require the THO complex for transcription Splicing-independent loading of TREX on nascent RNA is required for efficient expression of dual-strand piRNA clusters in The THO complex is required for nucleolar integrity in Drosophila spermatocytes Discovery and characterization of piRNAs in the human fetal ovary Evolutionary dynamics of gene and isoform regulation in Mammalian tissues a cardiac-specific isoform of the RNA helicase Mov10l1 First exon length controls active chromatin signatures and transcription silencing potential and evolutionary impact of promoter DNA methylation in the human genome The role of DNA methylation in mammalian epigenetics Multi-omic analysis of gametogenesis reveals a novel signature at the promoters and distal enhancers of active genes Histone acetylation increases chromatin accessibility Dynamic competing histone H4 K5K8 acetylation and butyrylation are hallmarks of highly active gene promoters Intracellular crotonyl-CoA stimulates transcription through p300-catalyzed histone crotonylation Metabolic regulation of gene expression through histone acylations Identification of 67 histone marks and histone lysine crotonylation as a new type of histone modification Identification of mRNAs that are spliced but not exported to the cytoplasm in the absence of THOC5 in mouse embryo fibroblasts Transcriptional regulation of immediate-early gene response by THOC5 contributes to the M-CSF-induced macrophage differentiation THOC5 controls 3′end-processing of immediate early genes via interaction with polyadenylation specific factor 100 (CPSF100) Correlated 5-hydroxymethylcytosine (5hmC) and gene expression profiles underpin gene and organ-specific epigenetic regulation in adult mouse brain and liver Uncovering the role of 5-hydroxymethylcytosine in the epigenome Role of Tet proteins in enhancer activity and telomere elongation Activation of the protein deacetylase SIRT6 by long-chain fatty acids and widespread deacylation by mammalian sirtuins DNA methylation and histone deacetylation in the control of gene expression: basic biochemistry to human development and disease DNA methylation: roles in mammalian development Promoter directionality is controlled by U1 snRNP and polyadenylation signals A 5’ splice site enhances the recruitment of basal transcription initiation factors in vivo Recruitment of the human TREX complex to mRNA during splicing Promoter proximal splice sites enhance transcription The Drosophila HP1 homolog rhino is required for transposon silencing and piRNA production by dual-strand clusters Cutoff suppresses RNA polymerase II termination to ensure expression of piRNA precursors The cutoff protein regulates piRNA cluster expression and piRNA production in the Drosophila germline The genome of the Hi5 germ cell line from Trichoplusia ni an agricultural pest and novel model for small RNA biology Stalled spliceosomes are a signal for RNAi-mediated genome defense Strand-specific libraries for high throughput RNA sequencing (RNA-Seq) prepared without poly(A) selection Cellular source and mechanisms of high transcriptome complexity 1033 in the mammalian testis RNF17 blocks promiscuous activity of PIWI proteins in mouse testes RNF8 and SCML2 cooperate to regulate ubiquitination and H3K27 acetylation for escape gene activation on the sex chromosomes SFMBT1 functions with LSD1 to regulate expression of canonical histone genes and chromatin-related factors Dynamic reorganization of open chromatin underlies diverse transcriptomes during spermatogenesis A comparative encyclopedia of DNA elements in the mouse genome The sequence alignment/map format and SAMtools HTSeq—a Python framework to work with high-throughput sequencing data Bismark: a flexible aligner and methylation caller for Bisulfite-Seq applications Aligning short sequencing reads with Bowtie Sequence features that drive human promoter function and tissue specificity Download references and Theurkauf laboratories for their critical comments This work was supported in part by Chinese National Natural Science Foundation grants (31571362 and 31871296) to Z.W and National Institutes of Health grant P01 HD078253 to W.E.T Present address: Oncology Drug Discovery Unit These authors contributed equally: Tianxiong Yu The School of Life Sciences and Technology Program in Bioinformatics and Integrative Biology University of Massachusetts Medical School performed the Rhesus H3K27ac ChIP-seq experiment Peer review information Nature Communications thanks Benjamin Czech Download citation DOI: https://doi.org/10.1038/s41467-020-20345-3 Metrics details The PIWI-interacting RNA (piRNA) pathway prevents endogenous genomic parasites from damaging the genetic material of animal gonadal cells are thought to define each species’ piRNA repertoire and therefore its capacity to recognize and silence specific transposon families The unistrand cluster flamenco (flam) is essential in the somatic compartment of the Drosophila ovary to restrict Gypsy-family transposons from infecting the neighbouring germ cells Disruption of flam results in transposon de-repression and sterility yet it remains unknown whether this silencing mechanism is present more widely we systematically characterise 119 Drosophila species and identify five additional flam-like clusters separated by up to 45 million years of evolution Small RNA-sequencing validated these as bona-fide unistrand piRNA clusters expressed in somatic cells of the ovary where they selectively target transposons of the Gypsy family our study provides compelling evidence of a widely conserved transposon silencing mechanism that co-evolved with virus-like Gypsy-family transposons Retrotransposons replicate via RNA intermediates and are further subdivided into non-LTR elements including short interspersed nucleotide elements (SINEs) and long interspersed nucleotide elements (LINEs) which share similarity to endogenous retroviruses (ERVs) LTR transposons and ERVs both encode gag and pol open reading frames (ORFs) with ERVs and specialised retroelements (also known as errantiviruses) such as gypsy and ZAM also possessing an envelope (env) gene The env gene allows virus-like particle formation and cell-to-cell transposition in addition to the “copy-and-paste” mobilisation mechanism intrinsic to all LTR TEs their function and content varies with not all species showing enrichment of transposon remnants a Cartoon of a developing Drosophila egg chamber with an active transposon invasion from the soma (top) Somatic follicle cells lining the egg chamber are shown in green and germ cells are shown in beige Transposon transcripts (purple) originating from somatic cells enter the germ cells (bottom Once reverse transcribed and transported into the nucleus Transposon copy number increases over multiple generations until a transposon is inserted in antisense orientation into flam (step 2 b Cartoon of a Drosophila egg chamber in which transposon invasion is halted (top) A piRNA precursor transcript is produced from the flam locus in somatic cells (bottom The precursor is processed into piRNAs and loaded into Piwi proteins (step 4 The Piwi-piRNA complex enters the nucleus where it recognises transposon transcripts by sequence complementarity and instruments their co-transcriptional repression (step 5 here we systematically searched for flam-like unistrand piRNA clusters within the Drosophila subgenera Sophophora and Drosophila Our results highlight their unique characteristic architecture and specificity in regulating somatically active LTR elements particularly those carrying an envelope protein that facilitates transfer to germ cells our study suggests a conserved and essential role of somatically expressed unistrand piRNA clusters in the suppression of ERVs across the entire Drosophila genus a Cartoon showing synteny analysis pipeline melanogaster (dm6 genome) flam region with transposon annotation by RepeatMasker (RM) displaying some neighbouring genes used for synteny analysis The pie chart to the right indicates LTR content per strand in the cluster region c MCScan plot showing gene and flam synteny between D biarmipes (GCF_018148935 genome) flam region e Phylogenetic tree (left) representation of the melanogaster indicating the respective size of their flam-syntenic loci in kb (right) Source data are available in the source data file In conclusion, the flam locus likely appeared between 13.3 and 15.1 million years ago (MYA), following the emergence of the elegans/rhopaloa subgroups, and was detected in 12 species (Fig. 2e) despite largely conserved gene synteny in the region and the widespread presence of Gypsy-family elements in Drosophilids prompted the question whether analogous flam-like unistrand piRNA clusters exist elsewhere in the genomes of these species a Genome-wide detection of flam-like loci in D ficusphila using a sliding window approach minus strand) and total repeat content (grey) is shown across the whole genome (100 kb bins) and the de novo identified flamlike1 region ficusphila (GCF_018152265 genome) flam-syntenic region (black bar) with transposon annotations by RepeatMasker (RM) and EDTA Uniquely mapping piRNA (cpm) and total RNA levels (ln(cpm+1)) are presented (green/orange c Relative piRNA size distribution of piRNAs mapping sense (light brown) and antisense (dark brown) to D d Ping-pong signature for piRNA pairs mapping onto the flam-syntenic region f Relative piRNA size distribution of sense (light brown) and antisense (dark brown) piRNAs mapping to D g Phasing signature (3’ end to 5’ end distance) for piRNAs mapping onto flamlike1 h Zoom-in on genic region indicating presence of a piRNA cluster in the flam-syntenic region in D i Macrosynteny plot indicating gene synteny between D ficusphila highlighting flam (red) and flamlike1 (blue) j Zoom-in on genic region indicating the absence of a piRNA cluster in flamlike1-syntenic region in D As D. ficusphila appears to possess a dual-strand cluster in place of the flam locus, it either lacks somatically expressed LTR transposons or controls these TEs by other means. The presence of Gypsy family elements in all investigated genomes strongly indicates that D. ficusphila has somatically expressed transposons (Fig. S3c) We therefore set out to identify non-syntenic unistrand piRNA clusters in D ficusphila that resemble flam in terms of its size Gypsy-family TE content and strong enrichment for transposon insertions to be oriented on one genomic strand oshimai flamlike2 region with transposon annotation by EDTA (blue e Phylogenetic tree representation highlighting flamlike1 flamlike2 and flamlike4 presence and flamlike3 and flamlike5 conservation across Drosophila species pseudoobscura flamlike5 syntenic dual-strand cluster consistent with expression in somatic cells that lack the machinery needed to express and export transcripts from dual-strand clusters since sRNA-seq from whole ovaries captures a mixture of both somatic and germline piRNAs it remained uncertain if these unistrand piRNA clusters actually operate in the soma g Phylogenetic tree summarising all studied somatic piRNA clusters across all analysed species (n = 119) Cluster size represents the mean across all assemblies Species names in bold have sRNA-seq data to validate their expression Interestingly, we found that D. yakuba and D. erecta deviated from this pattern, displaying somatic expression at the 5’ end and germline expression towards the 3’ end of the flam-syntenic region (Fig. S14b, c) this indicates that all identified flam-like loci produce antisense piRNAs capable of targeting transposons and that they are expressed primarily in the somatic follicle cells of the ovary this indicates a strong selective pressure to maintain production of transposon-targeting piRNAs in somatic follicle cells a Boxplot showing fraction of interspersed repeat content for the indicated repeat classes Each data point represents one species (n = 119) Species with multiple genome assemblies are represented by their mean b Boxplot showing the number of subfamilies detected per LTR family with either gag + pol (left) or gag + pol + env (right) ORFs Each data point corresponds to one species (n = 119) sense) to total transposon content across all annotated flam-like clusters Gypsy elements are shown in red (antisense) or blue (sense) and other LTR elements are shown in grey Clusters are grouped by synteny as indicated to the right Species and genome assembly (alphabetically sorted) are indicated to the left but showing LTR content across flam and major dual-strand clusters in D Cluster strand was defined according to total transposon content (light grey) e Boxplot showing strand bias defined as sense strand minus antisense strand contribution to total transposon content for transposons classified as LTR Strand bias is shown across all annotated flam-like clusters (left n = 48) or major dual-strand clusters in D melanogaster and proTRAC de novo predicted clusters (right The means were compared using a two-sided Student’s t Test f Boxplot displaying Gypsy versus other LTR coverage against the genomic average across different unistrand clusters Each point corresponds to one cluster in one genome assembly g Scatterplot showing Gypsy enrichment against env enrichment in unistrand clusters from the indicated species (see “Cluster content analyses” in the Methods for details) Only high-quality LTR transposons are included in the analysis (both gag and pol and at least one good genomic hit) suggesting that similarities in piRNA populations between species is indicative of shared transposon burden To further our understanding of how transposons are regulated by flam-like clusters we characterised the individual transposons that are controlled by each cluster These species were selected based on the availability of both whole ovary and soma-enriched sRNA-seq and RNA-seq As controls we used the dual-strand 42AB in D Genomic origin of piRNAs that are antisense to transposons in D Barplots (right) display the number of uniquely mappable piRNAs against each TE in soma-enriched (a The TEs are arranged in decreasing order following their somatic-to-germline enrichment The number of piRNAs that map to the indicated clusters are coloured according to cluster strand (sense red) and piRNAs mapping elsewhere in the genome are shown in grey Labels are shown for subfamilies that are exclusively controlled (>90% of piRNAs) by a cluster and best hit to known TEs are indicated if available (80/80/80 rule) Boxplots (left) summarise the fraction of piRNAs antisense to individual transposons derived from each cluster Total cluster-derived piRNA abundance (white) are further subdivided into the sense (blue) and antisense (red) cluster strands The number of transposon subfamilies covered by each cluster (>10 reads) are indicated under each boxplot Pooled counts from 2–4 biological replicates Our identification of flam within the suzukii subgroup together with the absence of any piRNA cluster at the flam-syntenic region in and beyond the rhopaloa subgroup places the emergence of flam between 13.3 and 15.1 MYA long before the melanogaster subgroup separated from the remainder of the melanogaster group around 6.8 MYA We speculate that all Drosophila species use flam-like piRNA clusters in a somatic branch of the pathway that specifically evolved to repress ERVs While flam-like clusters have not been detected in all species we have consistently identified somatic unistrand piRNA clusters in all species where we performed soma-enriched sRNA-seq we did not observe any Drosophila species lacking the presence of env-containing Gypsy-family elements More unistrand piRNA clusters are therefore likely to be discovered as we gain access to more sequencing data and improved genome assemblies in the future Most flam-like unistrand clusters reported here follow the pattern of a single large locus We hypothesise that in addition to being the most efficient way of stopping TE invasion as disruption of flam-like piRNA clusters likely result in sterility The recurring presence of unistrand clusters across the Drosophila genus strongly argues for an essential role of these loci perhaps as a means to produce piRNAs in the soma without access to the germline piRNA expression and export machinery Conversion between unistrand (a) and dual-strand (b) piRNA clusters Transposons are present either in sense (blue) or antisense (red) orientation relative to the cluster transcript(s) Produced piRNAs mapping to the sense (green) or antisense (orange) strand are shown Once a promoter active in the soma is gained (a) selection will favour antisense insertions to ensure that transposon-complementary piRNAs are produced the cluster can only be transcribed in germ cells where the germline-specific branch of the piRNA pathway produces transcripts from both strands The strand bias is therefore lost over evolutionary time d Selective constraints acting on unistrand piRNA clusters Transposon insertions in sense orientation are tolerated towards the 3ʹ end (c) but are rarely observed at the 5ʹ end (d) This may indicate that the region closer to the promoter is under stronger selective pressure insertions in sense orientation may introduce polyadenylation signals causing early transcription termination abolishing the production of essential piRNAs targeting specific TEs (d) Although their transcriptional regulation may differ the recurrent emergence of flam-like loci across the Drosophila genus and the wider presence of unistrand clusters within in the animal kingdom hints at convergent evolution where this mechanism is best equipped to antagonise TE mobilisation our study opens the door to understanding the co-evolution between virus-like Gypsy-family transposons and the host defence mechanisms that silence them Further characterisation of these novel piRNA clusters as well as the piRNA pathway machinery in these species will allow us and others to test several long-standing hypotheses regarding piRNA cluster emergence and the licensing of their transcripts for piRNA biogenesis Assemblies for 36 species annotated by the NCBI Eukaryotic Genome Annotation Pipeline (listed on https://www.ncbi.nlm.nih.gov/genome/annotation_euk/all) from any species within the Drosophila genus were downloaded on three separate occasions (2020-10-17 Only the most recently annotated genome assembly is listed for each species and as a result 19 species were represented by a single assembly and 15 species were represented by two different assemblies Since the clusters themselves are not conserved, we used a synteny analysis (see “Synteny_clusters” at https://github.com/susbo/Drosophila_unistrand_clusters) melanogaster genome as a reference and extracted the 20 unique up- and downstream genes we extracted the coding sequence (protein-coding genes) or the full transcript (all others) we mapped these sequences onto the genome of interest using blat (v36x6 -minIdentity=25) and filtered the results to keep the best hit (pslCDnaFilter we constructed a candidate list with all genomic regions that had at least two gene hits within 1 Mb These candidate regions were then manually inspected for the presence of a transposon-rich area at the expected syntenic location Clusters running into assembly breakpoints were labelled as either 5’ or 3’ depending on whether they were located next to up- or downstream genes De novo identified clusters (proTRAC) were converted from GTF to GenePred RNA-seq and sRNA-seq tracks were displayed as standard bigWig tracks produced by deepTools All genome browser shots shown in this study were made by exporting the assembly hub display as a pdf followed by manual refinement to enhance readability Mappability tracks for the sRNA-seq were constructed by generating all possible 26-mers from each genome (bedtools -S -n 2 -M 1 --best --strata --nomaqround --chunkmbs 1024 --no-unal) and converting the alignments to bigWig using deepTools bamCoverage (v3.3.2 --binSize 1 --normalizeUsing None --scaleFactor 0.038461) This will construct a per-nucleotide signal between 0 and 1 representing the ability to uniquely map reads to each position An initial de-novo transposon library was built using EDTA (v1.9.3, --sensitive 1 --anno 1 --evaluate 1)37 with transposons of all types being detected some runs failed when one of the types were missing and we manually resumed EDTA at the next type for these genomes and Zind-d101g_BS02) that failed to run with EDTA v1.9.3 did run successfully with v1.9.6 Dwas-d101g) that had problems with v1.9.3 still had to be resumed with v1.9.6 due to not detecting any LTR transposons To search for flam-like clusters, we developed a search strategy based on the known enrichment of LTR transposons arranged in the same orientation in flam (see “De-novo_clusters” at https://github.com/susbo/Drosophila_unistrand_clusters) repeat annotations from the EDTA were used The repeats were separated based on strand retaining either only LTR transposons or all transposons with a predicted class (i.e. Overlapping annotations were combined (bedtools merge) and strand-specific transposon coverage was computed (bedtools coverage) across the genome using a 100 kb sliding window with a 5 kb step size (bedtools makewindows Each genome was manually inspected for regions enriched in LTR transposons and located outside of centromeric or telomeric regions This analysis was strongly contingent on assembly quality but we nevertheless identified 15 clusters that fulfilled the outlined criteria including several corresponding to flam across the D Six of the initial candidates were found outside of the D two species had publicly available sRNA-seq data and both produced large amounts of piRNAs from one strand only We therefore concluded that the approach was working Synteny analysis using these five clusters as starting points (described below) revealed that D All Drosophila species were maintained at room temperature. The origin of each species and their food requirements are indicated in Supplementary Data 8 Small RNAs were isolated from 16 species (2–3 replicates each) using the TraPR Small RNA Isolation Kit (Lexogen; catalogue nr 128.24) following the manufacturer’s instructions sRNA libraries were generated using the Small RNA-Seq Library Prep Kit (Lexogen; catalogue nr Both primers A3 and A5 as well as the primer RTP were used at 0.5x Library size distribution was analysed on an Agilent TapeStation system using a High Sensitivity D1000 ScreenTape (Agilent Technologies; catalogue nr 5067-5584) with High Sensitivity D1000 Reagents (Agilent Technologies; catalogue nr Libraries were pooled in equal molar ratio quantified with KAPA Library Quantification Kit for Illumina (Kapa Biosystems; catalogue nr KK4873) and were sequenced 50 nt paired-end on an Illumina NovaSeq 6000 or 75 nt single-end on an Illumina MiSeq sequencing platform generating 33 (±20) million reads per library 75-100 ovary pairs were dissected in ice-cold PBS Ovaries were dissociated for 18 min in 0.25% Trypsin (Sigma-Aldrich; catalogue nr Dissociated tissue was pushed through a 40 µm nylon mesh (Greiner Bio-One; catalogue nr 542040) washed with equal volume Schneider 2 medium (Thermo Fisher Scientific; catalogue nr Pelleted cells were directly used as input for sRNA isolation using the TraPR Small RNA Isolation Kit (Lexogen; catalogue nr KK4873) and were sequenced 50 nt paired-end on an Illumina NovaSeq 6000 sequencing platform generating 43 (±25) million reads per library The BAM files were converted to bigWig using bamCoverage from deepTools71 (v3.3.2 --binSize 1 --ignoreForNormalization chrM --normalizeUsing CPM --exactScaling --skipNonCoveredRegions --minFragmentLength 23 --maxFragmentLength 30) and additionally ‘--filterRNAstrand’ to separate the two strands ‘--scaleFactor’ to scale counts per million to reflect all mapped reads and optionally ‘--minMappingQuality 50’ when extracting uniquely mapped reads RNA-seq libraries were produced using NEBNext Ultra Directional Library Prep Kit for Illumina (New England BioLabs; catalogue nr following the manufacturer’s instructions for rRNA depleted RNA KK4873) and sequenced paired-end 50 nt on an Illumina NovaSeq 6000 generating 25 (±11) million reads per library The soma-enrichment RNA-seq libraries were generated for 5 species (2 replicates each) Enrichment for somatic cells was done identically as described for the soma-enriched sRNA-seq libraries except that 35-50 ovary pairs were used as starting material Pelleted cells were directly used as input for RNA isolation using the TRIzol (Thermo Fisher Scientific; catalogue nr RNA was treated with DNase (New England BioLabs; catalogue nr M0303) followed by ribosomal RNA depletion using RiboPOOL (siTOOLs Biotech; catalogue nr dp-K024-000007) following the manufacturer’s protocol KK4873) and sequenced paired-end 50 nt on an Illumina NovaSeq 6000 generating 42 (±7.1) million reads per library we calculated the ping-pong signature using a 5’ end overlap score for overlap x nt as where ni is the number of 5’ ends mapping at the plus strand position i and mi+x is the number of 5’ ends mapping at the minus strand position i + x The fraction of overlapping reads involved in ping-pong was calculated as s10/(s1 + …+s20) A z10 score was defined as (s10-mean(s1,…,s9,s11,…,s20))/stdev(s1,…,s9,s11,…,s20) we calculated a 3ʹ to 5ʹ end score for distance y as where ni is the number of 3’ ends mapping at position i and mi+y is the number of 5’ ends mapping at position i + y at the same strand The fraction of closely mapped reads with phasing signature was calculated as h1/(h1 + …+h20) A z1 score was calculated as (h1-mean(h2,…,h20))/stdev(h2,…,h20) Phasing calculations were done for the plus and minus strand separately Synteny analysis for flam-like clusters was performed using the same strategy as for flam except that Augustus gene predictions (v3.3.2 --species=fly --UTR=off --singlestrand=true) were used instead of FlyBase annotations The MAKER-masked genome from the EDTA output was used as genome input to Augustus Full transcript and coding sequences were extracted from the annotations Sequences with strong hits to the raw transposon libraries were excluded (blat -q=dna -t=dna -minIdentity=25; pslCDnaFilter -minCover=0.2 -globalNearBest=0) and gene predictions shorter than 200 nt were excluded The blat identity threshold was reduced to 20 the closest flanking genes displayed good conservation and we used these to search for syntenic regions using our UCSC Genome Browser session ATAC-seq was performed for nine species similar as described in77 6-12 ovary pairs of yeast-fed flies were dissected in ice-cold PBS and centrifuged for 5 min at 500 g at 4 °C Ovaries were lysed in Resuspension Buffer (RSB 3 mM MgCl2 in nuclease free water) containing 0.1% NP40 and 0.01% Digitonin and washed out with cold RSB containing 0.1% Tween-20 The transposition reaction was performed with 0.33x PBS 1x TD buffer and 100 nM transposase (Illumina Tagment DNA Enzyme and Buffer Small Kit; catalogue nr Samples were incubated for 1 h at 37 °C in a thermomixer mixing at 1000 rpm The transposed fragments were isolated using the DNA Clean and Concentrator-5 Kit (Zymo Research; catalogue nr Library was PCR amplified for 5 cycles using the NEBNext High-Fidelity MasterMix (New England BioLabs; catalogue nr M0541S) followed by qPCR amplification to determine the exact number of additional cycles required for optimal library amplification Amplified DNA library was purified using the DNA Clean and Concentrator-5 Kit (Zymo Research; catalogue nr D4014) and further cleaned using AMPure XP beads (Beckman Coulter; catalogue nr 100-600 bp fragments were selected on a 2% agarose gel cassette using the Blue Pippin (Sage Science; catalogue nr Library size distribution was analysed on an Agilent 2100 bioanalyzer using the High Sensitivity DNA Kit (Agilent Technologies; catalogue nr KK4873) and were sequenced 50 nt paired-end on an Illumina NovaSeq 6000 platform or Illumina MiSeq sequencing platform generating 4.5-25.6 million paired-end reads per library with ‘-repeatmasker’ set to RepeatMasker annotations generated by EDTA and with ‘-geneset’ set to NCBI gene predictions Clusters within 40 kb from each other were combined for the analyses melanogaster clusters were mapped onto each genome using BLAT (v36x6 filtered to retain only the best hit (pslCDnaFilter -minCover=0.2 -globalNearBest=0.0) and the predicted clusters were subsequently annotated by how many of these genes that were within 1 Mb Cluster predictions used in the soma-enrichment analysis were performed using the same strategy but restricted to libraries generated for this study and using either only soma-enriched or only total sRNA-seq libraries (2–3 replicates per species and library type) Clusters identified using either somatic or total libraries were concatenated and any clusters within 40 kb from each other were merged Clusters of size <35 kb were discarded to enable analysis of strand biases across major clusters Total piRNA coverage per cluster was normalised to counts per million and calculated for soma-enriched and total libraries separately Somatic clusters were defined as clusters with at least 2-fold soma enrichment over total libraries and germline clusters were defined as being higher expressed in the total ovary libraries In addition to the consensus sequences obtained from EDTA we also used the RepeatModeler (v2.0.1) output within the EDTA folders to improve detection of LINE elements We reasoned that we could not provide a list of known LINEs to EDTA since that would mainly reflect melanogaster transposons and would bias the comparisons between the melanogaster subgroup and other species EDTA and RepeatModeler consensus sequences were combined and further processed using a custom pipeline (see “Transposon_libraries” at https://github.com/susbo/Drosophila_unistrand_clusters) and remaining sequences were clustered using cd-hit-est (v4.8.1 -G 0 -g 1 -c 0.90 -aS 0.90 -n 8 -d 0 -b 500) to combine any sequences with ≥90% identity across ≥90% of the length Custom scripts were used to select one representative sequence from each cluster maximising both the number of high-quality genomic hits (blastn filtered to cover at least 50% of the query sequence) and the length of the sequence Sequences with fewer than 2 high-quality genomic hits were removed from the transposon libraries To prioritise sequences and to detect known transposon domains gag and pol ORFs from RepeatPeps.lib in RepeatMasker (v4.1.2) using blastx (v2.10.0 Sequences covering at least 50% of the full peptide domain were considered true hits which were considered to belong to the same subfamily This was repeated using 90/80/90 and 80/80/80 thresholds to detect more distant similarities The curated and annotated transposon consensus sequences have been made available (https://github.com/susbo/Drosophila_TE_libraries) --max-seeds 100 -q -k 1 -p 10 --no-unal --new-summary --summary-file) the median alignment rate was subtracted from each library to reduce false hits driven by abundant non-coding RNAs To determine whether clusters were more likely to have captured Gypsy-family LTR transposons compared with other LTR transposons we defined a Gypsy enrichment ratio as Gypsy is the set of all Gypsy-family transposon subfamilies Captured is the set of all transposon subfamilies found inside the cluster region and Not captured is the set of all other transposon subfamilies Reads mapping uniquely to each genome were intersected with cluster coordinates using bedtools intersect Resulting counts were normalised to the total number of reads mapping to each genome For the cluster content analysis (Fig. 7) we considered only reads of length 24-28 nt that mapped uniquely to the curated transposon libraries a set of 100 piRNA-regulated transposons were defined for whole ovary and soma-enriched ovary by ranking the sequences in the curated transposon library by the number of piRNAs mapping to them across all replicates The rankings were highly similar between whole ovary and soma-enriched ovary and in total 116 to 128 transposon subfamilies were selected per species To enable comparison of the counts in soma-enriched and whole ovary libraries we derived cpm values by normalising the counts to the total number of reads mapping to the genome Soma-enrichment per transposon subfamily was calculated as the difference in cpm between the pooled soma-enriched and whole ovary libraries we further restricted the analysis to reads that also mapped uniquely to the genome assembly and used the read identifiers to assigned transposon identity and transposon strand to each genome-mapping read the reads were intersected to piRNA cluster coordinates (bedtools intersect v2.26.0) in strand-specific mode to allow determination of whether the reads originated from the sense or antisense strand of a cluster dual-strand clusters were assumed to be located on the + strand Metadata for the curated transposon libraries were obtained as described previously under “Construction of curated de novo transposon libraries” To avoid false hits to conserved protein domains and to increase sensitivity compared with sequence-based searches, we employed a synteny-based search strategy (see “Synteny_biogenesis_genes” at https://github.com/susbo/Drosophila_unistrand_clusters) melanogaster genome as a reference and extracted the 20 closest genes up- and downstream genes -maxIntron=500000 -minMatch=2 -minScore=30 -oneOff=1 -minIdentity=10) and filtered the results to keep the best hit (pslCDnaFilter we constructed a hit list with all genomic regions that had at least two hits within 1 Mb from another which was manually inspected for the presence of the gene of interest Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article The flamenco Locus Controls the gypsy and ZAM Retroviruses and Is Required for Drosophila Oogenesis piRNA-mediated regulation of transposon alternative splicing in the soma and germ line Genome surveillance by HUSH-mediated silencing of intronless mobile elements The hush complex cooperates with trim28 to repress young retrotransposons and new genes The role of KRAB-ZFPs in transposable element repression and mammalian evolution piRNA-guided genome defense: From biogenesis to silencing Discrete small RNA-generating loci as master regulators of transposon activity in drosophila A single unidirectional piRNA cluster similar to the flamenco locus is the major source of EVE-derived transcription and small RNAs in Aedes aegypti mosquitoes A role for Piwi and piRNAs in germ cell maintenance and transposon silencing in Zebrafish Adaptive evolution leads to cross-species incompatibility in the piRNA transposon silencing machinery Evolutionary dynamics of piRNA clusters in Drosophila Transcriptional properties and splicing of the flamenco piRNA cluster Export of piRNA precursors by EJC triggers assembly of cytoplasmic Yb-body in Drosophila a gene controlling the gypsy retrovirus of Drosophila melanogaster Gypsy transposition correlates with the production of a retroviral envelope-like protein under the tissue-specific control of the Drosophila flamenco gene The beta heterochromatic sequences flanking the I elements are themselves defective transposable elements Drosophila germline invasion by the endogenous retrovirus gypsy: Involvement of the viral env gene Infection of the germ line by retroviral particles produced in the follicle cells: a possible mechanism for the mobilization of the gypsy retroelement of Drosophila and diversity of retrotransposons at the flamenco locus reflect the regulatory properties of piRNA clusters Recurrent insertion and duplication generate networks of transposable element sequences in the Drosophila melanogaster genome Functional adaptations of endogenous retroviruses to the drosophila host underlie their evolutionary diversification Highly contiguous assemblies of 101 drosophilid genomes Highly contiguous genome assemblies of 15 drosophila species generated using nanopore sequencing History of the discovery of a master locus producing piRNAs: the flamenco/COM locus in Drosophila melanogaster Channel nuclear pore complex subunits are required for transposon silencing in Drosophila Benchmarking transposable element annotation methods for creation of a streamlined The absence of core piRNA biogenesis factors does not impact efficient transposon silencing in Drosophila proTRAC-a software for probabilistic piRNA cluster detection Poised for contagion: evolutionary origins of the infectious abilities of invertebrate retroviruses Evolution and phylogeny of insect endogenous retroviruses Gypsy endogenous retrovirus maintains potential infectivity in several species of Drosophilids Trapping a somatic endogenous retrovirus into a germline piRNA cluster immunizes the germline against further invasion Reactivation of a somatic errantivirus and germline invasion in Drosophila ovaries on the neo-Y chromosome of Drosophila miranda Requirements for multivalent Yb body assembly in transposon silencing in Drosophila a major site for Piwi-associated RNA biogenesis and a gateway for Piwi expression and transport to the nucleus in somatic cells Dynamics of Transposable Element Invasions with piRNA Clusters Large Drosophila germline piRNA clusters are evolutionarily labile and dispensable for transposon regulation Natural variation of piRNA expression affects immunity to transposable elements Rapid evolution of piRNA clusters in the Drosophila melanogaster ovary The Cutoff protein regulates piRNA cluster expression and piRNA production in the Drosophila germline Daedalus and gasz recruit armitage to mitochondria Rapid evolutionary diversification of the flamenco locus across simulans clade Drosophila species Widespread introgression across a phylogeny of 155 Drosophila genomes a database of repetitive elements in eukaryotic genomes Adaptive seeds tame genomic sequence comparison DrosoPhyla: Resources for drosophilid phylogeny and systematics Maternally deposited germline piRNAs silence the tirant retrotransposon in somatic cells Rapid evolutionary dynamics of an expanding family of meiotic drive factors and their hpRNA suppressors Deep experimental profiling of microRNA diversity Run or Die in the Evolution of New MicroRNAs-Testing the Red Queen Hypothesis on De Novo New Genes miRBase: from microRNA sequences to function melanogaster modENCODE transcriptome annotation The genome of drosophila innubila reveals lineage-specific patterns of selection in immune genes Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype PiRNA-guided transposon cleavage initiates Zucchini-dependent An improved ATAC-seq protocol reduces background and enables interrogation of frozen tissues Fast and accurate short read alignment with Burrows-Wheeler transform and validation of bias in ATAC-Seq data with ataqv Considering transposable element diversification in de novo annotation approaches A unified classification system for eukaryotic transposable elements van Lopik, J. et al. Unistrand piRNA clusters are an evolutionarily conserved mechanism to suppress endogenous retroviruses across the Drosophila genus, Drosophila_TE_libraries, Zenodo, https://doi.org/10.5281/zenodo.8377332 (2023) van Lopik, J. et al. Unistrand piRNA clusters are an evolutionarily conserved mechanism to suppress endogenous retroviruses across the Drosophila genus, Drosophila_unistrand_clusters, Zenodo, https://doi.org/10.5281/zenodo.8377341 (2023) Download references We thank Hannon group members for fruitful discussions Holly Goodrick for help with library preparation and Emma Kneuss for providing early access to unpublished sRNA-seq libraries We thank the Scientific Computing core at the CRUK Cambridge Institute for HPC resources and the Genomics core for sequencing services GJH is a Royal Society Wolfson Research Professor (RSRP\R\200001) by Cancer Research UK (G101107) and the Wellcome Trust (110161/Z/15/Z) The following species were kindly provided by the indicated laboratories: D virilis from Simon Collier at the Department of Genetics These authors contributed equally: Jasper van Lopik Susanne Bornelöv & Benjamin Czech Nicholson designed the experiments and interpreted the results performed ATAC-seq experiments and analysis performed all other wet-lab work and preliminary computational analysis except the MCScan analysis that was performed by MAT All authors read and approved the final version Download citation DOI: https://doi.org/10.1038/s41467-023-42787-1 Cellular and Molecular Mechanisms of Brain-aging Volume 15 - 2023 | https://doi.org/10.3389/fnagi.2023.1157818 are caused by neuronal loss and dysfunction Despite remarkable improvements in our understanding of these pathogeneses serious worldwide problems with significant public health burdens are remained new efficient diagnostic and therapeutic strategies are urgently required PIWI-interacting RNAs (piRNAs) are a major class of small non-coding RNAs that silence gene expression through transcriptional and post-transcriptional processes Recent studies have demonstrated that piRNAs are also produced in non-gonadal somatic cells and further revealed the emerging roles of piRNAs we aimed to summarize the current knowledge regarding the piRNA roles in the pathophysiology of neurodegenerative diseases we first reviewed on recent updates on neuronal piRNA functions We also discuss the aberrant expression and dysregulation of neuronal piRNAs in neurodegenerative diseases we review pioneering preclinical studies on piRNAs as biomarkers and therapeutic targets Elucidation of the mechanisms underlying piRNA biogenesis and their functions in the brain would provide new perspectives for the clinical diagnosis and treatment of AD and various neurodegenerative diseases piRNA-mediated regulation of gene expression (A) The current model for piRNA-mediated RNA silencing mechanism piRNA guides PIWI protein to the complementary target RNA to catalyze the endonucleolytic cleavage (slicing) PIWI-piRNA complexes recognize nascent RNAs and recruit epigenetic modifiers thereby modifying the chromatin state and gene expression (B) A gene regulation via piRNAs during neuronal differentiation of NT2 cells This review focuses on the neuronal expression and functions of piRNAs the implication of dysregulation of neuronal piRNAs in various human neurodegenerative diseases and piRNAs as potential biomarkers and therapeutic targets against these diseases we summarize multiple neuronal functions of piRNAs and their biogenesis factors These observations indicated the presence of piRNA-mediated regulation of neuropathic pain We have reviewed the association between piRNAs and neurodegenerative diseases (Table 1) suggesting the presence of consistently dysregulated piRNAs in AD brains and their utility as potential biomarkers for AD Sun et al. (2018) showed that the transcription of specific subsets of transposons such as human endogenous retroviruses (HERVs) and retrotransposons is enhanced in AD brains and brains with progressive supranuclear palsy (PSP) Pathogenic tau aggregates may promote neuronal cell death through heterochromatin decondensation and aberrant expression of PIWI and piRNAs caused by transposon dysregulation in AD and neurodegenerative tauopathies piRNAs could be candidate biomarkers for AD and the CSF-derived exosomal sncRNA signature may have the potential to identify AD individuals with high sensitivity and specificity PD is the second most common neurodegenerative disease after AD with major neuropathological features, including the gradual loss of dopaminergic neurons in the substantia nigra pars compacta and the presence of Lewy bodies and Lewy neurites in the neurons of PD brains (Bernheimer et al., 1973; Kalia and Lang, 2015; Bloem et al., 2021) Moreover, the expression levels of six piRNAs, piR-has-92056, piR-hsa-150797, piR-hsa-347751, piR-hsa-1909905, piR-hsa-2476630, and piR-hsa-2834636 in blood small extracellular vesicles showed the highest relevance to PD, with an AUC value of 0.89 using a sparse partial least square discriminant analysis (sPLS-DA), suggesting that these piRNAs can be potential noninvasive biomarkers for PD diagnosis (Zhang and Wong, 2022) these observations imply that dysregulation of piRNAs is linked to the pathogenesis of ALS and that piRNAs can be potential diagnostic biomarkers and therapeutic targets of ALS suggesting a possible role of the brain piRNAs in HD pathogenicity yet the involvement of neuronal piRNAs in transposon activation remains elusive Further investigation of neuronal piRNA activity and function could uncover their exact target genes and their contribution to regulatory gene networks associated with neuronal processes and diseases This also leads to an understanding of the precise mechanisms that help develop novel therapeutic 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This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) *Correspondence: Satoshi Inoue, c2lub3VlQHRtaWcub3IuanA= Metrics details Piwi-interacting RNAs are small regulatory RNAs with key roles in transposon silencing and regulation of gametogenesis The production of mature piwi-interacting RNAs requires a critical step of trimming piwi-interacting RNA intermediates to achieve optimally sized piwi-interacting RNAs The poly(A)-specific ribonuclease family deadenylase PNLDC1 is implicated in piwi-interacting RNA trimming in silkworms The physiological function of PNLDC1 in mammals remains unknown here we show that PNLDC1 is required for piwi-interacting RNA biogenesis Pnldc1 mutation in mice inhibits piwi-interacting RNA trimming and causes accumulation of untrimmed piwi-interacting RNA intermediates with 3′ end extension leading to severe reduction of mature piwi-interacting RNAs in the testis Pnldc1 mutant mice exhibit disrupted LINE1 retrotransposon silencing and defect in spermiogenesis these results define PNLDC1 as a mammalian piwi-interacting RNA biogenesis factor that protects the germline genome and ensures normal sperm production in mice Here we demonstrate the physiological function of PNLDC1 in mice We show that PNLDC1 is required for mammalian piRNA 3′ trimming in vivo and is essential for transposon silencing and spermatogenesis in mice Pnldc1 mutation causes piRNA 3′ extension and great reduction of mature piRNAs by analyzing the accumulated pre-piRNAs in Pnldc1 mutant mice we report the existence of previously speculated phased piRNA biogenesis in mouse pachytene piRNAs These findings underscore the importance of the piRNA length maturation in mammalian germline genome defense and development This indicates that the piRNA pathway in PNLDC1-deficient neonatal male germ cells is still at least functional for LINE1 silencing despite defective piRNA trimming and partial MIWI2 mislocalization we show that PNLDC1 is essential for mammalian piRNA 3′ end trimming Defective pre-piRNA trimming results in compromised piRNA production and function which contributes to transposon upregulation and block in spermatogenesis We envision that targeting the mammalian piRNA trimming machinery especially the enzymatic activity of PNLDC1 could serve as a novel method for development of small-molecule inhibitors for animal sterilization and human male contraception All the animal procedures were approved by the Institutional Animal Care and Use Committee of Michigan State University (AUF 10/16-173-00) All experiments with mice were conducted ethically according to the Guide for the Care and Use of Laboratory Animals and institutional guidelines A total of five first-generation (F0) Pnldc1 mutant male mice were identified and used for analyses after reaching adulthood A stable mutant mouse line harboring an exon 1–exon 9 deletion was established by breeding a female Pnldc1 mutant founder mouse with a wild-type C57BL/6J male Subsequent intercrossing generated homozygous Pnldc1 mutant mice with exon 1–exon 9 deletions Neonatal and adult mutant male mice and their respective littermate controls were used for analyses Mouse testes and epididymides were fixed in 4% PFA or Bouin’s fixative in PBS at 4 °C overnight and embedded in paraffin For the histological and morphological analysis sections were stained with hematoxylin and eosin after dewaxing and rehydration Complimentary DNA corresponding to LINE1 ORF1 222–357 aa (L1Md-A2 GenBank: M13002.1) was cloned into pET-28a (His-tag) vectors His-tagged recombinant protein was used as an antigen to generate rabbit anti-ORF1 polyclonal antisera (Pacific Immunology) Testes were fixed in 4% PFA in PBS overnight at 4 °C and embedded in paraffin Antigen retrieval was performed by microwaving the sections in 0.01 M sodium citrate buffer (pH 6.0) tissue sections were blocked in 5% normal goat serum (NGS) for 30 min Testis sections were then incubated with anti-MIWI (1:50; 2079 or FITC-conjugated mouse anti-γH2AX (1:500; 16–202A sections were incubated with Alexa Fluor 555 goat anti-rabbit IgG (1:500; A21429 Life Technologies) for 1 h and mounted using Vectorshield mounting media with DAPI (H1200 Fluorescence microscopy was performed using Fluoview FV1000 confocal microscope (Olympus Testes were fixed in 4% PFA in PBS overnight at 4 °C Sense and antisense DIG-labeled RNA probes were transcribed using DIG RNA Labeling Kit (Roche) from a linearized plasmid containing a full length of LINE1 ORF1 (nucleotides 1741–2814 After denaturing the probes for 10 min in hybridization cocktail solution (Amresco) the probes were added to the sections and incubated overnight at 65 °C sections were incubated with alkaline phosphatase conjugated goat anti-DIG Fab fragments (Roche) overnight The positive signal was visualized by adding BM Purple (Roche) Total RNA was extracted from mouse tissues using Trizol reagent (Thermo Scientific). For complimentary DNA (cDNA) synthesis, 1 μg of RNA was treated with DNase I (M0303S, NEB) and reverse transcribed with iScript cDNA Synthesis Kit (Bio-Rad). RT-PCR was performed using primers shown in Supplementary Table 1 Mouse testes were collected and homogenized using lysis buffer (20 mM HEPES pH 7.3 and 1 mM DTT) with protease inhibitor cocktail (Thermo Scientific) and RNase inhibitor (Promega) testis lysates were centrifuged at 12,000 rpm for 10 min The supernatants were pre-cleared using protein-A agarose beads (Roche) for 2 h Abcam) antibodies together with protein-A agarose beads were added to the lysates and incubated for 4 h The beads were washed in lysis buffer for five times Immunoprecipitated RNAs were isolated from the beads using Trizol reagent (Thermo Scientific) for piRNA labeling or small RNA library construction immunoprecipitated beads were boiled in protein loading buffer for 5 min Western blotting of MILI or MIWI was performed as described above Total RNA was extracted from mouse testes using Trizol reagent (Thermo Scientific) Total RNA (1 μg) or immunoprecipitated RNA (MILI or MIWI) was de-phosphorylated with Shrimp Alkaline Phosphatase (NEB) and end-labeled using T4 polynucleotide kinase (NEB) and [γ-32P] ATP 32P labeled RNA was separated on a 15% Urea-PAGE gel and signals were detected by exposing the gel on phosphorimager screen followed by scanning on Typhoon scanner (GE Healthcare) All sequencing data are deposited in the Sequence Read Archive of NCBI under the accession number SRP095532 All other data that support the findings of this study are available from the corresponding authors upon reasonable request Skeparnias, I., Alphanastasakis, D., Shaukat, A. N., Grafanaki, K. & Stathopoulos, C. Expanding the repertoire of deadenylases. RNA Biol., doi:10.1080/15476286.2017.1300222 (2017) Download references Cheng for critical reading of the manuscript This study was technically supported by the Research Technology Support Facility and Transgenic and Genome Editing Facility of Michigan State University was supported by a grant R24 OD-012221 from the Office of the Director Office of Research Infrastructure Programs This study was supported in part by Michigan State University funds and a grant R01HD084494 from the NIH to C.C The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health Jiali Liu and Kunzhe Dong contributed equally to this work Reproductive and Developmental Sciences Program analyzed the histological defects of Pnldc1 mutant mice The authors declare no competing financial interests Download citation DOI: https://doi.org/10.1038/s41467-017-00854-4 Volume 14 - 2023 | https://doi.org/10.3389/fgene.2023.1129194 This article is part of the Research TopicNon-Coding RNA Elements as Regulators of Host-Pathogen InteractionsView all 5 articles piRNAs function as genome defense mechanisms against transposable elements insertions within germ line cells Recent studies have unraveled that piRNA pathways are not limited to germ cells as initially reckoned but are instead also found in non-gonadal somatic contexts these pathways have also been reported in bacteria associated with safeguard of genomes against transposable elements regulation of gene expression and with direct consequences in axon regeneration and memory formation In this Perspective we draw attention to early branching parasitic protozoa whose genome preservation is an essential function as in late eukaryotes little is known about the defense mechanisms of these genomes We and others have described the presence of putative PIWI-related machinery members in protozoan parasites We have described the presence of a PIWI-like protein in Trypanosoma cruzi bound to small non-coding RNAs (sRNAs) as cargo of secreted extracellular vesicles relevant in intercellular communication and host infection we put forward the presence of members related to Argonaute pathways in both Trypanosoma cruzi and Toxoplasma gondii The presence of PIWI-like machinery in Trypansomatids and Apicomplexa could be evidence of an ancestral piRNA machinery that evolved to become more sophisticated and complex in multicellular eukaryotes We propose a model in which ancient PIWI proteins were expressed broadly and had functions independent of germline maintenance A better understanding of current and ancestral PIWI/piRNAs will be relevant to better understand key mechanisms of genome integrity conservation during cell cycle progression and modulation of host defense mechanisms by protozoan parasites PIWI-interacting RNAs (piRNAs) are single-stranded RNA molecules that range between 21–35 nucleotides with 2′-O-methyl-modified 3′ ends (Ozata et al., 2019) The best characterized function of piRNAs is in genome defense against TE insertion in germ line cells Akin to microRNAs (miRNAs), the main function of piRNAs is performed by Argonaute proteins of the PIWI subfamily clade (Aravin et al., 2006). Generally, piRNAs bind to PIWI proteins to target transcripts coming from piRNA clusters, TE regions or mRNAs via base-pair complementarity, inducing their endonucleolytic cleavage and generating, in turn, new piRNAs (reviewed in Yamashiro and Siomi, 2018) proposed model for Protozoa parasites -related piRNA pathways discussed in this perspective we can observe the somatic piRNAs at follicle cells where transcribed piRNA cluster by RNA Pol II RNA polymerase are processed in primary piRNAs binds to PIWI proteins and are guided to silence TE invaders or target mRNAs for post-transcriptional gene regulation the canonical piRNA pathway in germline cells from the ovary of D Primary piRNA biogenesis occurs in follicle cells at the nuage there is a piRNA amplification process called ping-pong cycle The piRNAs produced during this process are called secondary piRNAs) Primary piRNAs can also bind to PIWI protein and go back to the nucleus to remodel chromatin (B) Proposed model for Apicomplexa and Trypanosomatids PIWI-like proteins discussed in this perspective We proposed an intermediate mechanism between RNAi and piRNA biogenesis where mpiRNAs are transcribed in the nucleus to reach the cytoplasm to find Dicer or Ago/PIWI-like proteins they can either be secreted to mediate cell to cell and cell-host communication or target mRNAs to control gene expression mpiRNAs are composed mainly of tRNA halves recruited by TcPIWI-tryp and in T The components of piRNA pathways are localized to different subcellular regions depending on their function. For example proteins that participate in piRNA biogenesis -including Vasa (a DDX-4 homolog of mammals) and Armitage, among others-, are localized near the nuage in fly germ cells or Yb bodies in the surrounding somatic follicular cells (Rogers et al., 2017) these differences among models may reflect the diversity of PIWI/piRNAs machinery components it would be interesting to analyze whether the pathway has disappeared over time from early to modern eukaryotes (Metazoans) we highlight the expression of Argonaute and piRNA pathway-related proteins in two protozoan parasites with relevance in human health: Trypanosoma cruzi and Toxoplasma gondii The Apicomplexa phylum is exclusively composed of parasitic protists. Among others, the phylum encompasses species of Plasmodium, and Toxoplasma gondii, the agents of malaria and toxoplasmosis, respectively. Similar to Trypanosomatids, some organisms from the Apicomplexa family do not present RNAi or Argonaute orthologs. That is the case of P. falciparum where dsRNAs are able to trigger gene silencing (Riyahi et al., 2006) Toxoplasma has a complex life cycle with different stages Sexual forms (macro- and microgametes) are only present within the guts of Felid species (their definitive host) Sexual recombination takes place in order to generate a diploid oocyst which upon sporulation gives rise to eight sporozoites This raises the possibility that TgAgo might share germline-associated functions similar to those reported for metazoan PIWI in the context of oocysts The resemblance of PIWI-tryp and TgAgo to the PIWI subfamily led us to revisit phylogenetic structural and sequence alignment analyses in selected organisms to update our understanding of the matter We looked for specific members related to AGO/PIWI proteins in Protists, Algae, Plants, Metazoan and Prokaryotes in order to compare sequence alignments for the presence or absence of different domains (Figure 1) We further performed sequence homology analyses to determine the presence of conserved members of the piRNA pathways in Toxoplasma gondii. We found homology with proteins such as Dicer (Braun et al., 2010) Tudor and several others related to RNA metabolism Further studies on Argonaute proteins from other members of the Apicomplexa clade would be required to functionally classify them as PIWI-like or under the recently defined WAGO proteins Understanding the evolution and relevance of PIWI proteins and pathways in Apicomplexan could have implication for targeted drug discovery as they might relate to the ones of plants and be involved in parasite division or differentiation Our previous reports on these organisms show that the gene expression regulation and the regulation of TE mediated by the PIWI protein were already an ancestral characteristic of the protein uncovering an early evolutionary origin for somatic functions of the piRNA pathway Within the germline of animals, both PIWI and piRNAs are highly abundant, ensuring genome integrity of germ cells. We propose here that PIWI machinery could represent an evolutionary conserved pathway, consequence of an arms race between genomes against invading pathogens and the hosts, demonstrated by its expression in early branches of evolution (Figure 2A) non-gonadal expressed PIWI proteins recruit certain cellular transcripts in the absence of canonical piRNAs We pose here several unanswered questions in this field In a scenario with no canonical piRNA pathways would the ancestral Argonaute protein genes be defending the genome against a TE invasion From it being absent in the organism to being complex with more than one protein acting as a key player We analyzed here two examples of vastly studied protozoan models from Apicomplexa and Trypanosomatid groups with implications for human and animal health they share complex life cycles that alternate between different stages of intracellular and extracellular forms they have intermediate and definitive hosts and treatments are either not effective or there exists resistance to drugs it would be relevant to find molecular targets in these parasites with no homology when comparing to their hosts We then propose the Argonaute family of proteins as potential specific candidates to be explored in the quest for new therapies to control parasitic infections Ongoing research aims to better understand the evolutionary origins of RNAi pathways which will lead to better understanding of their functions PIWI-piRNAs pathways remain a puzzling group of sRNAs with emerging novel mechanisms and functions Why were Argonaute proteins lost in some lineages What would be the driving force behind the specialization process of the PIWI family of proteins Future research tackling these open questions will shed light onto these ill-understood aspects of RNAi pathways of gene expression regulation The original contributions presented in the study are included in the article/supplementary material further inquiries can be directed to the corresponding author performed experiments and prepared figures MEF wrote and revised this manuscript and AC conceptualized the manuscipt All authors have read and agreed to the published version of the manuscript MRGS, AC and MEF are PEDECIBA-Biología and Sistema Nacional de Investigadores researchers. MLC is a PhD student from PEDECIBA: The authors would like to thank the Advanced Bioimaging Unit at Institute Pasteur Montevideo, for their support in this project. 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This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use *Correspondence: M. R. Garcia-Silva, cmdhcmNpYUBwYXN0ZXVyLmVkdS51eQ== Metrics details PIWI-interacting small RNAs (piRNAs) protect the germline genome and are essential for fertility piRNAs originate from transposable element (TE) RNAs or 3´ untranslated regions (3´UTRs) of protein-coding messenger genes with the last being the least characterized of the three piRNA classes we demonstrate that the precursors of 3´UTR piRNAs are full-length mRNAs and that post-termination 80S ribosomes guide piRNA production on 3´UTRs in mice and chickens when other co-translational RNA surveillance pathways are sequestered piRNA biogenesis degrades mRNAs right after pioneer rounds of translation and fine-tunes protein production from mRNAs Although 3´UTR piRNA precursor mRNAs code for distinct proteins in mice and chickens they all harbor embedded TEs and produce piRNAs that cleave TEs we discover a function of the piRNA pathway in fine-tuning protein production and reveal a conserved piRNA biogenesis mechanism that recognizes translating RNAs in amniotes Despite these piRNA precursors being annotated as lncRNAs rather than directly channeling to RNA granules for processing we recently demonstrated that ribosomes translate their upstream open reading frames (uORFs) transcription and translation of mammalian piRNA precursors utilize conventional machineries therefore leaving the features that distinguish them from other lncRNAs and mRNAs elusive which explains why eukaryotic mRNAs are generally monocistronic the presence of long ORF on piRNA precursors should inhibit ribosome-guided piRNA biogenesis It is thus mechanistically essential for both piRNA biogenesis and translational regulation to test whether ribosome-guided biogenesis can only occur after the translation of a short ORF The lack of understanding of 3′UTR piRNAs hindered our efforts to identify either common features that mark such transcripts for piRNA biogenesis and/or machinery that sorts diverse RNAs for piRNA biogenesis Here we characterize the biogenesis of 3′UTR piRNAs in mice demonstrating that their precursors are full-length protein-coding mRNAs We further show that piRNA biogenesis from these precursors is coupled with efficient translation and that ribosomes guide piRNA precursor fragmentation on mRNA 3′UTRs We demonstrate that this tight coupling of ribosome binding and piRNA biogenesis fine-tunes protein synthesis from mRNAs Ribosome-guided piRNA processing occurs at the meiotic stage when ribosome recycling factors and NMD are temporally inhibited we demonstrate that 3′UTR ribosome-guided piRNA processing also occurs in chickens Although 3′UTR piRNAs are derived from distinct sets of genes in mice and chickens we found the presence of TE sequences to be a shared feature that serves to produce anti-sense TE piRNAs that cleave TEs post-transcriptionally indicating that TE suppression is a conserved evolutionary force driving 3′UTR piRNA production we find that a general and conserved piRNA biogenesis pathway recognizes translating RNAs regardless of their ORF length a The distance from the annotated transcription start site of each mRNA that comes from 3′UTR piRNA-producing loci (uppl) (left n = 30) and from the 5′-ends of uppl mRNA 3′UTRs (right n = 30) to the nearest A-MYB ChIP-seq peak the MYB motif from the mouse UniPROBE database MEME-identified sequence motif in the A-MYB ChIP-seq peaks near the uppl mRNA transcription state sites E-value computed by MEME measures the statistical significance of the motif The information content is measured in bits and A position in the motif at which all nucleotides occur with equal probability has an information content of 0 bits while a position at which only a single nucleotide can occur has an information content of 2 bits c Boxplots showing the abundance of uppl mRNA (left) and 3′UTR piRNA (right) per uppl gene in A-Myb mutants (red) and their heterozygous littermates (blue) in testes at 14.5 and 17.5 dpp Box plots show the 25th and 75th percentiles whiskers represent the 5th and 95th percentiles d Aggregated data for 3′UTR piRNA abundance (upper) and uppl mRNA abundance (bottom) on uppl mRNAs (10% trimmed mean) from adult Mov10l1CKO/∆ testes (left) and Mov10l1CKO/∆ Neurog3-cre (right) Signals are aligned to the transcriptional start site (5′cap) and site of polyadenylation (3′PolyA) and further aligned to the ORF regions Dotted lines show the translation start codon (ORF Start) and stop codon (ORF End) The x-axis shows the median length of these regions our results suggest that the increased steady-state abundance of uppl mRNAs is due to the lack of 3′UTR piRNA biogenesis our results indicate that 3′UTR piRNAs come from full-length uppl mRNAs not from isoforms derived from uppl 3′UTRs aggregation plots of piRNA reads surrounding the 5′ and 3′ splice sites of uppl mRNAs from the adult mouse testes the signal was calculated for non-genome matching piRNA reads mapping to exon–exon junction sequences b Boxplots showing spliced RNA transcript length distributions c Boxplots showing exon length distributions d Boxplots showing ORF and UTR length distributions e Discrete Fourier transformation of the distance spectrum of 5′-ends of RPFs across ORFs (gold) and 3′UTRs (green) of uppl mRNAs in adult wild-type testes f Boxplots showing translational efficiency (RPF abundancy divided by mRNA abundancy) in adult wild-type testes Box plots in (b–d and f) show the 25th and 75th percentiles suggesting that the unique feature of piRNA precursors can be traced back to exon–intron structures and the selection of poly-A cleavage sites uppl mRNAs are efficiently translated on their main ORFs without obvious signs of aberrant initiation or elongation distinguishing them from control mRNAs uppl mRNAs function to produce both piRNAs and proteins a Normalized reads of anti-HA-immunoprecipitated RPFs and piRNAs mapping to four representative uppl genes in adult testes b Residual plot of partial correlation between piRNA and RPF abundance conditional on RNA-seq abundance at the uppl ORFs (left) and 3′UTRs (right) in adult testes Linear regression was performed between piRNA and RNA-seq abundance and between RPF and RNA-seq abundance separately and residuals were plotted with smooth parameter “method = lm” Data are presented as mean values ± standard deviation shown as error bands c Distance spectrum of 5′-ends of RPFs (26–32 nts) from ORFs (left) and from 3′UTRs (right) that overlap piRNAs in adult testes (sample size n = 3 independent biological replicates) uppl 3′UTRs bound by ribosomes are processed into piRNAs a Sequence logos depicting nucleotide (nt) bias at 5′-ends and 1 nt upstream of 5′-ends anti-HA-immunoprecipitated 5′P RNA species b Boxplots of the ratios of 5′P RPF versus 5′OH RPFs in adult wild-type testes c Boxplots of the ratios of RPF abundance in monosome fractions versus those in polysome fractions in adult wild-type testes d Boxplots of the ratios of RNA abundance in CBP80 IP versus eIF4E IP in adult wild-type (upper) and Mov10l1 CKO mutant (lower) testes e Aggregated data for RPF abundance from untreated adult testes (top) and from harringtonine-treated adult testes (bottom) across 5′UTRs and 3′UTRs of the uppl mRNAs (left) and control mRNAs (right) and the y-axis represents the 10% trimmed mean of relative abundance f piRNA abundance on uppl mRNAs (10% trimmed mean) in adult testes Box plots in (b–d) show the 25th and 75th percentiles This indicates that RPFs predominantly present with 5′P ends in 3′UTRs at steady state suggesting that in vivo cleavage occurs efficiently on ribosome-bound 3′UTRs indicating that 3′UTR RPFs are cleaved by the piRNA processing machinery piRNA processing machinery generates the 5′P in vivo cleavage products with a ribosome bound at their 5′ extremities and these 5′P ends become the 5′-ends of future piRNAs while the distribution of control mRNAs was unaffected (p = 0.32) Our results indicate that our failure to detect appreciable translation of eIF4E-bound uppl mRNAs is due to their processing into piRNAs while they are CBP80-bound suggesting that uppl mRNA translation and piRNA biogenesis are temporally coupled and carried out using the same uppl RNA molecule 3′UTR ribosomes migrate from upstream long ORFs rather than loading internally Unlike the conventional initiation mechanisms on mRNA ORFs after uORF that require a short uORF length our study suggests compromised post-termination recycling which underlies the coupling between translation at ORFs and piRNA processing at 3′UTRs indicating a lack of a biogenic relationship between ribosome-bound ORFs and piRNAs our results indicate that ribosome-guided piRNA processing occurs at uppl 3′UTRs but not at uppl ORFs the lack of 5′ overlap between the cleavage products and uppl ORF piRNAs indicates that the cleavage products are inefficiently processed into piRNAs Relative abundance of AGO2 were calculated as the abundance of AGO2 normalized by the abundance of TUBULIN (sample size n = 4 independent biological samples) Data are mean ± standard deviation; *p = 0.048 e Boxplots of the 5′P decay intermediates per mRNA in adult wild-type testes 3′UTR piRNA processing affects the level of proteins from essential genes the increased protein level of AGO2 in Mov10l1 CKO mutants leads to increased activity of miRNAs expressed during pachynema Given the sensitivity to gene dosage and the essential functions of these target genes increased miRNA-guided AGO2-mediated cleavage of their transcripts and decreased protein synthesis may contribute to the infertility of Mov10l1 CKO mutants Considering that AGO2 is just one of the uppl mRNA protein products our data support the biological significance of 3′UTR piRNA biogenesis in fine-tuning protein abundance during normal development a Normalized reads of RPFs and piRNAs mapping to four representative chicken uppl genes in adult rooster testes both strands of uppl mRNAs code for piRNAs; blue represents Watson strand mapping reads; red represents Crick strand mapping reads b Discrete Fourier transformation of the distance spectrum of 5′-ends of RPFs across ORFs (gold) and 3′UTRs (green) of chicken uppl mRNAs in adult rooster testes c Distance spectrum of 5′-ends of RPFs from uppl ORFs (left) and from 3′UTRs (right) that overlap piRNAs in adult rooster testes (sample size n = 3 independent biological replicates) d Sequence logos depicting nucleotide bias at 5′-ends and 1 nt upstream of 5′-ends of the following species from adult rooster testes We used these 23 chicken transcripts as non-piRNA-producing control mRNAs in the following analyses in chickens While none of the mouse uppl mRNAs come from sex chromosomes (X or Y chromosomes) explained by meiotic sex chromosome inactivation 8 out of 23 (35%) of the chicken uppl genes mapped to the Z chromosomes (the bird sex chromosomes that are not inactivated because rooster is the homogametic sex) which are significantly enriched compared to all the mRNA-encoding genes we assembled for rooster testes (5.7% mouse and chicken 3′UTR piRNAs are derived from diverse the majority of which are essential for viability and fertility a Boxplots showing the fraction of transcript exon sequence correspondence to sense (blue) and antisense (red) transposon sequences in mouse genome b Sequence logo showing the nucleotide composition of antisense SINE-piRNA species that uniquely map to mouse uppl mRNAs c The 5′–5′ overlap between piRNAs from opposite strands of SINE consensus sequences was analyzed to determine whether antisense SINE piRNAs from mouse uppl mRNAs display Ping-Pong amplification in trans The number of pairs of piRNA reads at each position is reported (sample size n = 3 independent biological replicates) The Z score indicates that a significant ten-nucleotide overlap (Ping-Pong) was detected Z score = 1.96 corresponds to p value = 0.025 d The RNA abundance of each TE superfamily in testes at the five developmental time points e Boxplots showing the fraction of transcript exon sequence correspondence to sense (blue) and antisense (red) transposon sequences in chicken genome f Sequence logo showing the nucleotide composition of antisense LINE-piRNA species that uniquely map to chicken uppl mRNAs g The 5′–5′ overlap between piRNAs from opposite strands of LINE consensus sequences was analyzed to determine whether antisense LINE-piRNAs from chicken uppl mRNAs display Ping-Pong amplification in trans (sample size n = 3 independent biological replicates) our data indicate that a subset of piRNAs produced from mouse uppl mRNAs post-transcriptionally silence TEs which may accommodate rapidly changing populations of TEs our data show that the use of mRNAs embedded with TEs to produce antisense TE piRNAs that cleave TEs post-transcriptionally is a common strategy in amniotes we find that ribosomes guide piRNA biogenesis downstream of ORFs regardless of ORF length Similar to other co-translational mRNA quality-control pathways piRNA processing from mRNAs fine-tunes the protein products from these mRNAs This co-translational processing of piRNAs is found in both mice and chickens we reveal a general and conserved mechanism by which post-termination ribosomes guide piRNA 5′-end formation from non-protein-coding regions of RNAs in amniotes the TE piRNAs produced from uppl mRNAs may lead to a cascade of events to regulate mRNA stability Given the rapid changes in TE families in each species over evolutionary time the shared mechanisms for producing TE piRNAs from distinct gene subsets in divergent lineages suggest the biological significance of this strategy although fine-tuning protein is unlikely to represent the primary selective force that marks a subset of mRNAs as piRNA precursors we expect the better fitness gained by the rapid turn-over of these essential mRNAs to further reinforce their piRNA precursor identity the regulation of ribosome recycling occurs during meiosis prophase in cells that are not terminally differentiated as spermatocytes will undergo two more rounds of cell division This ribosome recycling regulation is specific to uppl mRNAs and may be due to the localization of uppl mRNAs in proximity to the mitochondria where piRNAs are processed by the reprogramming of RNA metabolism at pachynema including the inhibition of ribosome recycling and the NMD pathway allowing for robust piRNA production in a short but critical time window during spermatogenesis Our study indicates that reprogramming of ribosome recycling can occur locally and stage-specifically to enable biologically significant processes both lncRNA piRNA precursors and mRNA piRNA precursors exhibit longer first exons suggesting that unique exon–intron structure could be one unique feature given the translation of short upstream ORFs is insufficient to mark a transcript for piRNA biogenesis and the uppl mRNAs do not exhibit faulty translation on their main ORFs the translation intermediates with post-termination ribosomes on a long 3′UTR is a prime candidate for further testing if the TE-rich prenatal piRNAs target and initiate the processing of the 3′UTR piRNA precursors the embedding of TE elements could also serve as a determining feature of a transcript for piRNA processing the study of 3′UTR piRNAs allows a more comprehensive investigation into the unique features defining piRNA precursors consistent with the recent appearance of TDRD5 ribosome-guided piRNA biogenesis is unlikely to be conserved in invertebrates and further studies are required to trace its evolutionary origins we reveal a conserved and general piRNA biogenesis mechanism that recognizes translating RNAs regardless of whether they harbor long ORFs or not The assembly of 80S ribosomes on non-coding regions of RNA is not restricted by the length of the upstream ORFs and is temporally staggered with translation-dependent RNA quality-control pathways The coupling of piRNA biogenesis with translation fine-tunes the abundance of proteins that are critical for spermatogenesis in both mice and chickens Comparisons of compound mutants and controls involving Mov10l1 CKO mutation were performed using siblings from individual litters White Leghorn testes of the Cornell Special C strain from 1-year-old roosters were used according to guidelines for animal care of the NIH and the University Committee on Animal Resources at the University of Rochester Spike-in RNA) was used as a spike-in control testis lysates were resolved by electrophoresis on 10% sodium dodecyl sulfate (SDS)-polyacrylamide gels The proteins were transferred to a 0.45 µm polyvinylidene difluoride membrane (EMD Millipore and the blot was probed with anti-AGO2 mouse monoclonal antibody (Wako Pure Chemical Corporation and then detected with sheep anti-mouse immunoglobulin G–horseradish peroxidase (IgG-HRP; GE Healthcare and donkey anti-rabbit IgG-HRP (GE Healthcare Western blotting images were taken using Azure c300 imaging system Five A260 absorbance units were loaded on a 10–50% (w/v) linear sucrose gradient prepared in buffer (20 mM HEPES-KOH 100 μg/ml cycloheximide) and centrifuged in a SW-40ti rotor at 154,348 × g for 2 h 40 min at 4 °C Samples were collected from the top of the gradient using a gradient Fractionation system (Brandel USA; BR-188) while monitoring absorbance at 254 nm Synthetic spike-in RNAs were added to each collected fraction before RNA purification USA) using the Direct-zol™ RNA MiniPrep plus Kit (Zymo Research Ribo-seq was performed as previously described45 Cleared testis lysates were incubated with 60 units of RNase T1 (Fermentas USA) and 100 ng of RNase A (Ambion) per A260 unit for 30 min at room temperature the fractions corresponding to 80S monosomes were recovered for library construction (iii) The unligated RNAs proceed to the conventional Ribo-seq library construction (iv) The ligated RNAs directly proceed to the reverse transcription steps for library construction RNA-seq data were generated on HiSeq 2000 instrument (Illumina cDNA was amplified by PCR using KAPA HIFI Hotstart polymerase (Kapa Biosystems and 250–350 nts double-stranded DNA was isolated on 8% native PAGE gels testes were fixed in Bouin’s solution overnight and then stained with hematoxylin and eosin Seminiferous tubules were fixed in 2% paraformaldehyde containing 0.1% Triton X-100 for 10 min at room temperature placed on a slide coated with 1 mg/ml poly-L-lysine (Sigma) with a small drop of fixative The coverslip was removed after freezing in liquid nitrogen The slides were later rinsed three times for 5 min in phosphate-buffered saline (PBS) and incubated for 12 h at 4 °C with rabbit anti-PELOTA antibody (1:50 dilution; Thermo Secondary antibodies conjugated with Alexa Fluor 488 (Molecular Probes Histology and immunostaining images were taken using Leica DM4000 B LED microscope system with the Leica software: Leica Application Suite X v1.1.0.12420 The mice were anesthetized with ketamine/xylazine mixture (ketamine 100 mg/kg; xylazine 25 mg/kg) via intraperitoneal injection testes were exteriorized with a longitudinal incision around 1 cm at the center of abdomen The tunica albuginea was penetrated using a sharp 26 G needle (BD and the needle was withdrawn to generate a path for introducing a blunt end Hamilton needle (Hamilton PBS containing 0.02% Fast Green FCF (Thermo Fisher Scientific 0.5 μg/μl in a total volume of 10 μl) or with Okadcid Acid (LX Laboratories 16 nM per testis in a total volume of 10 μl) was slowly injected using a Hamilton microsyringe (1705RN) into one testis and a vehicle control without the drug was injected into the other testis of the same animal The needle was held in place for 30 s before removal to prevent backflow of the solution Successful completion of injection was indicated by testis filled with green solution The testes were returned to the abdominal cavity after injection the mice were euthanized by cervical dislocation We report piRNA abundance either as parts per million reads mapped to the genome (ppm) or as reads per kilobase pair per million reads mapped to the genome (rpkm) using a pseudo count of 0.001 a piRNA is counted only when the 5′-end of the piRNA maps to the ORF or 3′UTR of a transcript we identified 44,856 mRNA transcripts and considered the remaining 36,223 transcripts as lncRNAs and their relative positions on transcripts were reported For alternative transcription with overlapping annotations We calculated the distance spectrum of 5′-ends of Set A (RPFs or degradome reads) that overlapped with Set B (piRNAs or simulated sequences) as follows: for each read b in Set B we identified all the reads in Set A whose 5′-ends overlapped within the 50-nt region upstream of b including the 5′-end of b and 50 nts downstream of b (200-nt window of b reads) We assigned the b spectrum as the fractions of 5′-ends of a reads distributed across the 100-nt window of b reads The height of the b spectrum at each nucleotide position in the 100-nt window of b reads was based on the number of a reads whose 5′-ends overlapped at this position divided by the total number of a reads whose 5′-ends overlapped with the 100-nt window of b reads The sum of all b spectra was then divided by the total number of reads in Set B We defined this average fraction of 5′-ends of a reads that overlapped with the 100-nt window of b reads as the distance spectrum of 5′-ends of Set A that overlap Set B The Z score for overlap at the 5′-end position was calculated using the spectral value at positions −50–−1 and 2–50 as background The relative spectral density was calculated by normalizing to the value at the first position We generated a random pool of 28-mer sequences using a sliding window of 1 nucleotide from 5′ to 3′ of the piRNA precursors We then sampled from this 28-mer pool to match the first nucleotide composition of the real reads These simulated sequences from piRNA precursors were used as random controls for piRNAs (source code available upon request) protein concentration was determined by BCA (Thermo Scientific) Samples were then diluted to 1 mg/ml in 5% SDS 25 µg of protein from each sample was reduced with DTT to 2 mM followed by incubation at 55 °C for 60 min Iodoacetamide was added to 10 mM and incubated in the dark at room temperature for 30 min to alkylate the proteins followed by six volumes of 90% methanol and 100 mM TEAB The resulting solution was added to S-Trap micros (Protifi) and centrifuged at 4000 × g for 1 min The S-Traps containing trapped proteins were washed twice by centrifuging through 90% methanol and 100 mM TEAB One microgram of trypsin was brought up in 20 µl of 100 mM TEAB and added to the S-Trap followed by an additional 20 µl of TEAB to ensure the sample did not dry out The cap to the S-Trap was loosely screwed on but not tightened to ensure the solution was not pushed out of the S-Trap during digestion Samples were placed in a humidity chamber at 37 °C overnight the S-Trap was centrifuged at 4000 × g for 1 min to collect the digested peptides Sequential additions of 0.1% trifluoroacetic acid (TFA) in acetonitrile and 0.1% TFA in 50% acetonitrile were added to the S-trap Samples were frozen and dried down in a Speed Vac (Labconco) then re-suspended in 0.1% TFA prior to analysis Peptides were loaded onto a 100 µm × 30 cm C18 nano-column packed with 1.8 µm beads (Sepax) using an Easy nLC-1200 HPLC (Thermo Fisher) connected to a Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher) and solvent B was 0.1% formic acid in 80% acetonitrile Ions were delivered to the mass spectrometer using a Nanospray Flex source operating at 2 kV Peptides were eluted off the column using a multi-step gradient then ramped up to 90% B in 6 min and held there for 4 min to wash the column before returning to starting conditions in 2 min The column was re-equilibrated for 7 min for a total run time of 180 min The Fusion Lumos was operated in data-dependent mode performing a full scan followed by as many MS2 scans as possible in 3 s The full scan was done over a range of 375–1400 m/z with a resolution of 120,000 at m/z of 200 Peptides with a charge state between 2 and 5 were selected for fragmentation Precursor ions were fragmented by collision-induced dissociation using a collision energy of 30 and an isolation width of 1.1 m/z MS2 scans were collected in the ion trap with the scan rate set to rapid Raw data were searched using SEQUEST within the Proteome Discoverer software platform v2.2 (Thermo Fisher) employing the SwissProt mouse database along with a custom fasta database that included both test and control proteins Trypsin was selected as the enzyme allowing up to 2 missed cleavages Carbamidomethyl on cysteine was selected as a fixed modification Oxidation of methionine was set as a variable modification A percolator was used as the false discovery rate calculator filtering out peptides with a q value > 0.01 Label-free quantitation was performed using the Minora Feature Detector node with a minimum trace length of 5 The Precursor Ions Quantifer node was then used to calculate protein abundance ratios using only unique and razor peptides The summed abundance-based method was employed which sums the peak areas for all the peptides for a given protein to determine protein ratios Random sequences were generated using the Markov method and eCAI was estimated at 99% level of confidence and 99% coverage CDSs of housekeeping genes were used as the reference set for all the calculations Further information on research design is available in the Nature Research Reporting Summary linked to this article Antisense RNA: function and fate of duplex RNA in cells of higher eukaryotes Small RNA silencing pathways in germ and stem cells The growing catalog of small RNAs and their association with distinct Argonaute/Piwi family members The biogenesis and function of PIWI proteins and piRNAs: progress and prospect Zygotic amplification of secondary piRNAs during silkworm embryogenesis The RNase PARN-1 trims piRNA 3´ ends to promote transcriptome surveillance in C Identification and functional analysis of the pre-piRNA 3’ trimmer in silkworms mediates 2′-O-methylation of Piwi- interacting RNAs at their 3′ ends and migration of the primordial germ cells in the mouse embryo A signaling principle for the specification of the germ cell lineage in mice Long first exons and epigenetic marks distinguish conserved pachytene piRNA clusters from other mammalian genes NMD: a multifaceted response to premature translational termination Surveillance pathways rescuing eukaryotic ribosomes lost in translation Ribosomes guide pachytene piRNA formation on long intergenic piRNA precursors What determines whether mammalian ribosomes resume scanning after translation of a short upstream open reading frame Early origins and evolution of microRNAs and Piwi-interacting RNAs in animals A regulatory circuit for piwi by the large Maf gene traffic jam in Drosophila A broadly conserved pathway generates 3’UTR-directed primary piRNAs Single-molecule long-read sequencing reveals a conserved intact long RNA profile in sperm Exon size distribution and the origin of introns Alternative cleavage and polyadenylation in spermatogenesis connects chromatin regulation with post-transcriptional control UPF2-dependent nonsense-mediated mRNA decay pathway is essential for spermatogenesis by selectively eliminating longer 3’UTR transcripts MicroRNAs control mRNA fate by compartmentalization based on 3’ UTR length in male germ cells ALKBH5-dependent m6A demethylation controls splicing and stability of long 3’-UTR mRNAs in male germ cells Selective 40S footprinting reveals cap-tethered ribosome scanning in human cells Functional 5’ UTR mRNA structures in eukaryotic translation regulation and how to find them Translation efficiency is determined by both codon bias and folding energy The codon Adaptation Index–a measure of directional synonymous codon usage bias An improved implementation of codon adaptation index E-CAI: a novel server to estimate an expected value of Codon Adaptation Index (eCAI) ppcor: An R package for a fast calculation to semi-partial correlation coefficients The pioneer round of translation: features and functions Evidence for a pioneer round of mRNA translation: mRNAs subject to nonsense-mediated decay in mammalian cells are bound by CBP80 and CBP20 Temporal and spatial characterization of nonsense-mediated mRNA decay The exon junction complex is detected on CBP80-bound but not eIF4E-bound mRNA in mammalian cells: dynamics of mRNP remodeling Inhibition of translation in eukaryotic systems by harringtonine Reinitiation and other unconventional posttermination events during eukaryotic translation Dom34 rescues ribosomes in 3’ untranslated regions Modified ribosome profiling reveals high abundance of ribosome protected mRNA fragments derived from 3’ untranslated regions Rli1/ABCE1 recycles terminating ribosomes and controls translation reinitiation in 3’UTRs in vivo Dynamic regulation of a ribosome rescue pathway in erythroid cells and platelets eIF5A functions globally in translation elongation and termination Nuclear localization of EIF4G3 suggests a role for the XY body in translational regulation during spermatogenesis in mice Analysis of Dom34 and its function in no-go decay The Hbs1-Dom34 protein complex functions in non-stop mRNA decay in mammalian cells Upf1 senses 3’UTR length to potentiate mRNA decay How retroviruses escape the nonsense-mediated mRNA decay Readthrough of stop codons under limiting ABCE1 concentration involves frameshifting and inhibits nonsense-mediated mRNA decay Nonsense-mediated mRNA decay - mechanisms of substrate mRNA recognition and degradation in mammalian cells The antagonistic gene paralogs Upf3a and Upf3b govern nonsense-mediated RNA decay Quality and quantity control of gene expression by nonsense-mediated mRNA decay The DEAD-box protein Dbp5p is required to dissociate Mex67p from exported mRNPs at the nuclear rim The DEAD-box protein Dbp5 controls mRNA export by triggering specific RNA:protein remodeling events RAFT1 phosphorylation of the translational regulators p70 S6 kinase and 4E-BP1 The mammalian phenotype ontology: enabling robust annotation and comparative analysis High-throughput discovery of novel developmental phenotypes Mice deficient for spermatid perinuclear RNA-binding protein show neurologic Functional analysis of Asb-1 using genetic modification in mice Gene deletion of inositol hexakisphosphate kinase 1 reveals inositol pyrophosphate regulation of insulin secretion Targeted inactivation of testicular nuclear orphan receptor 4 delays and disrupts late meiotic prophase and subsequent meiotic divisions of spermatogenesis is essential for development and appears not to be involved in DNA methylation An essential role for an inositol polyphosphate 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recycle post-termination 40S subunits in vivo DENR promotes translation reinitiation via ribosome recycling to drive expression of oncogenes including ATF4 40S ribosome profiling reveals distinct roles for Tma20/Tma22 (MCT-1/DENR) and Tma64 (eIF2D) in 40S subunit recycling PIWI slicing and RNA elements in precursors instruct directional primary piRNA biogenesis Somatic primary piRNA biogenesis driven by cis-acting RNA elements and trans-acting Yb Cell-type-specific isolation of ribosome-associated mRNA from complex tissues Neurogenin 3-expressing progenitor cells in the gastrointestinal tract differentiate into both endocrine and non-endocrine cell types Selective depletion of rRNA enables whole transcriptome profiling of archival fixed tissue Comparative analysis of RNA sequencing methods for degraded or low-input samples Squash procedure for protein immunolocalization in meiotic cells The UCSC Genome Browser Database: update 2006 Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks Ribosome footprint profiling of translation throughout the genome TopHat: discovering splice junctions with RNA-Seq R: A Language and Environment for Statistical Computing (R Foundation for Statistical Computing StringTie enables improved reconstruction of a transcriptome from RNA-seq reads De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis Identifying periodically expressed transcripts in microarray time series data DAMBE7: new and improved tools for data analysis in molecular biology and evolution CAIcal: a combined set of tools to assess codon usage adaptation Comprehensive modeling of microRNA targets predicts functional non-conserved and non-canonical sites ImageJ2: ImageJ for the next generation of scientific image data Download references and members of the Li laboratory for discussions; G Lozada for help with editing the manuscript; and J and UR Genomics Research Center for help with the experiments This work was supported by National Institutes of Health grant R35GM128782 and Agriculture and Food Research Initiative Competitive Grant no 2018-67015-27615 from the USDA National Institute of Food and Agriculture to X.Z.L Center for RNA Biology: From Genome to Therapeutics Zheng analyzed the data with input from C.Z. performed the experiments with input from E.P.R designed the study and drafted the manuscript and all authors contributed to the preparation of the manuscript Peer review information Nature Communications thanks Haruhiko Siomi and Shu-Bing Qian for their contributions to the peer review of this work Download citation DOI: https://doi.org/10.1038/s41467-021-26233-8 Cellular and Molecular Life Sciences (2024) Metrics details PIWI proteins are therefore better equipped than AGO proteins to target newly acquired or rapidly diverging endogenous transposons without recourse to new small RNA guides the minimum requirements for PIWI slicing are sufficient to avoid inadvertent silencing of host RNAs Our results demonstrate the biological advantage of PIWI over AGO proteins in defending the genome against transposons and suggest an explanation for why the piRNA pathway was retained in animal evolution Mean and standard deviation of data from three independent trials are shown (b,c (left) GTSF1 accelerates the otherwise slow target cleavage by PIWIs by 10–100-fold probably by stabilizing the catalytically competent conformation of PIWI proteins Why PIWI slicing evolved to require an auxiliary protein is unknown the requirements for guide:target complementarity are relaxed for the PIWI proteins MILI (also known as PIWIL2) and MIWI (also known as PIWIL1) from mouse (Mus musculus) and Piwi from freshwater sponge (Ephydatia fluviatilis; hereafter denoted EfPiwi) PIWI proteins bind RNAs both with and without complementarity to the canonical 5′ seed of their guide PIWI-catalysed slicing requires at least 15 contiguously paired nucleotides and longer extents of complementarity tolerate guide:target mismatches at essentially any position including those that flank the scissile phosphate (t10 and t11) Although pairing to at least four  piRNA 5′ terminal nucleotides facilitates target finding in vitro and in vivo abundant piRNAs direct slicing of targets that lack 5′ complementarity the minimum 15-nucleotide stretch of complementarity that licenses piRNA-guided target cleavage is sufficient to distinguish host transcripts from transposon RNAs These findings suggest that the catalytic properties of PIWI proteins evolved to prevent transposons from escaping piRNA silencing through mutation while simultaneously retaining sufficient specificity to spare self-transcripts from inappropriate repression which suggests that the weaker affinity of PIWI proteins for the canonical seed sites will result in lower occupancy of such targets Our data therefore disfavour a model in which PIWI proteins find and productively regulate targets through seven-nucleotide canonical seed pairing We conclude that PIWI proteins are more flexible than AGO proteins in the types of sites they can bind but require longer complementarity for high-affinity binding we found that extending pairing beyond piRNA nucleotide g20 enabled MILI and MIWI to tolerate guide:target mismatches MILI and EfPiwi pre-steady-state cleavage rates (k) for targets of L1MC piRNA containing a single unpaired nucleotide Position and identity of mononucleotide mismatch in targets (indicated in blue) of L1MC piRNA (indicated in red) are on the top of the chart can efficiently cleave partially paired RNAs with mismatches anywhere in a target site Sequencing the 3′ products of piRNA-directed, MIWI-catalysed slicing showed that piRISC invariably hydrolysed the RNA at the canonical scissile phosphodiester bond, between target nucleotides t10 and t11, even when both g10 and g11 were unpaired or when contiguous pairing did not start until g11 (Extended Data Fig. 5c) These data suggest that piRNA–target base pairing near the cleavage site has little if any role in positioning the scissile phosphate within the MIWI catalytic centre PIWI inherently accommodates unpaired target nucleotides and that GTSF1 only accelerates cleavage piRNAs directed MILI and MIWI to cleave targets with as few as 15–19-nucleotide complementary nucleotides in vivo in mouse primary spermatocytes Schematic of the strategy used to identify 3′ cleavage products of piRNA-guided PIWI-catalysed slicing and to measure the fraction of targets cleaved by PIWI proteins in FACS-purified mouse primary spermatocytes Fraction of cleaved MILI and MIWI targets in FACS-purified mouse primary spermatocytes for contiguous pairing from nucleotide g2 Fraction of cleaved targets in FACS-purified mouse primary spermatocytes for perfect matches (indicated in blue) and for pairing containing a single-nucleotide mismatch (indicated in pink) Horizontal dotted lines indicate the medians for perfect matches and EfPiwi pre-steady-state cleavage rates in vitro for all possible stretches of ≥6-nucleotide contiguous pairing starting from nucleotides g2–g15 of L1MC piRNA Fraction of cleaved targets in FACS-purified mouse primary spermatocytes for 14-nucleotide contiguous pairing starting from nucleotides g2 to g5 Data are binned by piRNA intracellular concentration (<30 box plots show IQR and median; 95% CI was calculated with 10,000 bootstrapping iterations; n = 16 permutations of 4 control (C57BL/6) and 4 pi2−/−pi9−/−pi17−/− animals Cleavage by MILI or MIWI is indistinguishable in our data thus \({f}_{{\rm{c}}{\rm{l}}{\rm{e}}{\rm{a}}{\rm{v}}{\rm{e}}{\rm{d}}}^{{\rm{g}}2-{\rm{g}}\,X}\) corresponds to the sum of targets sliced in mouse primary spermatocytes by both PIWI proteins Note that pairing longer than g2–g20 contained too few data points to measure the corresponding \({f}_{{\rm{c}}{\rm{l}}{\rm{e}}{\rm{a}}{\rm{v}}{\rm{e}}{\rm{d}}}^{{\rm{g}}2-{\rm{g}}\,X}\) The lower median fcleaved values for targets mismatched to piRNA 5′ sequences compared with other piRNA regions may reflect slower on-rates for piRNAs of low intracellular concentration (see also the next section) the median \({f}_{{\rm{cleaved}}}^{{\rm{mismatches}}\,{\rm{to}}\,{\rm{g3}}{\rm{-g8}}}\) = 0.10 compared with \({f}_{{\rm{cleaved}}}^{{\rm{mismatches}}\,{\rm{to}}\,{\rm{g9}}{\rm{-g18}}}\) = 0.18 the median \({f}_{{\rm{cleaved}}}^{{\rm{mismatches}}\,{\rm{to}}\,{\rm{g3}}{\rm{-g8}}}\) = 0.06 compared with \({f}_{{\rm{cleaved}}}^{{\rm{mismatches}}\,{\rm{to}}\,{\rm{g9}}{\rm{-g19}}}\) = 0.21 a wide variety of piRNA–target pairing patterns can efficiently direct MILI and MIWI to cleave targets unlike the relatively limited pairing configurations tolerated by AGO-clade Argonaute proteins our data identified piRNA-directed cleavage in vivo in mouse primary spermatocytes of targets for which pairing to the guide starts at g3 We conclude that because pairing to piRNA 5′ terminal nucleotides is dispensable for both target finding and slicing piRISC efficiently cleaves targets that lack full complementarity to the canonical 5′ seed target nucleotides t9–t15 face the protein surface which makes insertions likely to distort the catalytic centre We speculate that piRNA guide bulges between t11 and t15 specifically affect PIWI proteins because they impair interactions with GTSF1 Decision function coefficients for 400 logistic function fits (regression models) using around 3,500 distinct piRNA–target pairs detected in mouse primary spermatocytes n = 16 permutations of 4 control (C57BL/6) and 4 pi2−/−pi9−/−pi17−/− animals × 5-repeated × 5-fold cross validation Number of piRNAs and siRNAs predicted to cleave mutated versions of the L1Md AI transposon sequence Data are median and IQR from 100 independent simulations AGO and PIWI proteins use different rules to find and slice targets The features with the lowest median coefficients were the identity of nucleotide t1 and the location of the target site within a transcript (−0.18 to +0.13; Fig. 4a) This result suggests that these factors are not rate-determining for piRNA-guided cleavage in vivo these analyses show that simple biochemical principles are sufficient to predict efficient piRNA-directed cleavage in vivo piRNA concentration determines how frequently a target encounters piRISC and therefore the concentration of the piRISC–target complex tighter guide:target base pairing (binding energy) extends the lifetime of the piRISC–target complex which increases the likelihood of cleavage and any change in the 5′ position of a siRNA duplex can invert which strand becomes a guide for an AGO protein will require full complementarity between their guides and targets Our data suggest that the catalytically competent geometry of PIWI proteins does not intrinsically rely on perfect complementarity between the target and piRNA near the cleavage site was favoured by evolution as the mainstay of transposon defence in animals No statistical method was used to determine the sample size the maximum possible sample size (n = 4–12) was used for each type of data which ensured that variability arising from all accountable sources was incorporated in the analyses (animal Randomization is not relevant to this study because it did not involve treatment or exposure of animals to any agent untreated wild-type mice were compared with untreated mutant mice lacking piRNAs from four genomic loci Blinding is not relevant to this study because during analyses wild-type control and mutant datasets were easily identified Blinding was not performed during data acquisition and/or analysis Cells were collected at 70% confluency using a TC cell scraper (ThermoFisher 50809263) into ice-cold PBS and collected by centrifugation at 500g and the pellet was stored at −80 °C until lysed in 10 ml of 30 mM HEPES-KOH 15% (v/v) glycerol and 1× protease inhibitor cocktail (1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (Sigma E3132) and 10 μM leupeptin hemisulfate) per g frozen cells Cell lysis was monitored by staining with trypan blue Crude cytoplasmic lysate was clarified at 20,000g flash frozen in liquid nitrogen and stored at −80 °C After five washes in 2 M potassium acetate extract buffer and five washes in 100 mM potassium acetate extract buffer MILI or MIWI piRISC was eluted from the beads twice with 200 ng µl–1 3×Flag peptide in 100 µl of 100 mM potassium extract buffer with rotation for 1 h at room temperature The combined 200 µl eluate was used immediately for capture oligonucleotide affinity purification or flash frozen in liquid nitrogen and stored at −80 °C The supernatant containing eluted piRISC was then incubated for 30 min at room temperature with 300 µl Dynabeads MyOne Streptavidin T1 paramagnetic beads (prewashed according to the manufacturer’s instructions followed by two washes in 100 mM potassium extract buffer) to remove excess competitor DNA oligonucleotide 20 μl anti-Flag M2 paramagnetic beads (Sigma M8823) was added and incubated with the supernatant for 4 h rotating at 4 °C to isolate piRISC from testis lysate Beads were then washed four times with 2 M potassium acetate extract buffer and four times with 100 mM potassium acetate extract buffer piRISC was eluted from the beads twice with 200 ng µl–1 3×Flag peptide in 100 µl of 100 mM potassium extract buffer with rotation for 1 h at room temperature Dissected animal tissue samples were homogenized at 4 °C in 5 volumes of 30 mM HEPES-KOH 1 mM DTT and 15% (v/v) glycerol in a dounce homogenizer using 10 strokes of the loose-fitting pestle A followed by 20 strokes of tight-fitting pestle B to generate crude lysate S20 was prepared by clarifying the crude lysate at 20,000g The protein concentration was estimated using a BCA assay (ThermoFisher Crude and fractionated testis lysate were flash frozen in liquid nitrogen and stored at −80 °C Each 750 ml culture of Sf9 cells was pelleted and resuspended in 25 ml lysis buffer (50 mM Tris pH 8.0 300 mM NaCl and 0.5 mM TCEP) and lysed using a high-pressure (18,000 p.s.i.) microfluidizer (Microfluidics M100P) and the clarified lysate was incubated with 1 ml Ni-NTA resin (Qiagen followed by washing twice in nickel wash buffer (50 mM Tris pH 8.0 The resin was then washed once with wash buffer supplemented with 5 mM CaCl2 in preparation for micrococcal nuclease treatment to degrade co-purifying cellular RNAs The washed resin was resuspended in nickel wash buffer supplemented with 5 mM CaCl2 (final volume of 20 ml) 2910A) was added per 750 ml culture and incubated at room temperature for 1  h inverting gently every 15 min to resuspend the resin After three washes with nickel wash buffer without CaCl2 protein was eluted with 6× column volumes of nickel elution buffer (wash buffer supplemented with 300 mM imidazole) Eluted protein was supplemented with 5 mM EGTA to chelate any remaining calcium and dialysed (10,000 MWCO) against 50 mM Tris pH 8.0 an aliquot of EfPiwi (1/50 of the protein yield from 750 ml of Sf9 culture) was incubated with a synthetic piRNA guide (15 µM f.c.) for 15 min at room temperature and then dialysed into 50 mM Tris pH 8.0 0.02% CHAPS buffer overnight at 4 °C (12,000 MWCO) To prepare for capturing guide-loaded EfPiwi 2.5 nmol of biotinylated capture oligonucleotide was incubated with 40 µl high capacity neutravidin resin (ThermoFisher 29204) in 1 ml wash A buffer (30 mM Tris pH 8.0 0.02% CHAPS and 0.5 mM TCEP) for 30 min at 4 °C followed by two washes with 2 ml wash A buffer EfPiwi–guide complex was captured by incubating with the capture oligonucleotide-conjugated neutravidin resin at room temperature for 1.5 h with rotation The resin was then washed three times with 2 ml wash A buffer four times with 2 ml wash B buffer (30 mM Tris pH 8.0 and three times with 2 ml wash C buffer (30 mM Tris pH 8.0 The resin was then resuspended in 250 µl wash C buffer containing biotinylated competitor oligonucleotide (50 µM f.c.) and incubated with rotation at room temperature for 3 h To remove excess competitor oligonucleotide the supernatant was incubated for 30 min at 4 °C with 60 µl fresh neutravidin resin (prewashed twice in wash C buffer) and the supernatant was dialysed overnight at 4 °C into extract buffer (30 mM HEPES-KOH 15% (v/v) glycerol and 0.01% (v/v) Triton X-100) The dialysed EfPiwi–guide RNA complex was aliquoted pCold-GST GTSF-expression vectors were transformed into Rosetta-Gami 2 competent cells (Sigma Cells were grown to an OD600 of 0.6–0.8 in the presence of 1 μM ZnSO4 at 37 °C then chilled on ice for 30 min to initiate cold shock Protein expression was induced with 0.5 mM IPTG for 18 h at 15 °C washed twice with PBS and cell pellets were flash frozen and stored at −80 °C Cell pellets were resuspended in lysis/GST column buffer containing 20 mM Tris-HCl pH 7.5 5% (v/v) glycerol and 1× protease inhibitor cocktail (1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride (Sigma Cells were lysed by a single pass at 18,000 p.s.i through a high-pressure microfluidizer (Microfluidics and the resulting lysate clarified at 30,000g for 1 h at 4 °C Clarified lysate was filtered through a 0.22 µm Millex Durapore low-protein-binding syringe filter (EMD Millipore) and applied to glutathione Sepharose 4b resin (Cytiva 17075604) equilibrated with GST column buffer the resin was washed with 50 column-volumes of GST column buffer To elute the bound protein and cleave the GST tag in a single step 1 mM DTT and 5% (v/v) glycerol was added to the column and the column sealed and incubated for 3 h at 4 °C the column was drained to collect the cleaved protein The eluate was diluted to 50 mM NaCl and further purified using a HiTrap Q (Cytiva 29051325) anion exchange column equilibrated with 20 mM Tris-HCl The bound protein was eluted using a 100–500 mM NaCl gradient in the same buffer Peak fractions were analysed for purity by SDS–PAGE and the purest were pooled and dialysed into storage buffer containing 30 mM HEPES-KOH Aliquots of the pooled fractions were flash frozen in liquid nitrogen and stored at −80 °C muelleri was used to design the expression construct of Ephydatia sp The EmGtsf1 expression vector was transformed into BL21(DE3) cells (NEB Transformed cells were grown in LB medium supplemented with 1  μM ZnSO4 at 37 °C until an OD600 of 0.6–0.8 The incubation temperature was lowered to 16 °C and protein expression was induced by the addition of 1 mM IPTG for 16 h Cells were collected by centrifugation and cell pellets flash frozen in liquid nitrogen and stored at −80 °C Thawed cell pellets were resuspended in lysis buffer (50 mM Tris 300 mM NaCl and 0.5 mM TCEP) and passed through a high-pressure (18,000 p.s.i.) microfluidizer (Microfluidics The lysate was clarified by centrifugation at 30,000g for 20 min at 4 °C Clarified lysate was applied to Ni-NTA resin (Qiagen) and incubated for 1 h The resin was washed with nickel wash buffer (300 mM NaCl Protein was eluted in four column volumes of nickel elution buffer (300 mM NaCl TEV protease was added to the eluted protein to remove the amino-terminal His6 and MBP tags The resulting mixture was dialysed against HiTrap dialysis buffer (300 mM NaCl The dialysed protein was then passed through a 5 ml HiTrap chelating column (Cytiva) and the unbound material collected Unbound material was concentrated and further purified by size-exclusion chromatography using a Superdex 75 Increase 10/300 column (Cytiva) equilibrated in 50 mM Tris Peak fractions were analysed for purity by SDS–PAGE in vitro transcribed with T7 RNA polymerase purified using a 7% denaturing polyacrylamide gel and capped using α-[32P]GTP (Perkin Elmer) and a Vaccinia Capping System (NEB Unincorporated α-[32P]GTP was removed using a G-25 spin column (Cytiva target RNA was purified using a 7% denaturing polyacrylamide gel eluted overnight with rotation in 0.4 M NaCl at 4 °C and collected by ethanol precipitation Radiolabelled target (10 nM f.c.) was added to a mix of purified piRISC and GTSF1 (500 nM f.c.) to assemble a 30 μl cleavage reaction a 5 μl sample was quenched in 280 μl 50 mM Tris-HCl then proteinase K (1 mg ml–1 f.c.) was added and the mix incubated at 45 °C for 15 min followed by extraction with phenol–chloroform–isoamyl alcohol (25:24:1 RNA was resuspended in 10 μl 95% (v/v) formamide 0.025% (w/v) bromophenol blue and 0.025% (w/v) xylene cyanol and resolved on a 7% denaturing polyacrylamide gel exposed to a storage phosphor screen and imaged on a Typhoon FLA 7000 (GE) The raw image file was used to quantify the substrate and product bands Data were fit to the burst-and-steady-state equation to determine the concentration of active piRISC (see equation and fitting procedure in the section ‘Analysis of CNS data’) PCR products were purified with a 2% agarose gel and sequenced on a NextSeq 550 (Illumina) to obtain 79-nucleotide the two membranes were washed with 100 ml wash buffer for 3 s Membranes were air-dried and signals detected by phosphorimaging to monitor binding The nitrocellulose membrane areas containing piRISC-bound RNA were excised and incubated with 1 mg ml–1 proteinase K in 100 mM Tris-HCl 150 mM sodium chloride and 1% (w/v) SDS for 1 h at 45 °C shaking at 300 r.p.m After phenol–chloroform extraction and ethanol precipitation amplified and sequenced as described above for the input RNA pool The assay also included a control reaction using piRISC storage buffer Binding reactions were incubated at 33 °C for 2 h RNA binding was measured by capturing protein–RNA complexes on Protran nitrocellulose (GE GE10600002) and unbound RNA on a Hybond-XL (Cytiva 45-001-151) in a Bio-Dot apparatus (Bio-Rad) membranes were washed with 10 μl equilibration buffer (30 mM HEPES-KOH Membranes were air-dried and signals detected by phosphorimaging all binding data were fit to the following equation using IgorPro 6.11 (WaveMetrics): [ST] is the total RNA target concentration and Kd is the apparent equilibrium dissociation constant To the ssDNA oligonucleotide pool of Cleave-’n-Seq (CNS) targets (Extended Data Fig. 3b) obtained from TWIST Bioscience, a T7 promoter was added by PCR (primers listed in Supplementary Table 4) The PCR products were in vitro transcribed with T7 RNA polymerase then treated with TURBO DNase (ThermoFisher and the CNS target RNA library was purified using a 7% denaturing polyacrylamide gel PCR products were purified using a 2% agarose gel and sequenced on a NextSeq 550 (Illumina) to obtain 60-nucleotide both the 0–8 min and the 0–960 min subsets) were sequenced in the same NextSeq 550 run Data from three trials of each of the 0–8 min and 0–960 min subsets were combined to estimate pre-steady-state cleavage rates The 355 nm laser was used to excite Hoechst 33342; the 488 nm laser was used to record forward and side scatter and to excite propidium iodide Propidium iodide emission was detected using a 610/20 bandpass filter Hoechst 33342 emission was recorded using 450/50 and 670/50 band pass filters All samples used in this study met the following criteria: spermatogonia 95–100% pure with ≤5% pre-leptotene spermatocytes; primary spermatocytes 35–40% diplotene spermatocytes; secondary spermatocytes the PCR product was purified in a 2% agarose gel Small RNA sequencing (RNA-seq) libraries samples were sequenced using a NextSeq 550 (Illumina) to obtain 79 nucleotide RNA-seq libraries were sequenced using a NextSeq 550 (Illumina) to obtain 79+79 nucleotide The PCR product was purified from a 1% agarose gel and sequenced using a NextSeq 550 or NovaSeq 6000 (Illumina) to obtain 79+79 nucleotide or 150+150 nucleotide To analyse RBNS31 data the sequence of the 3′ adapter (5′-TGGAATTCTCGGGTGCCAAGG-3′) was removed using fastx toolkit (v.0.0.14) then each sequencing read in the RNA input library and piRISC-bound libraries was interrogated for the presence of all binding sites of interest The entire single-stranded 20 nucleotide random-sequence region flanked by four nucleotides of constant primer-binding sequence on either side (GATCNNNNNNNNNNNNNNNNNNNNTGGA) was searched for the presence of piRISC-binding sites The sequencing depth of the input library (about 50 × 106 reads) allowed measurement of input frequencies for ≤12 nucleotide motifs To interrogate non-overlapping target sets each ≤10 nucleotide contiguous binding site was required to be flanked by nucleotides that not complementary to the guide: for example a g4–g12 contiguous target site did not pair to guide positions g3 and g13 Each 11-nucleotide-long contiguous complementary site was required to be flanked by a non-matching nucleotide only at its 5′ end: for example a g4–g14 contiguous target site did not pair to guide position g3 To eliminate interference from potential piRISC cleavage activity GTSF1 was omitted from binding reactions; we also relied on the fact that we do not interrogate sites that are long enough (≥15 nucleotides) to be cleaved by piRISC Sequencing data (representing the abundance of uncleaved targets) were first normalized to the sequencing depth (parts per million (ppm)) To adjust for the decrease in total abundance of the library over the course of cleavage reaction each ppm value was divided by the sum of ppm values of targets that contained ≤7 nucleotide complementarity to the piRISC piRNA guide the relative abundance of cleaved product at non-zero time points was inferred as follows: [Prelative] = (ppm0 min − ppmX min)/ppm0 min [Prelative] ranged from 0 (no cleaved product) to 1 (all substrate cleaved) The combined [Prelative] data from three independent trials of each 0–8 min and 0–960 min subsets (that is 480 and 960 min) were used to fit the burst-and-steady-state scheme \(E+S\mathop{\mathop{{\rm{\rightleftharpoons }}}\limits^{{k}_{1}}}\limits_{{k}_{-1}}ES\mathop{\to }\limits^{{k}_{2}}EP\mathop{\to }\limits^{{k}_{3}}E+P\) The resulting (k2 + k3) was reported as the pre-steady-state cleavage rate (k) Mouse AGO2 CNS data for let-7a and miR-21 RISCs are from a previous study3 and mouse AGO2 CNS data for L1MC RISC was generated for this study piRNAs were considered undetectable in pi2−/−pi9−/−pi17−/− mutants if their mean abundance in mutants (n = 9) was ≤0.1 ppm only unambiguously mapping 5′-monophosphorylated RNA species for which abundance was ≥0.04 ppm were used For 5′-monophosphorylated RNAs mapped in annotated transcripts the nucleotide sequence of the corresponding transcript was used to find piRNAs potentially explaining the cleavage and we used the genomic sequence for 5′-monophosphorylated RNAs mapped outside any annotated transcript To ensure that piRNA–target combinations for all pairing configurations did not overlap the piRNA nucleotide immediately after the paired region was required to be unpaired with the target: for example g11 was unpaired and thus did not overlap with g2–g11 Calculating of the fraction of cleaved sites was performed for a collapsed even if a cleavage site was explained by several piRNAs Cumulative abundance of all piRNAs explaining each site was used to assess the effect of piRNA concentration For each of the 16 permutations of 4 C57BL/6 control and 4 pi2−/−pi9−/−pi17−/− mutant primary spermatocyte datasets we identified 3,150–3,750 5′-monophosphorylated RNAs (that is potential 3′ cleavage products of piRNA-guided slicing) for which abundance was ≥0.1 ppm and that were explained by ≥19 paired nucleotides between g2 and g25 of pi2 pi9 and pi17 piRNAs (target insertions or deletions were not allowed) Note that although abundance and binding energy remained the best predictive features regardless of the minimum number of paired nucleotides used as a threshold requiring <19 paired nucleotides produced too few piRNA–target data points to inform the importance of pairing to piRNA 5′ terminal nucleotides if the abundance of the 5′-monophosphorylated RNAs decreased by ≥8-fold in pi2−/−pi9−/−pi17−/− mutants compared with C57BL/6 controls All other sites were assigned as uncleaved rescaled to [0,1] (use of the negative of ΔG0 creates a positive relationship between strength of binding and probability of cleavage) 0 if t1H; x32: equals 1 target site is in the 5′ UTR 0 if outside the 5′ UTR; x33: equals 1 if the target site is in the ORF 0 if outside the ORF; x34: equals 1 if the target site is in the 3′ UTR 0 if outside the 3′ UTR; x35: equals 1 if the target site is in lncRNA weights inversely proportional to class frequencies were used RepeatedStratifiedKFold and cross_validate from scikit-learn were used to perform 5× repeated 5-fold cross validation resulting in 5 × 5 = 25 logistic function fits for each of the 16 permutations of 4 control 4 mutant datasets generating the total of 25 × 16 = 400 logistic regression models area under the precision-recall curve (AUC) for each of the 400 logistic functions was calculated either with the corresponding pi2−/−pi9−/−pi17−/− dataset (400 AUC values total) or with each of the 16 permutations of 4 C57BL/6 and 4 pi7−/− mutant datasets (6,400 AUC values total) Only synonymous substitutions were allowed in LINE ORFs and 100 independent simulations were performed for each consensus sequence piRNAs from fetal mouse testis (embryonic day 16.5) were sequenced and used for the analyses and 21 nucleotide siRNAs were simulated using piRNA 5′ prefixes piRNA and siRNA guides were predicted to cleave the mutated transposon sequence using the following rules: ≤6 total mismatches at any position were allowed for 26 nucleotide piRNAs; ≤5 total mismatches g11 and g13 were allowed for 21 nucleotide siRNAs Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article Code used in this work has been deposited at GitHub (https://github.com/ildargv/Gainetdinov_et_al_2023) High-throughput analysis reveals rules for target RNA binding and cleavage by AGO2 Xiao, Y. et al. Structural basis for RNA slicing by a plant Argonaute. Nat. Struct. Mol. Biol. https://doi.org/10.1038/s41594-023-00989-7 (2023) Argonaute proteins confer immunity in all domains of life piRNA-mediated gene regulation and adaptation to sex-specific transposon expression in D PIWI slicing and EXD1 drive biogenesis of nuclear piRNAs from cytosolic targets of the mouse piRNA pathway Transcriptional silencing of transposons by Piwi and maelstrom and its impact on chromatin state and gene expression Piwi induces piRNA-guided transcriptional silencing and establishment of a repressive chromatin state piRNA pathway targets active LINE1 elements to establish the repressive H3K9me3 mark in germ cells RNA Bind-n-Seq: quantitative assessment of the sequence and structural binding specificity of RNA binding proteins Principles and pitfalls of high-throughput analysis of microRNA-binding thermodynamics and kinetics by RNA Bind-n-Seq Endogenous siRNA and miRNA targets identified by sequencing of the Arabidopsis degradome Expanding the microRNA targeting code: functional sites with centered pairing Single-molecule imaging reveals that Argonaute reshapes the binding properties of its nucleic acid guides An integrated map of genetic variation from 1,092 human genomes Cellular abundance shapes function in piRNA-guided genome defense A ubiquitin ligase mediates target-directed microRNA decay independently of tailing and trimming The ZSWIM8 ubiquitin ligase mediates target-directed microRNA degradation and PIWI-protein identity determine piRNA stability Role of temperature in regulation of spermatogenesis and the use of heating as a method for contraception Novel method for measuring polyuridylic acid binding to ribosomes A double-filter method for nitrocellulose-filter binding: application to protein–nucleic acid interactions Flow cytometric characterization of viable meiotic and postmeiotic cells by Hoechst 33342 in mouse spermatogenesis Elimination of PCR duplicates in RNA-seq and small RNA-seq using unique molecular identifiers Strand-specific libraries for high throughput RNA sequencing (RNA-seq) prepared without poly(A) selection The biochemical basis of microRNA targeting efficacy 25 years of serving the community with ribosomal RNA gene reference databases and tools Tailor: a computational framework for detecting non-templated tailing of small silencing RNAs Transcript-level expression analysis of RNA-seq experiments with HISAT a database of eukaryotic repetitive elements MIWI2 and MILI have differential effects on piRNA biogenesis and DNA methylation Differences between germline and somatic mutation rates in humans and mice Download references We thank staff at the University of Massachusetts FACS Core for help sorting mouse germ cells; staff at the University of Massachusetts Transgenic Animal Modeling Core for help generating pi2−/− pi9−/− and pi17−/− mice; and members of the Zamore Laboratory for discussions and critical comments on the manuscript This work was supported in part by NIGMS R35 GM136275 grant to P.D.Z and 1S10 OD028576 to the University of Massachusetts Flow Cytometry Core Facility is an investigator of the Howard Hughes Medical Institute RNA Therapeutics Institute and Howard Hughes Medical Institute University of Massachusetts Chan Medical School Department of Integrative Structural and Computational Biology (purification and loading MILI and MIWI; loading EfPiwi; determination of fraction active for all piRISCs; analyses of RBNS data; CNS experiments and analyses; FACS; RNA 5′-monophosphate RNA library preparation and analyses; logistic regression classifier; and in silico mutagenesis); J.V.-B generation of HEK293 cells overexpressing MIWI and MILI); and P.-H.W All the authors discussed the results and approved the manuscript Nature thanks Claus Kuhn, Taiowa Montgomery and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available Measurements of MIWI piRISC affinity for its targets (0.1 nM) using nitrocellulose filter binding assay Mean and the standard deviation of the data from three independent trials are shown and mouse AGO2 binding affinities for ≥11-nt complementary stretches contiguously paired from all guide nucleotides Mean and standard deviation from three independent trials are shown Kruskal-Wallis test (one-way ANOVA on ranks) p-values are < 10−15 for data with one and two mismatches binding affinities (KD) for a g2–g8 match with different t1 nucleotide identities mouse AGO2 pre-steady-state cleavage rate for one or two consecutive mismatches between g2–g20 Box plots show IQR and median: for one mismatch n = 24 (three geometries × four piRNAs × MILI and MIWI) n = 6 for EfPiwi (three geometries × two piRNAs) n = 21 for AGO2 (three geometries for L1MC guide and three geometries × three contexts for let-7a and miR-21 guides); for two consecutive mismatches n = 8 (one geometry × four piRNAs × MILI and MIWI) n = 2 for EfPiwi (one geometry × two piRNAs) n = 19 for AGO2 (one geometry for L1MC RISC and nine geometries for let-7a and miR-21 RISCs) Benjamini-Hochberg corrected p-values for post hoc pairwise two-tailed Mann-Whitney tests for difference in pre-steady-state cleavage rate of targets with a mononucleotide mismatch either at g10 or g11 among EfPiwi Kruskal-Wallis test (one-way ANOVA on ranks) p-value = 10−5 for mismatch at g10 and p-value = 4.1 × 10−6 for mismatch at g11 Kruskal-Wallis test (one-way ANOVA on ranks) p-value = 0.0004 Benjamini-Hochberg corrected p-values for post hoc pairwise Mann-Whitney tests are shown Relative abundance of 3′ cleavage products generated by L1MC-guided MIWI (2  h at 33 °C) All data and the median from three independent trials are shown Data are also shown for spike-in RNAs that contained no target sites and were added to the reaction to account for 5′-to-3′ exonucleolytic trimming or non-templated addition of nucleotides to RNA 5′ ends Box plots show IQR and median; 95% confidence interval was calculated with 10,000 bootstrapping iterations Steady-state abundance of piRNA precursor transcripts and mature piRNAs in FACS-purified mouse primary spermatocytes Fraction of cleaved targets in FACS-purified mouse primary spermatocytes for pairing containing a single mononucleotide mismatch and EfPiwi pre-steady-state cleavage rates in vitro for all possible stretches of ≥ 6-nt contiguous pairing starting from nucleotides g2–g15 of piRNA #1 Intracellular concentration of pachytene piRNAs in mouse primary spermatocytes Data are the mean of 12 biologically independent samples Change in pre-steady-state cleavage rate for mononucleotide target insertions (a) or target deletions (b) Target insertion data are for 16 or 40 targets: four insertion geometries for ten piRISCs (MILI and EfPiwi for pairing up to g26,or MIWI for pairing up to g30) Guide bulge data are for four or ten targets: one deletion geometry for ten piRISCs (MILI and EfPiwi for pairing up to g26 or MIWI for pairing up to g30) Fraction of cleaved targets in FACS-purified mouse primary spermatocytes for pairing containing a single mononucleotide bulge in target or guide sequence Area under the precision-recall curves for random control 400 logistic regression classifier models trained with pi2−/−; pi9−/−; pi17−/− data and 6,400 tests of the 400 models using pi7−/− data Kruskal-Wallis test (one-way ANOVA on ranks) p-value = 4.5 × 10−12 FDR (Benjamini-Hochberg) corrected p-values for post hoc pairwise Mann-Whitney tests are shown Number of piRNAs and siRNAs predicted to cleave L1Md_Gf and L1Md_Tf transposon sequences Sequence overlap between transpositionally active families of LINEs and LTR-transposons and exons or introns of mouse mRNAs and lncRNAs Binding affinities and cleavage rates for MILI FACS gating strategy to purify mouse primary germ cells Source data for analyses of in vivo cleavage by MILI and MIWI (number of putative cleavage sites) Mouse strains and sequences of oligonucleotides used in this study Amount of spike-in RNAs used to prepare small RNA-seq and RNA-seq libraries in this study expected 3′ cleavage product and the corresponding 8 nucleotide barcode for data in Extended Data Fig Source data for analyses of in vivo cleavage by MILI and MIWI (coordinates and identity of putative cleavage sites) Download citation DOI: https://doi.org/10.1038/s41586-023-06257-4 Metrics details PIWI proteins and the associated PIWI-interacting RNAs (piRNAs) protect genome integrity by silencing transposons Here we report the extensive sequence and quantitative correlations between 2′,3′-cyclic phosphate-containing RNAs (cP-RNAs) and piRNAs in the Bombyx germ cell line and mouse testes The cP-RNAs containing 5′-phosphate (P-cP-RNAs) identified by P-cP-RNA-seq harbor highly consistent 5′-end positions as the piRNAs and are loaded onto PIWI protein suggesting their direct utilization as piRNA precursors We identified Bombyx RNase Kappa (BmRNase κ) as a mitochondria-associated endoribonuclease which produces cP-RNAs during piRNA biogenesis BmRNase κ-depletion elevated transposon levels and disrupted a piRNA-mediated sex determination in Bombyx embryos indicating the crucial roles of BmRNase κ in piRNA biogenesis and embryonic development Our results reveal a BmRNase κ-engaged piRNA biogenesis pathway in which the generation of cP-RNAs promotes robust piRNA production In contrast to the well-studied downstream pathway after pre-piRNA production the mechanisms involved in the upstream pathway of Zuc- or PIWI/piRNA-mediated processing of single-stranded precursor RNAs remain unclear we have comprehensively characterized the cP-RNA expression in Bombyx BmN4 cells and mouse testes using cP-RNA-seq and its advanced version P-cP-RNA-seq and we have demonstrated extensive correlative profiles of cP-RNAs and piRNAs suggesting direct utilization of cP-RNAs as piRNA precursor molecules We identified that Bombyx RNase Kappa (BmRNase κ) a mitochondria-associated endoribonuclease is responsible for cP-RNA production and robust piRNA generation Our in vitro analysis demonstrated cP-end formation by BmRNase κ-mediated RNA cleavage BmRNase κ preferably cleaved in-between cytidine and adenosine which matches the nucleotide composition biases of cP-RNAs endogenously expressed in BmN4 cells and mouse testes BmRNase κ-depleted Bombyx embryo exhibited enhanced transposon levels and failure of feminization both of which are indicative of a disrupted piRNA pathway These results collectively revealed that piRNA biogenesis is intermediated by BmRNase κ-generated cP-RNAs The generation of cP-RNAs would enable germ cells to implement the robust piRNA production that is required for embryonic development a Schematic representation of the procedures of piRNA-seq b BmN4 RNAs were gel-purified and subjected to the three sequencing methods Amplified cDNAs were developed by native PAGE The cP-RNA-seq and P-cP-RNA-seq methods mainly amplified approximately 150–180 bp cDNA products (length of inserts without adapter sequences: ~32–62 bp) whereas the sequencing procedure without CIP and T4 PNK treatments did not amplify these cDNAs The piRNA-seq amplified ~145 bp cDNA products (insert: ~27 bp) were gel-purified and subjected to Illumina sequencing c Proportion of tRNA-derived reads classified into the indicated subgroups of tRNA-derived non-coding RNAs The reads mapped to cyto tRNAAspGUC are indicated with dotted lines d The regions from which tdpiRAspGUC (blue) and P-cP-RNAs (magenta) were derived are shown in the cloverleaf secondary structure of cyto tRNAAspGUC Averages of three experiments with SD values are shown cP-RNAs were 3.5- and 5.7-fold more abundant than OH-RNAs Along with the extensive sequence overlaps and quantitative correlations between cP-RNAs and piRNAs these results suggest that cP-RNAs are used as precursor molecules for piRNA generation c Read length distributions of the obtained sequences from two biological replicates d Total piRNAs or Mili-bound piRNAs were mapped to the total cP-RNAs or Mili-bound P-cP-RNAs The obtained mapping ratios are shown (#1 and #2: biological replicates) e Scatter plots showing the correlations between the read numbers of piRNAs and cP-RNAs/P-cP-RNAs which were mapped to each pachytene piRNA cluster f Nucleotide compositions around the 5′- and 3′-ends of the indicated RNAs A dashed line separates upstream (−) and downstream (+) positions for the 5′- and 3′-ends representing the cleavage site that produces the RNAs g The terminal positions of Mili-bound piRNA reads and their mapped cP-RNA/P-cP-RNA reads were compared and the matched rates for the nucleotide positions of the mapped cP-RNA/P-cP-RNA sequences from 5′-end (blue) and 3′-end (red) are shown h The alignment patterns of the RNAs in the indicated pachytene piRNA cluster loci Cumulated sequence reads are shown in capital blue while their upstream and downstream sequences are shown in lowercase gray these results suggest that the cP-RNAs generated by specific C–A cleavage are further cleaved at their 5′-end to harbor a 5′-terminal U which are loaded onto Mili as piRNA precursors i Control or BmRNase κ KD cells were transfected with pre-piR-I or its mutant pre-piR-nc (negative control) and de novo production of piR-J was analyzed by stem-loop RT-qPCR Averages of three independent KD experiments with SD values are shown j BmN4 cells were subjected to KDs of BmPapi Total RNAs were extracted from the KD cells and control cells and were subjected to TaqMan RT-qPCR for indicated cP-RNAs k Total RNAs extracted from control- or BmRNase κ KD cells were subjected to piRNA-seq The distances between 5′-ends of the transposon-mapped piRNA read across opposite genomic strands were plotted (ping-pong analysis) 70% of reads obtained from control- or BmRNase κ KD cells commonly conformed to a separation preference of 10 nt (ping-pong signal) l Total RNAs extracted from control- or BmRNase κ KD cells were subjected to piRNA-seq with spike-in control RNAs The relative abundance of transposon-mapped piRNA reads was calculated by normalization using spike-in reads suggesting that BmRNase κ-mediated cP-RNA production occurs upstream of the piRNA biogenesis steps involving BmPapi BmRNase κ KD does not appear to affect piRNA profiles but causes global reduction of piRNA levels The ligation product of cleaved substrate and 3′-AD was detected as a 79-bp cDNA band (shown in an arrowhead) the RNAs were ligated to 3′-AD only after they were treated with T4 PNK validating that the cleavage products of BmRNase κ contain cP and that BmRNase κ is an endonuclease which produces cP-RNAs The preference of BmRNase κ for C–A sequences is consistent with the nucleotide compositions of endogenously expressed cP-RNAs identified by cP-RNA-seq a A control siRNA (siCtrl) or two different siRNAs against BmRNase κ (siRNκ #1 and #2) were injected into Bombyx embryos The RNAs extracted from the embryos were subjected to RT-qPCR for quantification of the indicated three Bombyx transposons The average expression levels in siCtrl samples were normalized to the levels of Rp49 mRNA c The siRNκ #1 samples were subclassified into the following two groups: UP (n = 9) the samples whose transposons (at least one of the three examined transposons) were upregulated by more than twofold compared to the average of siCtrl samples; and N.C the indicated cP-RNAs (b) and piRNAs (c) were quantified by TaqMan RT-qPCR and stem–loop RT-PCR *p < 0.05; ** p < 0.01; and *** p < 0.001 The exact p values are provided in a Source Data file d Spearman correlation analysis was performed for UP samples of siRNκ #1 Blue and red indicate positive and negative correlations and the size of each circle is proportional to the correlation coefficients female (F) embryos were identified based on Fem RNA expression (siCtrl: n = 6; siRNκ #1: n = 8) and were subjected to evaluation of the expression of male (M)-specific ImpM mRNA f A proposed model for cP-RNA generation in piRNA biogenesis pathway suggesting the inhibition of feminization due to the reduction of Fem piRNA the BmRNase κ KD in the Bombyx embryos decreased the levels of cP-RNAs and piRNAs which resulted in the enhanced expression of transposons and disruption of the sex determination pathway This confirmed the crucial roles of BmRNase κ and its generated cP-RNAs in piRNA production and sexual development A less prominent 5′-U enrichment in Mili-loaded P-cP-RNAs than that of Mili-loaded piRNAs might result from the 3′-end endonucleolytic cleavage or other mechanisms which are involved in the processing of PIWI-loaded precursors into mature piRNAs cP-RNAs are expressed more abundantly than OH-RNAs and not all cP-RNAs become sources of piRNAs It will be intriguing to analyze how specific cP-RNAs selectively enter into the piRNA biogenesis pathway Analyses of the endogenous cP-RNA species generated by RNase κ and their biological functions in somatic cells might unveil previously uncharacterized noncoding RNA pathways The BmN4 cells were cultured at 27 °C in an Insect-Xpress medium (Lonza). The mitochondrial fraction of the cells were prepared as described previously15 the gel-purified ~30–70-nt RNAs or the RNAs of Mili-immunoprecipitates were first subjected to 5′-AD ligation by incubating the RNAs in a reaction mixture containing 5′-AD T4 RNA Ligase (T4 Rnl; Thermo Fisher Scientific) After purification using CENTRI-SPIN 20 (Princeton Separation) the ligated RNAs were subjected to cP-RNA-seq (without 5′-AD ligation step) The amplified cDNAs were gel-purified and sequenced using NextSeq 500 (Illumina) The piRNA libraries from Mili-immunoprecipitates were obtained not by piRNA-seq but by standard small RNA-seq procedure (without NaIO4 oxidization) using the TruSeq Small RNA Sample Prep Kit (Illumina) because piRNAs are expected to be highly enriched in the fraction The expression levels were normalized to those of 5S rRNA After total RNA was treated with RQ1 DNase (Promega), the target sequences were reverse-transcribed using RevertAid Reverse Transcriptase (Thermo Fisher Scientific). The cDNA was subjected to real-time PCR using the SYBR Green PCR Master Mix (Applied Biosystems). The primers used are shown in Supplementary Table 3 The expression levels of mRNAs and transposons were normalized to those of Rp49 mRNA Anti-BmRNase κ antibody was prepared by immunizing the rabbits with a synthetic peptide (CEDLPFDEKNPPHSI; Genscript) The whole IgG was purified from the sera using Protein G Agarose (Sigma) to obtain the “BMK5263” anti-BmRNase κ antibody anti-β-Tubulin (Developmental Studies Hybridoma Bank and piR-t was detected by the probe 5′-GCATTCTGACGGAACTTGTAATGGTA-3′ the first methionine in the ORF of GFP was replaced with alanine to avoid initiation of translation from this site The resultant plasmids were transfected into BmN4 cells using Escort IV Transfection Reagent (Sigma) the cells were plated on a slide glass chamber (Lab-Tek) and cultured for 18 h The cells were then incubated with 100 nM of MitoTracker Red CMXRos (Life Technologies) for 30 min at 27 °C After DNA mounting with ProLong Gold Antifade Reagent containing DAPI (Life Technologies) images were acquired using a Nikon A1R Confocal Laser Microscope System The fluorescence signal intensities were quantified using ImageJ software (ver 1.48) after background subtraction with a rolling ball radius of 50 pixels The coding region of the BmRNase κ cDNA was cloned into the pGEX-6P-2 plasmid which expresses N-terminal GST-tagged and C-terminal 6× His-tagged BmRNase κ protein A plasmid for K9A mutant BmRNase κ was produced through site-directed mutagenesis The wild-type or K9A mutant BmRNase κ was expressed in E coli BL21 Star (DE3) at 16 °C for 16 h in the presence of 0.5 mM IPTG The harvested cells were suspended in the lysis buffer containing 50 mM Tris-HCl pH7.5 5 mM MgCl2 and 5 mM ATP were added to the lysate BmRNase κ protein was purified using Glutathione Sepharose 4B (GE healthcare) The GST-tag was cleaved using PreScission Protease (GE healthcare) The protein was further purified by Superdex 200 Increase 10/300 GL (GE Healthcare) and the cleaved products were treated with CIP or T4 PNK (no treatment was used as a control) The treated samples were subjected to RT-qPCR using the One Step PrimeScript RT-PCR Kit (TaKaRa) and the following primers: forward The amplified cDNAs were resolved on 10% native PAGE All experiments in this study were performed at least twice or more times independently to confirm that they yielded identical/similar results Image quantitation was performed using Image J Statistical analyses were performed by using Microsoft Excel or R Bar charts with error bars denote mean values ± S.D Further information on research design is available in the Nature Research Reporting Summary linked to this article Recruitment of Armitage and Yb to a transcript triggers its phased processing into primary piRNAs in Drosophila ovaries Mitochondrial protein BmPAPI modulates the length of mature piRNAs The mouse homolog of HEN1 is a potential methylase for Piwi-interacting RNAs Hen1 is required for oocyte development and piRNA stability in zebrafish Generation of 2’,3’-cyclic phosphate-containing RNAs as a hidden layer of the transcriptome The biogenesis pathway of tRNA-derived piRNAs in Bombyx germ cells Roles for the Yb body components Armitage and Yb in primary piRNA biogenesis in Drosophila Sex hormone-dependent tRNA halves enhance cell proliferation in breast and prostate cancers Selective amplification and sequencing of cyclic phosphate-containing RNAs by the cP-RNA-seq method The Bombyx ovary-derived cell line endogenously expresses PIWI/PIWI-interacting RNA complexes Genome-wide screening and characterization of transposable elements and their distribution analysis in the silkworm Transcriptome profiling reveals infection strategy of an insect maculavirus Genome-wide identification of short cyclic phosphate-containing RNAs and their regulation in aging Increasing cell density globally enhances the biogenesis of Piwi-interacting RNAs in Bombyx mori germ cells Artificial “ping-pong” cascade of PIWI-interacting RNA in silkworm cells Respective functions of two distinct Siwi complexes assembled during PIWI-interacting RNA biogenesis in Bombyx germ cells Analysis of catalytic residues in enzyme active sites A single female-specific piRNA is the primary determiner of sex in the silkworm Unique sex determination system in the silkworm Transgenic expression of the piRNA-resistant masculinizer gene induces female-specific lethality and partial female-to-male sex reversal in the silkworm The endosymbiotic bacterium wolbachia selectively kills male hosts by targeting the masculinizing gene Hierarchical roles of mitochondrial Papi and Zucchini in Bombyx germline piRNA biogenesis 3’ end formation of PIWI-interacting RNAs in vitro Cc RNase: the Ceratitis capitata ortholog of a novel highly conserved protein family in metazoans Molecular cloning and characterization of the human RNase kappa Essential cysteine residues for human RNase kappa catalytic activity RNASEK Is a V-ATPase-associated factor required for endocytosis and the replication of rhinovirus Flavivirus internalization is regulated by a size-dependent endocytic pathway The lupus autoantigen la prevents mis-channeling of tRNA fragments into the human microRNA pathway in Ribonucleases: structures and functions (eds Alessio Characteristic ribonucleolytic activity of human angiogenin Role of glutamine-117 in the ribonucleolytic activity of human angiogenin Genomic structure and expression analysis of the RNase kappa family ortholog gene in the insect Ceratitis capitata Morphological and histomorphological structures of testes and ovaries in early developmental stages of the silkworm Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells Download references We are grateful to Anastasios Vourekas (Louisiana State University) for the instructions on mouse testes dissection and to Keisuke Shoji (The University of Tokyo) and the members of Kirino lab for helpful discussions This study was supported by the National Institutes of Health grant (GM106047 American Cancer Society Research Scholar Grant (RSG-17-059-01-RMC Grant-in-Aid for Scientific Research on Innovative Areas (26113007 to Y.T.) from MEXT Grant-in-Aid for Scientific Research (S) (18H05271 to Y.T.) from JSPS Grant-in-Aid for Scientific Research on Innovative Areas (17H06431 to S.K JSPS Postdoctoral Fellowship for Research Abroad (to M.S and Grant-in-Aid for Scientific Research (C) (grant 19K06484 to N.I.) from JSPS This research utilized the MetaOmics Core Facility and Bioimaging Facility at Sidney Kimmel Cancer Center in Thomas Jefferson University and was supported by the National Institutes of Health grant (P30CA056036) These authors contributed equally: Megumi Shigematsu Department of Agricultural and Environmental Biology Graduate School of Agricultural and Life Sciences designed the study with contributions from all other authors found BmRNase κ and performed a Trimming assay Kawamura performed experiments on BmRNase κ expression and KD in BmN4 cells and in vitro BmRNase κ cleavage assay prepared the paper with contributions from all other authors All authors have read and approved the final paper Peer review information Nature Communications thanks Katalin Fejes Tóth and other Download citation DOI: https://doi.org/10.1038/s41467-021-24681-w Metrics details and how pre-piRNAs are generated remains unclear we established a Trimmer-knockout silkworm cell line and derived a cell-free system that faithfully recapitulates Zucchini-mediated cleavage of PIWI-loaded pre-pre-piRNAs We found that pre-piRNAs are generated by parallel Zucchini-dependent and -independent mechanisms Cleavage by Zucchini occurs at previously unrecognized consensus motifs on pre-pre-piRNAs and is accompanied by 2′-O-methylation of pre-piRNAs slicing of pre-pre-piRNAs with weak Zucchini motifs is achieved by downstream complementary piRNAs producing pre-piRNAs without 2′-O-methylation Regardless of the endonucleolytic mechanism pre-piRNAs are matured by Trimmer and Hen1 Our findings highlight multiplexed processing of piRNA precursors that supports robust and flexible piRNA biogenesis The sequencing data reported in this paper are publicly available in DDBJ, under the accession number DRA008549 All other data are available from the authors upon reasonable request All code required for bioinformatics analysis in this paper is available at https://github.com/kshoji-nt/BmZuc_cleavage Identification and functional analysis of the pre-piRNA 3′ Trimmer in silkworms PNLDC1 is essential for piRNA 3′ end trimming and transposon silencing during spermatogenesis in mice An essential role for PNLDC1 in piRNA 3′ end trimming and male fertility in mice mediates 2′-O-methylation of Piwi-interacting RNAs at their 3′ ends couples piRNA amplification in Nuage to phased piRNA production on mitochondria Genetic and mechanistic diversity of piRNA 3′-end formation 3′ end formation of PIWI-interacting RNAs in vitro A genome-wide RNAi screen draws a genetic framework for transposon control and primary piRNA biogenesis in Drosophila Minotaur is critical for primary piRNA biogenesis Efficient chromosomal gene modification with CRISPR/Cas9 and PCR-based homologous recombination donors in cultured Drosophila cells CRISPR–Cas9-mediated knockout of factors in non-homologous end joining pathway enhances gene targeting in silkworm cells Characterization of Armitage and Yb containing granules and their relationship to nuage in ovary-derived cultured silkworm cell In vitro analysis of RNA interference in Drosophila melanogaster Improved Northern blot method for enhanced detection of small RNA Bias-minimized quantification of microRNA reveals widespread alternative processing and 3′ end modification High-quality genome assembly of the silkworm Structural basis for piRNA 2′-O-methylated 3′-end recognition by Piwi PAZ (Piwi/Argonaute/Zwille) domains Download references Tatsuke for providing BmArmi expression vectors Förstemann for providing hCas9 and sgRNA expression vectors Kiuchi for sharing unpublished data and helpful discussion A part of Illumina sequencing was performed in the Vincent J Coates Genomics Sequencing Laboratory at UC Berkeley supported by NIH S10 OD018174 Instrumentation Grant We also thank Life Science Editors for editorial assistance Zamore and members of the Tomari laboratory for critical comments on the manuscript This work was in part supported by a Grant-in-Aids for Scientific Research on Innovative Areas (grant 26113007 to Y.T.) from the Ministry of Education Science and Technology in Japan and a Grant-in-Aid for Scientific Research (S) (grant 18H05271 to Y.T.) Grant-in-Aid for Scientific Research (B) (grant 16KT0064 to Y.S Grant-in-Aid for Scientific Research on Innovative Areas (grant 17H06431 to S.K.) Grant-in-Aid for Young Scientists (B) (grant 17K17673 to N.I.) Grant-in-Aid for Scientific Research (C) (grant 19K06484 to N.I.) and a Grant-in-Aid for JSPS Fellows (grant 17J02408 to K.S.) These authors contributed equally: Natsuko Izumi Department of Agrobiology and Bioresources Department of Computational Biology and Medical Sciences Peer review information Nature thanks René Ketting and the other These two piRNA biogenesis pathways lead to target-dependent amplification of piRNAs (via the ping-pong cycle) and expansion of piRNA sequences (via trailing piRNA production) Schematic representation of the domain structure of Trimmer and the position of the sgRNA target site for CRISPR–Cas9 Genomic PCR of a region including the sgRNA target site two additional PCR products (i and iii) were detected only in Tri-KO#4 cells Genome sequences around the sgRNA target site in naive or Tri-KO#4 BmN4 cells Genomic sequencing revealed various mutations at the sgRNA target site suggesting a polyploid nature of the trimmer locus and/or imperfect cell cloning Western blot analysis of Trimmer in two different Tri-KO cell lines (#4 and #6) Western blot analysis of whole-cell lysate from naive or Tri-KO#4 BmN4 cells In vitro trimming assay for Siwi-loaded 1U50 RNA using 1,000g ppt from naive or two different Tri-KO cell lines SYBR Gold staining of total RNAs from naive or three different Tri-KO cell lines (#3 Total RNAs extracted from Tri-KO #4 cells overexpressing wild-type Trimmer (WT) or its catalytic mutant E30A (EA) were 5′ radiolabelled and detected by phosphor imaging Mock indicates transfection of a control plasmid Trimmer expression was analysed by western blotting (upper) Length distribution of small RNAs mapped to 3,236 piRNA loci in NaIO4-treated small RNA library from naive or Tri-KO BmN4 cells Relative fraction of 2′-O-methylated Tri-KO small RNAs in each length Peak length distribution of piRNAs mapped to 3,236 piRNA loci in the NaIO4-treated library from naive BmN4 cells Changes by the depletion of BmZuc in the length distribution of Type-N (lower) or Type-E (upper) NaIO4-treated small RNAs in Tri-KO cells Scatter plot showing normalized piRNA abundance co-immunoprecipitated with Siwi or BmAgo3 from naive BmN4 cells for each piRNA loci Siwi-dominant piRNA loci (n = 1,946); purple dots Peak length frequency of Tri-KO small RNAs for Siwi-dominant (left) or BmAgo3-dominant (right) piRNA loci Length distribution of Tri-KO small RNAs bearing the peak length of 35 or 36 nt (type-E) for Siwi-dominant (upper) or BmAgo3-dominant (lower) piRNA loci BmZuc knockdown abolished small RNAs with the peak lengths Siwi-dominant type-E pre-piRNAs show a stronger +1U preference than BmAgo3-dominant ones Siwi-dominant type-E pre-piRNAs show a greater tendency to have downstream trailing piRNAs than BmAgo3-dominant ones The 5′ ends of piRNAs mapped to 20−100 nt downstream of type-N (top) or type-E (bottom) Tri-KO small RNAs were mapped on the antisense strand separately for Siwi-dominant (left) and BmAgo3-dominant (right) piRNA loci Type-N piRNAs have more antisense piRNAs at ~41−52 nt from the 5′ ends than type-E piRNAs regardless of which PIWI protein they bind (two-sided Wilcoxon signed rank test Four representative type-E (piRNA-1528 and 66) and type-N (piRNA-2986 and 304) piRNA loci and their downstream genomic regions were mapped with the 5′ ends of sense (grey) and antisense (red) piRNAs Distribution of type-E and type-N piRNAs mapped to a transposon called MER85 An example of mixed modes of pre-piRNA production Pre-pre-piRNA-1249 contains a BmZuc cleavage site and a slicing site by an antisense piRNA-loaded PIWI protein which is possibly produced by another antisense piRNA-guided slicing locates in an unannotated genomic region and cannot be assigned Detailed protocol for in vitro recapitulation of BmZuc-mediated cleavage of Siwi-loaded pre-pre-piRNAs Siwi-loaded 28–80U RNA was incubated with Tri-KO 1,000g ppt with or without ATP and the ATP-regeneration system Siwi-loaded 111750 RNA was incubated with Tri-KO 1,000g ppt Mock indicates RNAi against Renilla luciferase (in c−e) Confocal images of BmN4 cells stably expressing GFP-tagged BmArmi in the presence or absence of BmGasz (scale bars Quantitative real-time PCR analysis of BmArmi and BmGPAT1 Tri-KO cells were depleted of BmGPAT1 or BmGasz by RNAi and the mRNA levels for BmArmi or BmGPAT1 were analysed by real-time PCR The graph shows the average of two independent experiments Siwi-loaded 1U50 RNA was incubated with Tri-KO 1,000g ppt 1U50 RNA was cleaved multiple sites within a region that is devoid of U in a manner dependent on the BmZuc activity Peak length distribution of small RNAs derived from the randomized sequence library The top 6 nucleotides in the BmZuc motif are shown in colour and their mutations are shown in black Siwi-loaded 111750-derived RNAs were incubated with Tri-KO 1,000g ppt Each gel image was adjusted to equalize the loading signal Schematic representation of the randomized sequence library analysis for Siwi- or BmAgo3-loaded pre-piRNAs cleaved by BmZuc Peak length distribution of Siwi- or BmAgo3-bound 2′-O-methylated small RNAs derived from the corresponding randomized sequence library Nucleotide composition around the 3′ ends of mature piRNAs in naive BmN4 cells (right) or type-E pre-piRNAs in Tri-KO cells (left) separately analysed for Siwi-dominant (top) and BmAgo3-dominant (bottom) piRNA loci The 6 nucleotides in the BmZuc motif are highlighted Schematic explanation for the weighted BmZuc motif (top) and the calculation of the BmZuc score in the 17-nt sliding window analysis (bottom) Similarity scores with the weighted BmZuc motif (BmZuc score) for Siwi were calculated for 111750 RNA and their mutant sequences in sliding windows and plotted as in c Box plots show the maximum similarity scores with the weighted BmZuc motif for Siwi or BmAgo3 within the positions of 19−45 nt of Siwi-dominant (top) or BmAgo3-dominant (bottom) type-N or type-E piRNA loci or the shuffled control sequences (a pool of 3,236 species of 27-nt scrambled sequences that have the average nucleotide composition of the silkworm genome) Type-E piRNA loci have significantly higher BmZuc scores than the shuffled control sequences for both Siwi- and BmAgo3-dominant piRNAs (Mann–Whitney U test) This file contains Supplementary Discussion This file contains uncropped blot/gel images used in the figures and extended data figures Download citation DOI: https://doi.org/10.1038/s41586-020-1966-9 Nature Reviews Molecular Cell Biology (2025) Reproductive Biology and Endocrinology (2024) Metrics details The small RNA (sRNA) pathways identified in the model organism Caenorhabditis elegans are not widely conserved across nematodes the PIWI pathway and PIWI-interacting RNAs (piRNAs) are involved in regulating and silencing transposable elements (TE) in most animals but have been lost in nematodes outside of the C and little is known about how nematodes regulate TEs in the absence of the PIWI pathway we investigated the role of sRNAs in the Clade IV parasitic nematode Strongyloides ratti by comparing two genetically identical adult stages (the parasitic female and free-living female) We identified putative small-interfering RNAs microRNAs and tRNA-derived sRNA fragments that are differentially expressed between the two adult stages Two classes of sRNAs were predicted to regulate TE activity including (i) a parasite-associated class of 21–22 nt long sRNAs with a 5′ uridine (21-22Us) and a 5′ monophosphate and (ii) 27 nt long sRNAs with a 5′ guanine/adenine (27GAs) and a 5′ modification The 21-22Us show striking resemblance to the 21U PIWI-interacting RNAs found in C overlapping loci and physical clustering in the genome we have shown that an alternative class of sRNAs compensate for the loss of piRNAs and regulate TE activity in nematodes outside of Clade V It remains unclear how nematodes outside of Clade V compensate for the loss of piRNAs to regulate TE activity The absence of piRNAs in nematodes outside of Clade V leads us to consider if an alternative sRNA pathway can compensate for the loss of piRNAs and regulate TE activity in nematodes in other clades It is therefore essential that we study nematodes outside of the C including both parasitic and free-living species to better understand the diversity of sRNAs involved in regulating TEs and the role of sRNAs in parasitism ratti includes genetically identical free-living (FLF) and parasitic (PF) adult female stages Direct comparison between these two adult stages can uncover genetic features associated with parasitism including differences in sRNA and TE activity only females exist and reproduce via parthenogenesis whereas the free-living generation reproduces via sexual mating between males and females A comparison of these two adult stages is therefore useful to understand how TE dynamics and regulation differ between sexual and asexual reproduction We have sequenced sRNAs that are expressed in PF and FLF of S We then classified the sRNAs into classes or subsets of classes of sRNAs and identified those differentially expressed between the PF and FLF We identified two classes of sRNAs that are predicted to target TEs that were differentially expressed between the two adult stages The sRNAs expressed by the parasitic stage shared multiple features in common with piRNAs including similar length (21–22 nt) physical clustering in the genome and an upstream AU-rich sequence; representing the first set of piRNA-like sRNAs outside of Clade V nematodes We also identified miRNA families more abundant in the parasitic stage and tRNA fragments expressed specifically in the free-living stage which indicates that specific sRNAs classes may be directly related to parasitism Understanding the mechanisms associated with these sRNAs can therefore help us understand parasitism and has the potential to lead to improved disease diagnostics and treatments sRNA classification and differential expression Classification of sRNAs expressed by parasitic female (PF) and free-living female (FLF) stages of S ratti including sRNAs (a) enriched for a 5′ monophosphate or (b) 5′ modification-independent sequences which includes sRNAs with a 5′ monophosphate or polyphosphate modifications Graphs on the top show the classification of sRNAs as either miRNAs tRNA-derived sRNAs (tRFs) or as putative siRNAs originating from either protein-coding genes (CDS or intronic regions) intergenic regions or transposable elements (TE) Graphs below the x-axis show the proportion of the first 5′ nucleotide for each length of sRNA Results from two biological replicates of each condition are shown in the figure (c) Differential expression of sRNAs with a 5′ monophosphate (top) and 5′ modification-independent library (bottom) Differentially expressed sequences are highlighted in pink (FLF-overexpressed) and blue (PF-overexpressed) (FDR of < 0.01 fold change > 2) and sequences that are not differentially expressed are shown in black (logRPM = log reads per million these results indicate that distinct sets of sRNAs are overexpressed in the PF and the FLF suggesting they have specific roles in these life cycle stages were found in the 5′pN enriched library but not the 5′ modification-independent library highlighting the importance of using multiple library preparation methods to investigate sRNA expression 21–22Us with a 5′ monophosphate associated with parasitism (a) Length distribution and 5′ starting nucleotide of 13–30 nt sRNAs with a 5' modification originating from either protein-coding genes intergenic spaces or transposable elements that are significantly more abundant in the PF (n = 6506 sequences) FLF (n = 1945 sequences) or not DE (n = 3549 sequences) (b) Predicted targets of significantly more abundant 21–22Us in the PF FLF and non-DE based on antisense sequence complementarity (c) Classification of predicted TE targets of 21–22Us in the PF Targets include TcMar-Mariner DNA transposon (48.5%) LTR retrotransposons (10.8%) and an unclassified family (38.6%) (d) Enriched GO terms based on the biological function (BF) of the 21–22U predicted target genes Clustering of the GO terms based on similar functions was carried out using REVIGO Size of circle indicates the number of genes and colour indicates p-value (e) Difference in expression level (RPKM: FLF minus PF) of genes putatively targeted by PF-overexpressed 21–22Us The targeted genes were more highly expressed in the FLF cf (f) Expression of TEs either predicted to be targeted or not targeted by PF-overexpressed 21-22Us RPKM = Reads per kilobase million; TPM = Transcripts per kilobase million indicating that the PF-targeted TEs are being regulated to have a similar expression to the FLF Chromosomal distribution and overlapping patterns of 21-22Us is similar to C (a) Distribution of overexpressed 21–22Us across the genome which consists of two autosomes (11.7 Mb chromosomes I and 16.7 Mb chromosome II) and the chromosome X (12.9 Mb made up of 10 scaffolds) (b) Distribution of 21–22U predicted TE targets across the genome (n = 20 TE targets) (c) Identification of an overlap signature in the 21–22Us Figure showing an example of a 21U originating from chromosome X and all overlapping 21–22U sequences Bottom left figure showing the number of 21–22Us that overlap with other 21-22Us (88.78%) Bottom right figure showing the overlap lengths of 21–22Us that overlap with other 21-22U sequences in the genome (d) Sequence logos of PF 21–22U upstream sequences versus the FLF 21–22Us to identify nucleotide richness based on the bits of each nucleotide (e) No dicer-processing signature in PF 21–22Us based on the predicted overhang of the passenger strand Passenger strands cleaved by the enzyme Dicer Pie chart shows the percentage of passenger strands a piRNA-associated motif was not found upstream of the S 27GA 5′-polyphosphate sRNAs overexpressed at the two adult stages sRNA sequences found in a 5pN library were filtered out from sRNAs identified in the 5′ modification-independent library (a) Length distribution and 5′ starting nucleotide of 13–30 nt sRNAs with a 5′ modification overexpressed in the PF (n = 288 sequences) FLF (n = 115 sequences) or those not DE (n = 26,816) 27GAs are the most abundantly expressed sequence (b) Putative targets of the 27GAs in the PF 5′UTR and 3′UTR) and TEs were predicted based on antisense sequence complementarity (c) Classes of TE antisense to PF-overexpressed (d) Enriched GO terms of the genes predicted to be targeted by PF-overexpressed 27GAs (left) or FLF-overexpressed 27GAs (right) Clustering of each GO term (biological function) based on similar functions was carried out using REVIGO (e) Predicted protein function of putative target genes and the number (n) of 27GAs (f) Difference in expression level (RPKM: FLF minus PF) of genes putatively targeted by PF-overexpressed demonstrating the diversity in TE expression levels Chromosomal distribution of overexpressed 27GAs in the PF (n = 193 sequences) FLF (n = 52 sequences) and non-DE (n = 14,047 sequences) within I (11.7 Mb) Differential expression of (a) miRNA sequences showing miRNAs significantly overexpressed in the PF (n = 12 sequences) FLF (n = 9 sequences) (FDR < 0.01) and (b) seed sequences overexpressed in the PF (n = 23) and FLF (n = 17) miRNAs with the seed sequence UUGCGAC were predominately overexpressed in the PF The seed families that were differentially expressed comprised between 1 and 21 miRNA sequences and included both miRNA families conserved across other species and uncharacterised seed families The miRNAs in the seed family with the most members (seed sequence UUGCGAC) were predominantly overexpressed in the PF and may therefore target a specific set of mRNAs important in parasitism The UUGCGAC miRNA family was not found in seven other nematode species where data was available on miRBase (Ascaris suum Pristionchus pacificus and Panagrellus redivivus) indicating that it is likely to be a Strongyloides-specific family We have investigated sRNAs in the gastrointestinal parasite S ratti by directly comparing sRNAs expressed in genetically identical PF and FLF stages We have identified two distinct classes of sRNAs predicted to target TEs including a (i) piRNA-like 21–22Us with a 5′pN which have distinct subsets of sequences overexpressed in the PF and FLF We have also identified miRNA sequences and miRNA families based on their seed sequence that are differentially expressed in the PF and FLF We propose that sRNAs expressed at higher levels in the PF are either directly related to parasitism or related to a feature associated with the parasitic generation like parthenogenetic reproduction then it is likely that 21–22Us could be acting to repress the expression of a subset of highly expressed TEs back to the ‘normal’ levels observed in FLF and non-targeted TEs It is important to note here that the analysis of TE transcript activity was based on polyA-selected RNAseq data and therefore is only informative about polyadenylated TEs e.g retrotransposons with a polyA sequence and the transposase component of DNA transposons ratti 21-22Us also predominantly target DNA transposons ratti 21–22Us may be the equivalent to the C elegans piRNAs and processed through a similar manner Although we have shown that 21–22Us originate from clusters found on the X-chromosome some of these clusters are found in intergenic regions as well as predominately targeting TEs clustered on the X-chromosome through perfect antisense complementarity This indicates that 21–22Us may be derived from TE regions that are required to be regulated and silenced and we speculate that based on their expression patterns these could be associated with 21–22Us in S Further work is required to identify if these Argonaute proteins are associated with 21–22Us and to identify other components of this pathway There could therefore be a specific role of 27GAs in regulating histone modification that is specific to the PF stage showed no evidence for Dicer-processing further supporting that they are likely to be processed in a similar manner to C indicating that the RdRP pathway is active While this may partially explain the differences in expression levels it does not appear to be a reasonable explanation for the predominance of a particular sRNA at either stage With the results presented in this study and the presence of two genetically identical adult stages with different reproductive strategies ratti offers a particularly interesting study platform to investigate the role of TEs and sRNAs in reproduction sexual development and the regulation of sex-chromosomal genes TEs are fast evolving sequences subject to higher mutation rates compared with other sequences in the genome It is therefore important that a regulatory system can respond and adapt rapidly to variation in these sequences to accurately regulate active TEs In this study we have looked at a single time point in the PF and FLF stages The 27GAs more abundant in the PF or FLF represent a response to the TEs that are upregulated in that snapshot of time the TEs that are active in the genome at any one time may vary and the 27GAs would subsequently vary in response to these TE sequences Interestingly there are clearly distinct trends between the TEs and TE-associated genes presumably targeted by the PF and FLF and this could mean that particular classes or TE are more likely to be active in either stage The differentially expressed miRNAs may be involved in regulating these ‘parasitism genes’ and further lab-based approaches are required to investigate this which suggests that increased expression in the FLF may be due to the stressful conditions of the environment tRFs have not been well characterised and relatively little is known about this class of sRNA More work in this area is required to elucidate the role of tRFs The library preparation methods used here enrich for RNA molecules with a 5′ monophosphate which are likely to represent ‘true’ sRNAs we cannot rule out that some of the reads represent degraded products of tRNA or other longer RNA molecules This is the first report of sRNAs expressed in the PF stage of S Most parasitic nematodes do not have genetically identical parasitic and free-living adult stages ratti therefore offers an almost unique opportunity to identify sRNAs specifically associated with parasitism and to investigate sRNA-mediated targeting of TEs in a parasitic nematode We directly compared the sRNAs expressed in the PF and FLF and key findings from this work are an identification of a novel family of 21–22U piRNA-like sRNAs in the parasitic stage of Strongyloides and differential expression of 27GAs in the two adult stages These putative siRNAs originate from the X-chromosome and were predicted to target X-chromosome associated TEs TE-targeting sRNAs were particularly evident in the PF suggesting increased levels of TE regulatory activity associated with the parasitic stage Approximately 3000 iL3s in PBS were injected subcutaneously into rats rats were sacrificed on 6 days post infection (dpi) and small intestines were collected The small intestines were developed longitudinally washed twice with prewarmed (37 °C) PBS and incubated in PBS at 37 °C for 2 h to isolate PF PF were washed with PBS before proceeding RNA extraction faeces collected from infected animals (8–10 dpi) was incubated at 23 °C for 3 days using 2% agar plates PF and FLF were transferred individually to a new tube containing TRIzol (ThermoFisher) using a needle picker after quick wash by PBS The worm samples were snap frozen in liquid nitrogen and stored at − 80 °C until required Mature miRNA sequences available in the miRBase database (Release 21) for ten other nematode species (Ascaris suum Pangarellus redivivus) used as input for mature sequences from related species miRNA sequences were quantified using the quantifier.pl script from miRDeep2 An estimated signal-to-noise value of 10 was used as a cut-off value and only miRNAs scoring above this threshold were used for further analysis The resulting list of miRNA sequences was used for classification of miRNA sequences Differentially expressed TEs were identified using edgeR with thresholds of count per million (CPM) > 1 in at least 2 samples The protein-coding genes (CDS and introns were separated) ratti (downloaded from WormBase version WS277) ratti miRNA sequences (see above) were used as reference databases (using the option -refseq) Sequences were first filtered to remove low complexity reads and were then identified as miRNAs based on mature and precursor reference sequences Sequences that were not identified as miRNAs were aligned to other reference categories At this step if a sequence aligns in more than one reference category Sequences that were not classified into any of the categories outlined above were assigned as sRNAs from intergenic regions First 5′ nucleotide data for sRNA sequences was also obtained from Unitas Only reads with more than 2 CPM in at least two 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Genome Res. https://doi.org/10.1101/gr.849004 (2004) R: A Language and Environment for Statistical Computing NIH Image to ImageJ: 25 years of image analysis Download references Genome data analyses were partly performed using the DDBJ supercomputer system We thank Simo Sun for assistance and comments VLH was funded by an Elizabeth Blackwell Institute fellowship a Japanese Society for the Promotion of Science Fellowship (PE16024) and a Wellcome Trust Sir Henry Dale Fellowship (211227/Z/18/Z) the author has applied a CC BY public copyright licence to any Author Accepted Manuscript version arising from this submission MS was funded by a URSA University of Bath PhD studentship TK was funded by Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Numbers 19H03212 and 17KT0013 These authors contributed equally: Mona Suleiman and Asuka Kounosu wrote the manuscript with inputs from others prepared biological samples and performed sequencing Download citation DOI: https://doi.org/10.1038/s41598-022-14247-1 Metrics details Schematic model of piRNA-guided de novo DNA methylation of transposons in mouse male germline in a time window from embryonic day 15.5 (E15.5) to postnatal day 3 (P3) MIWI2 engages in the ping–pong cycle with MILI leading to the initiation of effector piRNA production Loading of MIWI2 with resulting effector piRNAs induces its translocation into the nucleus The MIWI2/piRNA complex then targets young transposon RNA through high complementarity of base-pairing which subsequently licences the recruitment of SPOCD1 and the associated DNA methylation and chromatin remodeling machinery resulting in targeted DNA methylation of promotor elements upstream of transposon loci To explore the downstream mechanisms of how MIWI2/piRNA instructs de novo TE methylation first defined the interactome of MIWI2 in foetal gonocytes They generated a Miwi2HA allele by fusing the HA epitope tag to the N-terminus of endogenous fully functional MIWI2 and then used anti-HA immunoprecipitation coupled with quantitative mass spectrometry (IP–MS) to do proteomics analysis This approach identified 28 MIWI2-associated proteins To determine the nuclear proteins in directing de novo TE methylation expression pattern of the gene should be restricted to the period of de novo methylation the protein encoded by this gene should be located in the nucleus Only a single gene Spocd1 meets these two criteria These data indicated that a novel MIWI2 interactor SPOCD1 may be a promising candidate for the execution of nuclear MIWI2 function In order to decipher the potential function of SPOCD1 in the piRNA pathway authors engineered Spocd1 mutation (Spocd1−) in mice They observed that Spocd1−/− male were infertile and lacked spermatozoa in the epididymis The Spocd1−/− testes were substantially smaller than their wild-type counterparts the expression of long interspersed nuclear element-1 (LINE1) and intracisternal A-particle (IAP) which are normally silenced in adult wild-type testes can be detected in the testes of adult Spocd1−/− mutant a marker of the persistence and strength of the DNA damage response revealed the extensive unrepaired double-stranded breaks in Spocd1−/− meiocytes these phenotypes implied that SPOCD1 is required for spermatogenesis and transposon repression and are consistent with the ones caused by the loss of piRNA pathway activity piRNA pathway contributed to establish genomic methylation patterns on IAP and LINE1 elements.5 Zoch et al next set out to determine whether SPOCD1 is essential for piRNA-directed de novo DNA methylation Whole genome methylation sequencing (Methyl-seq) were performed using genomic DNA from wildtype Demethylation was detected specifically in IAPEy and MMERVK10C as well as the young LINE1 families but not collective transposons in Spocd1−/− spermatogonia which was consistent with the demethylation pattern of Miwi2-deficiency Metaplots further indicated that loss of methylation occurs specifically at TE promoter elements in Spocd1−/− spermatogonia which corresponds to characteristics of piRNA-directed methylation SPOCD1 plays a role in piRNA-guided DNA methylation To further explore the exact role of SPOCD1 in piRNA pathway either in piRNA biogenesis or alternatively in downstream of MIWI2 function first analysed expression of small RNAs in Spocd1+/− and Spocd1−/− E16.5 foetal testes No major changes were detected in piRNA length distribution MIWI2 exhibited the normal localization in the nucleus in the absence of Spocd1 confirming that SPOCD1 has no effect on the piRNA processing and implying its role in the downstream of nuclear MIWI2 function An alternative nuclear function could be acting as a transcription factor essential for either transposon or gene expression RNA-seq data from E16.5 foetal gonocytes ruled out this possibility To further investigate how SPOCD1 contributes to methylation of TEs authors then performed anti-HA IP–MS from Spocd1HA/+ E16.5 foetal testes to dig out interactome of SPOCD1-HA Components of the de novo methylation machinery such as DNMT3L and DNMT3A as well as several components of the repressive chromatin remodeling NURD and BAF complexes were detected using the same stringent association criteria NURD and BAF components can also be detected in the MIWI2 IP probably by recruiting DNA methyltransferases (DNMT3L and DNMT3A) and chromatin remodeling complexes (NURD and BAF) a novel nuclear protein SPOCD1 serves as an executor for MIWI2/piRNA-mediated de novo DNA methylation of mammalian young transposons This work will advance our basic understanding of piRNA function in fertility and epigenetic inheritance and may guide our understanding of how piRNA pathway directs DNA methylation of TEs in human beings Specification and epigenetic programming of the human germ line Download references This work was supported by the Startup Funding to E.Z.S Key Laboratory of Growth Control and Translational Research of Zhejiang Province Download citation DOI: https://doi.org/10.1038/s41392-020-00294-5