**(NOTE: This acquisition closed on February 28 KÖNGEN, Germany; Feb. 7, 2025 – Accenture (NYSE: ACN) has agreed to acquire Staufen AG a Germany-based management consulting firm The acquisition will expand Accenture’s capabilities to drive operational excellence and competitiveness in manufacturing and supply chains particularly for clients in discrete manufacturing industries including automotive Manufacturers are under pressure to mitigate supply chain disruptions geopolitical tensions and fluctuating tariffs while staying abreast of rapid technological advances Staufen brings deep operational excellence expertise to clients helping them optimize their entire value chains drive value with digital manufacturing initiatives and improve overall businesses performance The company’s service portfolio includes solutions for Industry 4.0 supply chain management and organizational change as well as data-driven tools continuous improvement techniques and lean management principles Its comprehensive approach enhances clients’ product design eliminating inefficiencies and optimizing production capacity Staufen also fosters talent and leadership growth among industry professionals through its academy Staufen improved an automotive supplier’s shopfloor management logistics and quality control processes more agile and efficient Its hands-on approach fostered a culture of collaboration among the workforce leading to better operational performance and financial results organizations need solutions that address every critical aspect of their operations from rising production costs to supply chain complexities The need for operational excellence is greater than ever Adding Staufen will help us unlock the full value of manufacturing and supply chain operations.” added: “We optimize our client’s entire value chains which is essential in today’s fierce competition disruptive innovations and global challenges working directly in our clients’ offices and factories to drive true change Joining Accenture will enable us to bring our three decades of hands-on expertise to even more clients to help them navigate complex transitions with customized digital solutions.” With more than 200 professionals in Germany, Italy, Switzerland, the U.S., Mexico, and Brazil, Staufen serves clients from mid-market companies to blue-chip organizations. The team will join Accenture’s supply chain and operations practice The terms of the acquisition were not disclosed and the transaction is subject to customary closing conditions 1 This does not include Staufen’s Chinese entities and Staufen.ValueStreamer GmbH Forward-Looking StatementsExcept for the historical information and discussions contained herein statements in this news release may constitute forward-looking statements within the meaning of the Private Securities Litigation Reform Act of 1995 Words such as “may,” “will,” “should,” “likely,” “anticipates,” “aspires,” “expects,” “intends,” “plans,” “projects,” “believes,” “estimates,” “positioned,” “outlook,” “goal,” “target” and similar expressions are used to identify these forward-looking statements These statements are not guarantees of future performance nor promises that goals or targets will be met uncertainties and other factors that are difficult to predict and could cause actual results to differ materially from those expressed or implied risks that: Accenture and Staufen AG will not be able to close the transaction in the time period anticipated which is dependent on the parties’ ability to satisfy certain closing conditions; the transaction might not achieve the anticipated benefits for Accenture; Accenture’s results of operations have been negative or uncertain economic and geopolitical conditions and the effects of these conditions on the company’s clients’ businesses and levels of business activity; Accenture’s business depends on generating and maintaining client demand for the company’s services and solutions including through the adaptation and expansion of its services and solutions in response to ongoing changes in technology and offerings and a significant reduction in such demand or an inability to respond to the evolving technological environment could materially affect the company’s results of operations; risks and uncertainties related to the development and use of AI could harm the company’s business damage its reputation or give rise to legal or regulatory action; if Accenture is unable to match people and their skills with client demand around the world and attract and retain professionals with strong leadership skills the utilization rate of the company’s professionals and the company’s results of operations may be materially adversely affected; Accenture faces legal reputational and financial risks from any failure to protect client and/or company data from security incidents or cyberattacks; the markets in which Accenture operates are highly competitive and Accenture might not be able to compete effectively; Accenture’s ability to attract and retain business and employees may depend on its reputation in the marketplace; if Accenture does not successfully manage and develop its relationships with key ecosystem partners or fails to anticipate and establish new alliances in new technologies the company’s results of operations could be adversely affected; Accenture’s profitability could materially suffer due to pricing pressure if the company is unable to remain competitive if its cost-management strategies are unsuccessful or if it experiences delivery inefficiencies or fail to satisfy certain agreed-upon targets or specific service levels; changes in Accenture’s level of taxes or changes in tax laws or in their interpretation or enforcement could have a material adverse effect on the company’s effective tax rate the company is more susceptible to certain risks; if Accenture is unable to manage the organizational challenges associated with its size the company might be unable to achieve its business objectives; Accenture might not be successful at acquiring its business could be adversely affected; Accenture may be subject to criticism and negative publicity related to its incorporation in Ireland; as well as the risks uncertainties and other factors discussed under the “Risk Factors” heading in Accenture plc’s most recent Annual Report on Form 10-K and other documents filed with or furnished to the Securities and Exchange Commission Statements in this news release speak only as of the date they were made and Accenture undertakes no duty to update any forward-looking statements made in this news release or to conform such statements to actual results or changes in Accenture’s expectations Contacts:Sumedha MahoreyAccenture+91 9820192152sumedha.mahorey@accenture.comLeonie AmtenbrinkAccenture+49 152 52507129leonie.amtenbrink@accenture.comCopyright ©2025 Accenture Accenture and its logo are registered trademarks of Accenture Deal adds ability for Accenture to drive competitiveness in clients’ manufacturing and supply chains AI PoweredThe consulting firm Accenture has acquired Staufen AG saying the move will expand Accenture’s capabilities to drive operational excellence and competitiveness in manufacturing and supply chains adding Staufen will help Accenture serve clients in discrete manufacturing industries including automotive According to Accenture it made the deal because manufacturers are under pressure to mitigate supply chain disruptions and fluctuating tariffs while staying abreast of rapid technological advances Staufen brings expertise in helping clients optimize their entire value chains Staufen’s service portfolio includes solutions for Industry 4.0 and organizational change as well as data-driven tools Its approach enhances clients’ product design “Manufacturers must continuously improve their entire value chains to stay competitive,” Matthias Hégelé Accenture’s supply chain and operations lead for Germany “The acquisition of Staufen aligns with our strategy to reinvent supply chains and manufacturing for clients We will combine Staufen’s proven expertise in operational excellence and value chain transformation with our capabilities in digital technologies digital twins and supply chain and manufacturing software platforms to help clients transform their core value chains In a move to meet rising demand for AI transformation, Accenture and Microsoft are launching a copilot business transformation practice to help organizations reinvent their business functions with both generative and agentic AI and with Copilot technologies The practice consists of 5,000 professionals from Accenture and from Avanade—the consulting firm’s joint venture with Microsoft. They will be supported by Microsoft product specialists who will work closely with the Accenture Center for Advanced AI that group will collaborate on AI and Copilot agent templates and connectors to help organizations leverage their data and gen AI to reduce costs Accenture and Avanade say they have already developed some AI tools for these applications a supplier discovery and risk agent can deliver real-time market insights which could result in up to 15% cost savings And a procure-to-pay agent could improve efficiency by up to 40% and enhance vendor relations and satisfaction by addressing urgent payment requirements and avoiding disruptions of key services Likewise, they have also built solutions for clients using Microsoft 365 Copilot technology they have created Copilots for a variety of industries and functions including finance Another part of the new practice will be educating clients how to use the technology, using an “Azure Generative AI Engineer Nanodegree program” to teach users how to design and operationalize AI-driven applications on Azure The online classes will teach learners how to use AI models to solve real-world problems through automation “We are pleased to deepen our collaboration with Accenture to help our mutual customers develop AI-first business processes responsibly and securely while helping them drive market differentiation,” Judson Althoff executive vice president and chief commercial officer at Microsoft “By bringing together Copilots and human ambition paired with the autonomous capabilities of an agent we can accelerate AI transformation for organizations across industries and help them realize successful business outcomes through pragmatic innovation.” Consulting firm Accenture has taken another step to bulk up its supply chain advisory capabilities, announcing Monday that it has acquired Allitix a California-based consulting and technology company specializing in Anaplan solutions with capabilities across financial planning and analysis Anaplan is a Florida provider of corporate performance management (CPM) systems which it defines as enterprise cloud software that empowers organizations to see and lead better business outcomes by aligning their strategic objectives and resources Allitix provides tailored Anaplan-based solutions across finance with specific competencies in the manufacturing “Demand for connected enterprise planning is on the rise given its ability to unlock business value and spur total enterprise reinvention,” David Leckstein and agile approach to implementation complements our broader digital capabilities and further expands our ability to deliver integrated enterprise planning transformations for our clients that drive better but Accenture said that the acquisition adds 73 employees including over 60 Anaplan functional and technical professionals to Accenture Technology in North America with expertise across solution architecture The top three corporate development priorities in consulting firm PwC’s current strategy are climate the company said in remarks today at an Orlando user conference for IFS software the Swedish firm which has added dozens of AI applications to its cloud-based enterprise resource planning (ERP) tools in recent months that firm said at its "IFS Unleashed" event in Orlando And underlying the industry’s rush to AI is the growing availability of massive amounts of data PwC analyst Matthew Duffy said in a session at the show data drives all the major technology changes that PwC advises businesses to examine: subscription or as-a-service models and conversions between business to business (B2B0 and business to consumer (B2C) approaches “Data availability now is greater than it’s ever been and that’s where AI comes into play,” Duffy said “It’s not just about driving cost efficiencies in an existing business model but understanding your customer and your customer’s customer so you can create a whole new value proposition.” In fact, he said that PwC is not just giving that advice to its clients but applying it to the firm’s own strategy as well. That can be seen in the firm’s move in recent years to build its “Connected Solutions” business unit. Consulting firm Accenture has acquires Boslan, a Spanish provider of management services for large infrastructure projects, saying the move will it to guide clients as they build net-zero infrastructure projects. Boslan helps its clients engineer and oversee the construction of infrastructure for the net-zero transition electric vehicle charging infrastructure and hydrogen plants It also supports the construction of data centers and critical infrastructure By applying artificial intelligence (AI) and other digital technologies to asset lifecycle management Accenture and Boslan will help clients optimize their project investments and become carbon-neutral faster “European companies in the energy and utilities sector are leading the charge in transitioning to renewable energy and ensuring low-carbon emission power is competitive,” Götz Erhardt “Boslan has helped pioneer many projects in this space Their engineering and industry expertise will bolster our capabilities to help clients in Europe and globally realize their net zero ambitions It will also grow our footprint as a global provider of infrastructure and capital projects services including digital and AI solutions for asset lifecycle management which are essential to the reinvention of infrastructure projects.” Accenture has been growing its Industry X arm through a series of acquisitions, including the Infor systems integrator Advoco in 2021 It is with sadness that we report on the passing last month of our friend and colleague Steve Geary Steve was a contributing editor to DC Velocity since its inception in 2003 as well as a columnist for Supply Chain Quarterly He regularly reported on supply chain trends and developments with particular expertise covering military and government logistics he additionally was president of Supply Chain Visions a leading consultancy in supply chain management and logistics Steve also served as an adjunct faculty member and researcher at the Haslam College of Business at the University of Tennessee Steve and his many contributions to the industry will certainly be missed In late 2024, Accenture and Nvidia announced a partnership focused on further developing genAI for the manufacturing industries This partnership between Accenture and Nvidia created the Nvidia Business Group designed to help manufaturers rapidly scale their AI adoption The two companies noted that this group will “help clients lay the foundation for agentic AI functionality using Accenture’s AI Refinery which uses the full Nvidia AI stack — including Nvidia AI Foundry Nvidia AI Enterprise and Nvidia Omniverse — to advance areas such as process reinvention and AI-powered simulation.” To further expand its manufacturing and supply chain expertise, Accenture has agreed to acquire Staufen AG The focus of this acquisition will be on the discrete manufacturing industries including automotive industrial goods and medical equipment.  The acquisition of Staufen marks the latest in a series of strategic investments Accenture has made related to its supply chain and operations capabilities including Joshua Tree Group and On Process Technology in the U.S. Staufen’s service portfolio includes technologies and services for Industry 4.0 Staufen improved an automotive supplier’s shopfloor management procesess Accenture noted that Staufen’s hands-on approach helped foster a culture of collaboration among the workforce Accenture’s supply chain and operations lead for Germany said: “We will combine Staufen’s proven expertise in operational excellence and value chain transformation with our capabilities in digital technologies autonomous systems.”     With more than 200 professionals in Germany Staufen’s team will join Accenture’s supply chain and operations practice.  Industrial automation insights from Accenture featured on Automation World: Thank you for downloading this report! You can view it at the link below:View PDFIf you can not access the report, please contact ariana.lynn@thefastmode.com Get updates and alertsdelivered to your inbox Outsourcing Calculator Roles Roles WebsiteThe Source NewsLatest outsourcing news in the BPO industry BPO DirectoryListing over 4000+ business process outsourcing companies ArticlesRead the best outsourcing articles that help businesses save up to 70% GuidesRead the best outsourcing guides that help businesses save up to 70% PodcastOutsourcing talk show that tackles one outsourcing topic at a time White PapersWide array of outsourcing white papers in the Philippines GlossaryThe most comprehensive list of BPO related terms and definition VideosGet an insiders look in to the outsourcing industry Get instant pricing for your offshore team Download the outsourcing toolkit – plus all the key resources you need List/claim your companyRegister or claim your BPO company page Submit Source articleSubmit a guest post article to 'The Source' Subscribe to Inside OutsourcingSubscribe to the leading outsourcing newsletter Submit press releaseSubmit a press release statement from your company Advertise with OAPromote your brand’s story Invite DG as keynote speakerInvite Derek Gallimore as a keynote and conference speaker See all servicesSee all partner services and media options The complete outsourcing toolkit – plus all the key resources you need OA's Zoom room is open! Come chat with our outsourcing expert now. Join now 3,000 firms Independent Trusted Save up to 70% on staff Get 3 Quotes News » Accenture acquires Staufen AG to boost manufacturing excellence Liezel Once Copied URL Photo from AccentureKÖNGEN GERMANY — In a move to strengthen its manufacturing and supply chain capabilities Accenture has announced its acquisition of German consulting powerhouse Staufen AG for an undisclosed amount.  This acquisition marks an expansion of Accenture’s operational excellence portfolio particularly in discrete manufacturing sectors Accenture a global professional services company with revenues exceeding $60 billion and a workforce of over 700,000 people aims to leverage Staufen’s expertise to address mounting challenges in manufacturing and supply chain operations.  The acquisition brings together Staufen’s specialized knowledge in operational excellence with Accenture’s advanced digital capabilities including AI and digital twin technologies brings more than 200 professionals across six countries – Germany The company has built a strong reputation for its hands-on approach to value chain optimization and digital manufacturing initiatives “The need for operational excellence is greater than ever, particularly in Germany, home to top manufacturers. Adding Staufen will help us unlock the full value of manufacturing and supply chain operations,” says Christina Raab Wilhelm Goschy, Staufen AG’s CEO, emphasizes their practical approach: “We optimize our client’s entire value chains The acquisition particularly strengthens Accenture’s position in discrete manufacturing industries Staufen’s comprehensive service portfolio encompasses Industry 4.0 solutions complemented by its professional academy for leadership development This latest move reinforces Accenture’s commitment to building resilient autonomous systems while supporting sustainable manufacturing practices an objective index of the world’s 500 top outsourcing companies Accenture delivered a solid performance in the first quarter of fiscal 2025, reporting revenues of $17.7 billion and 8% in local currency compared to the same quarter last year.  Outsource Accelerator is the leading Business Process Outsourcing (BPO) marketplace globally independent resource for businesses of all sizes to explore and embed outsourcing into their operations the platform covers all major outsourcing destinations “The BPO industry has historically been resilient during crises "Outsourcing is fundamentally countercyclical The industry can do well in recessions and depressions," Gallimore said.” “Outsource Accelerator has developed an objective methodology ranking the top 500 outsourcing firms globally and tracking firms across the $250 billion global outsourcing industry.” “If outsourcing will soon be the way of the world this book may help many get through it successfully A book of useful information for a changing employment landscape.” “These are challenges Derek Gallimore chief executive officer of Outsource Accelerator has been hearing about since day one of the pandemic His Manila-based firm helps companies build and operate their offshore teams.” “The best ways to free up your time involve regulating better workplace productivity and finding different strategies to efficiently get work done here are several steps you can take.” “The Great Resignation is unavoidable and it is still happening as employees continue to look for job opportunities with greater working conditions From The Great Resignation to The Great Talent Reshuffle — the key here is to retain employees.” outsourcing is still often looked down upon or misunderstood but Derek Gallimore sees it creating a seismic shift of how and where companies employ their workers.” Outsource Accelerator is the trusted source of independent information advisory and expert implementation of Business Process Outsourcing (BPO) Outsource Accelerator offers the world’s leading aggregator marketplace for outsourcing It specifically provides the conduit between Philippines outsourcing suppliers and the businesses – clients – across the globe The Outsource Accelerator website has over 5,000 articles and a comprehensive directory with 4000+ BPO companies… all designed to make it easier for clients to learn about – and engage with – outsourcing Derek Gallimore has been in business for 20 years and has been living in Manila (the heart of global outsourcing) since 2014 Derek is the founder and CEO of Outsource Accelerator and is regarded as a leading expert on all things outsourcing “Excellent service for outsourcing advice and expertise for my business.” Learn more Get 3 Free Quotes Verified Outsourcing Suppliers 3,000 firms.Just 2 minutes to complete. Get Started SAVE UP TO 70% ON STAFF COSTS Learn more Connect with over 3,000 outsourcing services providers Get 3 Quotes Transform your business with skilled offshore talent 3,000 firms Simple Transparent Book a call Explore outsourcing today Outsourcing Calculator Explore prices The Source Accenture plc: No escape from the economic climate Monday - Friday 9am-12pm / 2pm-6pm GMT + 1 All financial news and data tailored to specific country editions Add CommentACNAccenture PLC$303.50-0.60%Stock Score Locked: Want to See it?Benzinga Rankings give you vital metrics on any stock – anytime Accenture plc ACN shares are trading relatively flat on Friday The tech behemoth reportedly announced it will be scrapping its global diversity and inclusion goals, marking a significant shift in its policies CEO Julie Sweet informed staff in a memo that the company will begin “sunsetting” its diversity goals set in 2017, along with career development programs aimed at specific demographic groups, Financial Times reports This move follows an evaluation of the company’s internal policies in light of the evolving political climate in the U.S. including recent Executive Orders that Accenture must comply with in reevaluating or abandoning their D&I targets since the election of former President Donald Trump Also Read: Applovin Emerges As Nasdaq 100’s Dark Horse In A Market Fixated On Nvidia, Tesla, Palantir Join Plus500 today and get up to $200 to start trading real futures then jump into live markets with lightning-fast execution In addition to this shift in diversity policy, Accenture has announced its acquisition of Staufen AG, a German management consulting firm specializing in manufacturing and supply chains The deal will expand Accenture’s capabilities particularly for clients in industries such as automotive allowing the company to drive operational excellence and improve competitiveness in discrete manufacturing sectors According to Benzinga Pro, ACN stock has gained 6% in the past year. Investors can gain exposure to the stock via Trust for Professional Managers Jensen Quality Growth ETF JGRW. Price Action: ACN shares are trading lower by 0.19% to $386.60 at last check Friday. Stock Score Locked: Want to See it?Benzinga Rankings give you vital metrics on any stock – anytime Benzinga does not provide investment advice free reports and breaking news that affects the stocks you care about and trade ideas delivered to your inbox every weekday before and after the market closes Metrics details The mammalian Staufen proteins (Stau1 and Stau2) mediate degradation of mRNA containing complex secondary structures in their 3’-untranslated region (UTR) through a pathway known as Staufen-mediated mRNA decay (SMD) This pathway also involves the RNA helicase UPF1 which is best known for its role in the nonsense-mediated mRNA decay (NMD) pathway Here we present a biochemical reconstitution of the recruitment and activation of UPF1 in context of the SMD pathway a core NMD factor and a known activator of UPF1 UPF2 acts as an adaptor between Stau1 and UPF1 stimulates the catalytic activity of UPF1 and plays a central role in the formation of an SMD-competent mRNP Our study elucidates the molecular mechanisms of SMD and points towards extensive cross-talk between UPF1-mediated mRNA decay pathways in cells The asterisk (*) indicates contaminants in d The source data for this figure are provided as source data files we analyze the interactions between Stau1 and UPF1 in vitro with an aim to understand the mechanism of recruitment and activation of UPF1 in the SMD pathway We found that although Stau1 mediates direct interactions with UPF1 this is not sufficient to reconstitute an mRNP the binding of Stau1 does not stimulate the catalytic activity of UPF1 in vitro This raises the question of how UPF1 is recruited to an SBS-mRNA and activated in the SMD pathway Our biochemical reconstitution experiments suggest that the core NMD factor UPF2 plays an integral role in the SMD pathway We present here a mechanistic study that elucidates how UPF2 mediates the recruitment and activation of UPF1 in the context of SMD and highlights the role of UPF2 in facilitating mRNA decay in this pathway our results indicate that UPF2 acts as an adaptor between Stau1 and UPF1 and might play an important role in the recruitment of UPF1 to the SMD pathway The dsRBD3 domain of Stau1 is necessary for binding UPF2 although a strong interaction between UPF2 and Stau1 requires the presence of at least two such binding-competent dsRBDs Contaminants that co-purified with GST-UPF2 are indicated by asterisks (*) d Analytical size-exclusion chromatography (SEC) depicting stable complex formation between UPF1 and a Stau1 construct containing only the UPF2-binding site (dsRBD2-3-DD) The terms Abs and Vr denote the absorbance and retention volume of the proteins in this and all subsequent chromatograms The exclusion volume of the column is 0.8 mL The corresponding SDS-PAGE analysis of the peak fractions (indicated on the chromatogram) is shown on the right Formation of a stable Stau1-UPF complex is mediated by UPF2 The source data for this figure are provided as a source data file Although the dsRBD3 domain of Stau1 is essential for mediating its interaction with UPF2 it appears that the dsRBD4 domain might contribute additional binding sites to assemble a stable interaction platform Available X-ray crystal structures and distance restraints between pairwise cross-linked residues were used as inputs for prediction and inter-links are depicted as described above implying that binding of UPF2 to Stau1 does not affect its interaction with RNA Our results suggest that the topology of this ternary complex is compatible with RNA binding although certain rearrangements might occur upon interaction of the complex with RNA A pivotal role for UPF2 in the SMD pathway a GST-pulldown assays of Stau1 with GST-UPF1CH and GST-UPF2s in the absence and presence of dsRNA The dsRNA was partially labeled with 32P to enable its detection by autoradiography (lower panels of input and precipitate) proteins were detected by staining with Coomassie Brilliant Blue (CBB) The co-precipitation of dsRNA with UPF1 is significantly enhanced in the presence of UPF2 due to strong interactions between UPF2 and Stau1 in the presence of dsRNA b RNA-dependent ATPase of UPF1 in complex with UPF2 and Stau1 performed using an enzyme-coupled phosphate detection assay The ATPase activity of UPF1 in the presence of either UPF2 or Stau1 served as controls The data points and their error bars represent the mean values and standard deviation (s.d.) from three independent experiments c Quantitative (q) PCRs to determine levels of known ADAR1/Stau1 targets upon knockdown of SMD proteins in U2OS cells Target mRNA levels were normalized to that of the GAPDH transcript in every case The control siRNA refers to a scrambled sequence that does not specifically target any transcript Knockdown of UPF2 in combination with ADAR1 leads to increase in the levels of SMD target The ADAR1/Stau1 knockdown was performed as a positive control The data were obtained from duplicates of the indicated number of biological replicates with error bars denoting the standard deviation (s.d.) between the biological replicates Individual data points are represented as solid circles while the mean of each data series is represented as a column The differences in mRNA levels between the ADAR1 knockdown and the ADAR1/UPF2 or the ADAR1/Stau1 knockdown samples is significant as indicated by the p values obtained from unpaired t tests the activation of UPF1 in the presence of Stau1–UPF2 did not exceed that by UPF2 alone These observations underline the importance of UPF2 in assembling an SMD-competent mRNP in cells and highlight it as a major player of the SMD pathway UPF2 plays a key role in recruiting UPF1 to the SBS-mRNA and activating the helicase in the context of SMD Although our cross-linking mass spectrometric data suggest that the surface of the UPF2-MIF4G3 domain that contacts Stau1 is distinct from its UPF3-binding site it is possible that steric hindrances prevent UPF2 from simultaneously interacting with UPF3 and Stau1 We anticipate that the observed competition between the NMD and SMD pathways is not because of competition in binding to UPF1 but rather because of partial overlap in the UPF2-binding sites UPF2 is delivered or handed over from the translation termination complex to UPF3 which is bound to the exon-junction complex present downstream of the premature termination codon Since SMD also relies upon the presence of an upstream termination codon a similar hand-over mechanism can be envisaged for recruiting UPF2 to the Stau1-bound mRNA in this pathway Stau1 represents the first example of a dsRBD that is capable of concomitant binding to dsRNA and a protein factor Together with the aforementioned studies on dimerization and RNA binding our studies also highlight the scope of interactions mediated by dsRBDs and their versatility in cellular functions In summary, we have identified the NMD factor UPF2 as the pivotal component that is essential for recruiting the RNA helicase UPF1 to a Staufen-mRNP, activating it within this complex and mediating SMD (Fig. 6) Our findings highlight the mechanistic similarities between NMD and SMD and point to an extensive cross-talk between these and possibly other UPF1-dependent pathways in cells whether UPF2 is involved in every such pathway and how it might be recruited to these pathways remains a topic for further studies All human UPF1, UPF2, and Stau1 constructs used in this study were expressed as 6×-His, His-GST, or His-Thioredoxin (Trx) fusions (cleavable with TEV or 3C protease, please refer to Supplementary Table 1) in Escherichia coli BL21 (DE3) STAR pRARE cells at 18 °C for at least 15 h Cells expressing recombinant proteins were lysed using buffer A (20 mM Tris-HCl and 10% glycerol) supplemented either with 500 mM NaCl (for UPF1 and UPF2) or 1 M NaCl (for Stau1) The proteins were enriched from the crude lysate by Ni2+-affinity chromatography and eluted from the Ni2+-NTA resin using buffer A supplemented with an additional 240 mM imidazole and 150 mM NaCl (for UPF1 and UPF2) or 1 M NaCl (for Stau1) The affinity tags on UPF1 and UPF2 were not removed in any case except for His-GST-UPF2 (126–1227) where the N-terminal His-GST tag was removed by cleavage with TEV protease The His-Trx tags of the Stau1 constructs were cleaved by 3C protease during overnight dialysis at 4 °C against dialysis buffer (20 mM Tris-HCl All proteins were subjected to a further purification step using a HiTrap Heparin Sepharose HP column (GE Healthcare) GE Healthcare) was performed in buffer B (20 mM HEPES and 1 µM ZnCl2) as a final step of purification To purify a UPF1-UPF2 complex for analytical SEC assays equi-molar amounts of purified UPF1 and UPF2 were mixed and incubated at 4 °C for 16 h following which the mixture was injected on a Superdex 200 column in buffer B The UPF1-UPF2-Stau1 complex was formed by mixing the purified UPF1-UPF2 complex (described above) with Stau1 in a 1:1 molar ratio (considering Stau1 as a dimer) The protein mixture was incubated for 16 h at 4 °C in buffer B supplemented with 50 mM NaCl (final salt concentration of 200 mM NaCl) The resultant ternary complex was isolated by SEC (Superdex 200 column) in buffer B To check the presence of dsRNA in the input and precipitate samples the dsRNA was 5′ end labeled with [γ-32P]ATP using T4 polynucleotide kinase and a 1.2-fold molar excess of 28-mer dsRNA (of which approximately one-third consisted of the labeled dsRNA) was added to the protein mixture after the overnight incubation The reaction mixture was further incubated at 25 °C for 2 h Bound proteins and RNA were captured and eluted as described above One set of eluates were resolved by 7.5% SDS-PAGE and stained with Coomassie Brilliant Blue to visualize the proteins while the second set of eluates were resolved on a 20% Urea-PAGE and analyzed by autoradiography to detect the labeled dsRNA The unformatted gels for all GST-pulldown assays Seven hundred picomoles of the UPF1-UPF2 complex (described above) were mixed with 700 pmol of the dimeric Stau1 proteins (dsRBD2-3-DD and dsRBD2-3-4-dd) and with 1400 pmol of Stau1 dsRBD2-3-4 to a final volume of 40 µL in buffer A (supplemented with 50 mM NaCl) and incubated at 4 °C for 16 h The peak fractions were analyzed by SDS-PAGE followed by staining with Coomassie Brilliant Blue The end point of the ATPase reaction (20-min time-point) of the UPF1-UPF2 mixture was set to 100% and all other data points were normalized to this value The raw data for all ATPase assays are provided as a Source Data file U2OS cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Bio&SELL) and 100 μg/mL of streptomycin (Thermo Fisher Scientific) and UPF2 were procured from Thermo Fisher Scientific (see below) Twenty picomoles of each siRNA (alone or in combinations as indicated) were transfected into 0.15 × 106 U2OS cells using Lipofectamine 2000 (Thermo Fisher Scientific) Total RNA was extracted from U2OS cells 72 h post transfection using RNATri (Bio&SELL) Two hundred nanograms of total RNA was used for gene-specific complementary DNA synthesis by the MLV-reverse transcriptase (Qiagen) Quantitative PCRs (qPCRs) were performed using the PowerUp SYBR Green Master Mix (Thermo Fisher Scientific) on a Stratagene Mx3005P instrument The data represent the mean of duplicates of three biological replicates while the error bars denote the standard deviations Unpaired t tests were performed to calculate the p values with the significance threshold set at 0.05 The qPCR primers used in this study are described in Supplementary Table 2 The following siRNAs used in this study were obtained from Thermo Fisher Scientific: ADAR1 (catalog # S1008) The control siRNA had the following sequence: UUUGUAAUCGUCGAUA CCC-dTdT The U2OS and HEK 293T (used for co-IP assays see below) cell lines were a gift from Florian Heyd and were routinely tested for mycoplasma contamination in the course of the study Cell lines showed the expected morphology and were not authenticated by other methods The analysis was carried out with one sample each for the ternary and the UPF1-UPF2 binary complex (in one and three technical replicates for the ternary and binary complexes The modeling was performed in a sequential manner where cross-links between UPF1 and the UPF2-MIF4G3 were modeled first and the resultant output was used to map cross-links between Stau1 and UPF2-MIF4G3 The outputs with the lowest Haddock score were considered for analysis The final model was generated by superposing the UPF2-MIF4G3 domains from both runs HEK 293T cells were cultured in DMEM supplemented with 10% FBS (Bio&SELL) and Pen/Strep (Invitrogen) A total of 0.5 × 106 cells/ml were seeded in a 6-well plate format and HA-UPF2 were co-transfected using polyethyleneimine “Max” (Polysciences Inc. Transfected cells were incubated at 37 °C and harvested 48 h later Cells were lysed using 200 µL of RIPA buffer (10 mM Tris-HCl supplemented with protease inhibitors) and RNase A (final concentration of 60 µg/mL) Fifteen microliters of anti-Flag M2 resin (Merck catalog # A2220) was added to each cell lysate sample and incubated at 4 °C for 1.5 h Unbound proteins were washed with RIPA buffer and Flag-tagged baits and the associated proteins were eluted in non-reducing SDS sample buffer by incubating at 30 °C for 3 h The eluted samples were resolved by 10% SDS-PAGE and then transferred to a PVDF membrane for analysis by western blotting with mouse anti-HA (1:4000; Covance catalog # F1804) antibodies wherever indicated The secondary antibody horse anti-mouse horse radish peroxidase (1:4000; Cell Signaling Technologies catalog # 7076S) was used to enable detection of tagged proteins by chemiluminescence Further information on research design is available in the Nature Research Reporting Summary linked to this article The clothes make the mRNA: past and present trends in mRNP fashion Deciphering the mRNP code: RNA-bound determinants of post-transcriptional gene regulation Genome-wide analysis of Staufen-associated mRNAs identifies secondary structures that confer target specificity Staufen1 senses overall transcript secondary structure to regulate translation hiCLIP reveals the in vivo atlas of mRNA secondary structures recognized by Staufen 1 Distinct roles of two conserved Staufen domains in oskar mRNA localization and translation a gene required to localize maternal RNAs in the Drosophila egg mRNA–mRNA duplexes that autoelicit Staufen1-mediated mRNA decay Mammalian Staufen1 recruits Upf1 to specific mRNA 3’UTRs so as to elicit mRNA decay SMD and NMD are competitive pathways that contribute to myogenesis: effects on PAX3 and myogenin mRNAs Karousis, E. D. & Muhlemann, O. Nonsense-mediated mRNA decay begins where translation ends. Cold Spring Harb. Perspect. Biol. 11, https://doi.org/10.1101/cshperspect.a032862 (2019) Structural and functional insights into the human Upf1 helicase core NMD factors UPF2 and UPF3 bridge UPF1 to the exon junction complex and stimulate its RNA helicase activity Tight intramolecular regulation of the human Upf1 helicase by its N- and C-terminal domains Upf1 ATPase-dependent mRNP disassembly is required for completion of nonsense- mediated mRNA decay Molecular mechanisms for the RNA-dependent ATPase activity of Upf1 and its regulation by Upf2 Unusual bipartite mode of interaction between the nonsense-mediated decay factors Staufen2 functions in Staufen1-mediated mRNA decay by binding to itself and its paralog and promoting UPF1 helicase but not ATPase activity Structural and functional analysis of the three MIF4G domains of nonsense-mediated decay factor UPF2 Interaction between Nmd2p and Upf1p is required for activity but not for dominant-negative inhibition of the nonsense-mediated mRNA decay pathway in yeast A conserved double-stranded RNA-binding domain Mammalian staufen is a double-stranded-RNA- and tubulin-binding protein which localizes to the rough endoplasmic reticulum RNA recognition by a Staufen double-stranded RNA-binding domain Molecular mapping of the determinants involved in human Staufen-ribosome association The crystal structure of Staufen1 in complex with a physiological RNA sheds light on substrate selectivity Staufen1 dimerizes through a conserved motif and a degenerate dsRNA-binding domain to promote mRNA decay A conserved structural element in the RNA helicase UPF1 regulates its catalytic activity in an isoform-specific manner The structural basis for the interaction between nonsense-mediated mRNA decay factors UPF2 and UPF3 Human nonsense-mediated mRNA decay factor UPF2 interacts directly with eRF3 and the SURF complex and Upf3p are interacting components of the yeast nonsense-mediated mRNA decay pathway Identification and characterization of human orthologues to Saccharomyces cerevisiae Upf2 protein and Upf3 protein (Caenorhabditis elegans SMG-4) Structure of NF-kappa B p50 homodimer bound to a kappa B site The HADDOCK2.2 Web Server: user-friendly integrative modeling of biomolecular complexes ADAR1 controls apoptosis of stressed cells by inhibiting Staufen1-mediated mRNA decay ADAR1-mediated 3’ UTR editing and expression control of antiapoptosis genes fine-tunes cellular apoptosis response Emerging roles for ribonucleoprotein modification and remodeling in controlling RNA fate MOV10 Is a 5’ to 3’ RNA helicase contributing to UPF1 mRNA target degradation by translocation along 3’ UTRs Rarely at rest: RNA helicases and their busy contributions to RNA degradation The RNA helicase DHX34 functions as a scaffold for SMG1-mediated UPF1 phosphorylation The RNA exosome and proteasome: common principles of degradation control Regulated degradation of replication-dependent histone mRNAs requires both ATR and Upf1 Regnase-1 and Roquin regulate a common element in inflammatory mRNAs by spatiotemporally distinct mechanisms Upf1 senses 3’UTR length to potentiate mRNA decay and recycling phases of translation in eukaryotes Dual function of UPF3B in early and late translation termination Nuclear factor 90 uses an ADAR2-like binding mode to recognize specific bases in dsRNA Dimerization of ADAR2 is mediated by the double-stranded RNA binding domain A bimodular nuclear localization signal assembled via an extended double-stranded RNA-binding domain acts as an RNA-sensing signal for transportin 1 In-gel digestion for mass spectrometric characterization of proteins and proteomes A high-speed search engine pLink 2 with systematic evaluation for proteome-scale identification of cross-linked peptides xiNET: cross-link network maps with residue resolution an attenuator of the Hsp70 chaperone cycle Download references Elena Conti for some expression constructs of UPF1 and UPF2 and Gourisankar Ghosh and Christian Benda for the plasmids of the dimerization domains of mouse p50 and the Hsp70-interacting protein (Hip) We also thank Fabien Bonneau for help with ATPase assays and Marco Preußner and Florian Heyd for sharing their expertise on RT-qPCR assays We are grateful to the members of our laboratory and Markus Wahl for helpful discussions and for critical comments on the manuscript This study was supported by the Deutsche Forschungsgemeinschaft (CH1245/2-1 and CH1245/3-1 to S.C.) and by the Max Planck Gesellschaft (H.U.) are also supported by the DFG Priority Program SPP 1935 Present address: Max Planck Institute of Colloids and Interfaces Max Planck Institute for Biophysical Chemistry Bioanalytics Institute for Clinical Chemistry purified the proteins used in the study with assistance from S.K.K performed the biochemical analyses and the cell-based assays performed the cross-linking mass spectrometry analysis The authors declare no competing interests Peer review information Nature Communications thanks David Tollervey reviewer(s) for their contribution to the peer review of this work Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Download citation DOI: https://doi.org/10.1038/s41467-019-13080-x Anyone you share the following link with will be able to read this content: a shareable link is not currently available for this article Cellular and Molecular Life Sciences (2022) Sign up for the Nature Briefing newsletter — what matters in science A house in Staufen AG burned down on Wednesday evening one person has now been found dead in the rubble The dead person has not yet been identified said a media spokesperson for the Aargau cantonal police on request The "Blick" had first reported on this One member of the fire department was slightly injured during the extinguishing work All that could be seen at the scene was a "field of rubble" The fire was extinguished in the second half of the night The cause of the fire is being investigated The houses in the vicinity of the fire were temporarily evacuated the house was already fully engulfed in flames Several reports of explosions and the fire were received by the cantonal emergency call center at 8.15 p.m Around 100 firefighters were deployed in the Neudörfli residential area The investigation into the cause of the fire is ongoing Metrics details A Correction to this article was published on 19 January 2021 This article has been updated Staufen-1 (STAU1) is an RNA-binding protein that becomes highly overabundant in numerous neurodegenerative disease models including those carrying mutations in presenilin1 (PSEN1) We previously reported that elevations in STAU1 determine autophagy defects and its knockdown is protective in models of several neurodegenerative diseases Additional functional consequences of STAU1 overabundance We studied the role of STAU1 in the chronic activation of the unfolded protein response (UPR) a common feature among neurodegenerative diseases and often directly associated with neuronal death Here we report that STAU1 is a novel modulator of the UPR and is required for apoptosis induced by activation of the PERK–CHOP pathway STAU1 levels increased in response to multiple endoplasmic reticulum (ER) stressors and exogenous expression of STAU1 was sufficient to cause apoptosis through the PERK–CHOP pathway of the UPR Cortical neurons and skin fibroblasts derived from Stau1−/− mice showed reduced UPR and apoptosis when challenged with thapsigargin In fibroblasts from individuals with SCA2 or with ALS-causing TDP-43 and C9ORF72 mutations we found highly increased STAU1 and CHOP levels in basal conditions and STAU1 knockdown restored CHOP levels to normal these results show that STAU1 overabundance reduces cellular resistance to ER stress and precipitates apoptosis our study describes a novel connection between the RNA-granule protein STAU1 and ER-stress-induced apoptosis that can be targeted in neurological diseases their genetic mutation and repository identification number All mutations were verified by PCR sequencing Identity authentication of HEK293 cells and human fibroblasts was carried out by short tandem repeat analysis with the GenePrint 24 System (Promega USA) and mycoplasma testing was carried out regularly Cultures of cortical neurons were prepared from WT or Stau1−/− neonatal mice euthanized according to the IACUC approved protocols Cortices from 6 to 7 animals were isolated and incubated with 50 units of papain (Worthington Biochemical USA) in Earle’s balanced salt solution with 1.0 mM l-cysteine and 0.5 mM EDTA for 15 min at 37 °C Digested tissue was washed with EBSS and mechanical dissociation was performed with a 1 ml micropipette in the presence of 0.1 mg/ml of DNase1 (Sigma-Aldrich) Cell suspension was filtered through a 40 µm strainer (Corning) to remove any remaining aggregates Neurons were seeded on poly-l-ornithine (Sigma-Aldrich) and laminin coated plates at a density of 50,000 per cm2 in Neurobasal Plus medium containing 2% B27 Plus supplement To prevent proliferation of glial cells 1 µM cytosine arabinoside (Sigma-Aldrich) was added on day 2 and 90% of the media volume was changed after 24 h 60% of culture medium was replenished every 2–3 days Experiments were conducted on day 9–10 by replacing all culture media with fresh media containing thapsigargin or vehicle (DMSO) After incubation with the corresponding secondary antibody signal was detected using Immobilon Western Chemiluminescent HRP Substrate (EMD Millipore) or SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific) and photographed with a Bio-Rad ChemiDoc Analysis and quantification was performed with Image Lab software (Bio-Rad) Relative protein abundance was first normalized against actin band intensity and then expressed as the ratio to the normalized control Imaging was performed at the Fluorescence Microscopy Core Facility a part of the Health Sciences Cores at the University of Utah Gene expression was normalized to GAPDH levels and analyzed with the relative standard curve method Cytotoxicity was quantified with the CytoScanTM LDH Cytotoxicity Assay (G-Biosciences) WT and Stau1−/− fibroblasts were plated in 96-well plates and treated with thapsigargin for 24 h Cell culture supernatant was then collected and LDH was quantified following the manufacturer’s instructions for chemical compound induced cytotoxicity Results were normalized for each genotype against a maximum LDH release (cells incubated with lysis buffer) and a spontaneous LDH activity (untreated control cells) Cytotoxicity percentage was then calculated as ((compound treated − spontaneous LDH activity)/(maximum LDH release − spontaneous LDH activity)) × 100 All results are presented as mean ± standard error of the mean (SEM) unless noted otherwise Comparisons between groups were made using the Student’s t test in OriginPro 2017 software Levels of significance are noted as *p ≤ 0.05 a HEK293 cells were incubated with tunicamycin (0.1 and 0.5 µM) Levels of STAU1 and CHOP were evaluated by western blot Graph represents quantification of STAU1/actin from three independent experiments Single asterisk (*) or double asterisks (**) denote significantly different from untreated control b Relative STAU1 mRNA levels in HEK293 cells treated with thapsigargin (0.5 µM) for the times indicated c Levels of STAU1 protein in HEK293 cells treated with thapsigargin (0.5 µM) for the times indicated Graph represents quantification of STAU1/actin for three independent experiments *p < 0.05 and **p < 0.01 according to the paired sample t test Western blots of cultured cortical neurons (a) or skin fibroblasts (b) from WT or Stau1−/− mice incubated with thapsigargin (0.25 and 0.5 µM Graph represents quantification of target protein/actin from three independent experiments c Cytotoxicity assessment by quantification of LDH release in WT or Stau1−/− mouse fibroblasts exposed to indicated doses of thapsigargin for 24 h d Western blots of HEK293 cells transfected with siControl or siSTAU1 for 72 h and incubated with thapsigargin (0.5 and 1 µM) for 18 h e mRNA levels of CHOP and ATF4 in HEK293 72 h post transfection with siControl or siSTAU1 and f after treatment with thapsigargin (0.5 µM) for the times indicated or double daggers (††) denote significantly different from WT or siControl treated with the corresponding dose of thapsigargin To confirm that the role of STAU1 in ER-stress-induced apoptosis was general to ER stress and not specific to thapsigargin, we analyzed cells treated with tunicamycin or brefeldin A, which induce ER stress by vastly different mechanisms. We found that STAU1 knockdown also attenuated the UPR and apoptotosis (Supplementary Fig. 1a, b) indicating STAU1 has a role modulating life and death decisions when cells are faced with ER stress Assessment of cell death by quantification of LDH release caused by loss of plasma membrane integrity showed a 63.9% reduction in cytotoxicity in Stau1−/− fibroblasts when compared with WT (Fig. 2c) (20.9 ± 2.8 average cytotoxicity in WT vs 13.2 ± 3.2 in Stau1−/−) These results indicate that the difference in active caspase levels effectively translates to protection against cell death in Stau1−/− cells Their transcriptional induction after thapsigargin did not reach statistical significance (not shown) in agreement with previous reports showing their acute activity is regulated mainly posttranscriptionally ATF4 and CHOP transcription increased immediately after addition of thapsigargin and peaked at 4 h (Fig. 2f). In contrast, STAU1 protein and mRNA levels showed a delayed increase, only evident 4–8 h after addition of thapsigargin (Fig. 1b, c) The fact that silencing STAU1 decreased both basal and induced levels of ATF4 and CHOP mRNAs even before overabundance of STAU1 was evident suggests that baseline levels of STAU1 may play a role in the modulation of ATF4 and CHOP mRNA levels overabundance of STAU1 might not be necessary to mediate its proapoptotic effects our data show that STAU1 amplifies the activation of the UPR in a proapoptotic manner and knockdown or knockout of STAU1 is sufficient to prevent apoptosis during ER stress HEK293 cells were transfected with 3xFlag-STAU1 (3xF-STAU1) or empty vector control (3xF) with addition of siRNA directed at PERK (siPERK) after 24 h or the PERK inhibitor GSK2606414 (0.5 µM) after 48 h protein levels were analyzed by western blot Graphs represent the quantification of three independent experiments Single asterisk (*) or double asterisks (**) denote significantly different from 3xF control Single dagger (†) or double daggers (††) denote significantly different from same genotype control ∗∗ or ††p < 0.01 according to the paired sample t test These results indicate that phosphorylation of eIF2α is not required for apoptosis triggered by STAU1 a Western blot of proteins involved in the UPR and b caspase 3 and cleaved caspase 3 in ATXN2-Q22 and ATXN2-Q58 cells Graph represents quantification of three independent experiments c Western blot of fibroblasts derived from an SCA2 patient with ATXN2-Q45 mutation d Western blot of cerebellar tissue from WT ATXN2-Q127 mice and Stau1+/− haploinsufficient littermates at 34 weeks of age Graph represents quantification of three animals per genotype Single asterisk (*) or double asterisks (**) denote significantly different from ATXN2-Q22 or WT control ** or ††p < 0.01 according to the paired sample t test These results provide evidence that a proapoptotic signaling axis involving calcium alterations STAU1 and ER stress is active in this model of SCA2 and CHOP in fibroblasts derived from human subjects without disease-related mutation (normal) with TARDBP A382T (line 1) and one with TARDBP G298S (line 2) (b) and two individuals with C9ORF72 GGGGCC repeat expansion (lines 1 and 2) Single asterisk (*) or double asterisks (**) denote significantly different from control patient Absence of caspase 3 cleavage and lowered LDH release were consistent with resistance to ER-stress-induced apoptosis in STAU1 knockout and knockdown cells (Fig. 2a, b, c) Our results indicate that STAU1 overabundance increases cellular sensitivity to apoptosis as STAU1 overabundance increased both total caspase 3 and cleaved caspase 3 levels in baseline and stressed conditions whereas STAU1 knockout or knockdown decreased baseline transcript levels of CHOP As increased caspase 3 levels can decrease the apoptotic threshold when cells are exposed to stress lowering STAU1 could therefore constitute a strategy to increase resistance to proapoptotic stress by lowering total caspase level and transcripts of ATF4 the present work informs on the role of STAU1 in multiple diseases by showing that it is a key modulator of ER-stress-induced apoptosis STAU1 overabundance caused by ER stress or calcium alterations reduces cellular resistance to ER stress and precipitates apoptosis through the PERK–CHOP pathway By decreasing ER stress and reducing p-eIF2α targeting STAU1 could ameliorate proteostasis and aberrant SG phenotypes in diseases caused by ATXN2 and C9ORF72 mutations as well as other disease gene mutations or sporadic forms of neurodegenerative diseases Further understanding of the molecular mechanisms linking STAU1 and ER stress will provide insight needed to safely modulate death pathways for therapeutic benefit A Correction to this paper has been published: https://doi.org/10.1038/s41418-021-00734-x Paul S, Dansithong W, Gandelman M, Zu T, Ranum LPW, Figueroa KP, et al. Staufen blocks autophagy in neurodegeneration. https://doi.org/10.1101/659649 Staufen1 links RNA stress granules and autophagy in a model of neurodegeneration Systematic analysis of RNA regulatory network in rat brain after ischemic stroke Neuronal RNA granules: a link between RNA localization and stimulation-dependent translation A loss of function allele for murine Staufen1 leads to impairment of dendritic Staufen1-RNP delivery and dendritic spine morphogenesis Mammalian Staufen 1 is recruited to stress granules and impairs their assembly Staufen1-mediated mRNA decay functions in adipogenesis lncRNAs transactivate STAU1-mediated mRNA decay by duplexing with 3’ UTRs via Alu elements Interaction of Staufen1 with the 5’ end of mRNA facilitates translation of these RNAs The RNA-binding protein Staufen1 is increased in DM1 skeletal muscle and promotes alternative pre-mRNA splicing Hypoxia and ER stress promote Staufen1 expression through an alternative translation mechanism Targeting the unfolded protein response in disease Integrating the mechanisms of apoptosis induced by endoplasmic reticulum stress The unfolded protein response: from stress pathway to homeostatic regulation Cytoplasmic stress granules: dynamic modulators of cell signaling and disease Mechanistic insights into mammalian stress granule dynamics Genome engineering using the CRISPR-Cas9 system Extracellular ATP and the P2X7 receptor in astrocyte-mediated motor neuron death: implications for amyotrophic lateral sclerosis Changes in Purkinje cell firing and gene expression precede behavioral pathology in a mouse model of SCA2 P2X7 receptor-induced death of motor neurons by a peroxynitrite/FAS-dependent pathway Measuring ER stress and the unfolded protein response using mammalian tissue culture system Staufen1 promotes HCV replication by inhibiting protein kinase R and transporting viral RNA to the site of translation and replication in the cells Moderate expansion of a normally biallelic trinucleotide repeat in spinocerebellar ataxia type 2 Deranged calcium signaling and neurodegeneration in spinocerebellar ataxia type 2 Chronic suppression of inositol 1,4,5-triphosphate receptor-mediated calcium signaling in cerebellar purkinje cells alleviates pathological phenotype in spinocerebellar ataxia 2 mice Atxn2 knockout and CAG42-knock-in cerebellum shows similarly dysregulated expression in calcium homeostasis pathway Deranged calcium signaling in Purkinje cells and pathogenesis in spinocerebellar ataxia 2 (SCA2) and other ataxias Role of inositol 1,4,5-trisphosphate receptors in pathogenesis of Huntington’s disease and spinocerebellar ataxias Ectopic expression of CHOP (GADD153) induces apoptosis in M1 myeloblastic leukemia cells a novel developmentally regulated nuclear protein that dimerizes with transcription factors C/EBP and LAP and functions as a dominant-negative inhibitor of gene transcription CHOP is implicated in programmed cell death in response to impaired function of the endoplasmic reticulum Gadd153 sensitizes cells to endoplasmic reticulum stress by down-regulating Bcl2 and perturbing the cellular redox state Targeting the unfolded protein response for disease intervention Oral treatment targeting the unfolded protein response prevents neurodegeneration and clinical disease in prion-infected mice Partial restoration of protein synthesis rates by the small molecule ISRIB prevents neurodegeneration without pancreatic toxicity Type I interferons mediate pancreatic toxicities of PERK inhibition The unfolded protein response is required to maintain the integrity of the endoplasmic reticulum prevent oxidative stress and preserve differentiation in beta-cells Download references for providing Stau1tm1Apa(−/−) (Stau1−/−) mice Monica Vetter from the Department of Neurobiology and Anatomy and her laboratory staff for allowing the use of the Bio-Rad ChemiDoc Dr Clement Chow for insightful discussion of this project and Erika Aoyama and Brian Marshall for providing technical assistance This work was supported by National Institutes of Neurological Disorders and Stroke (NINDS) grants R37NS033123 to SMP and U01NS103883 and R21NS081182 to SMP and DRS The authors declare that they have no conflict of interest The original online version of this article was revised: There was a typo in a gene name in the results of this manuscript Download citation DOI: https://doi.org/10.1038/s41418-020-0553-9 Cellular and Molecular Life Sciences (2021) The dates displayed for an article provide information on when various publication milestones were reached at the journal that has published the article activities on preceding journals at which the article was previously under consideration are not shown (for instance submission All content on this site: Copyright © 2025 Elsevier B.V. Metrics details During the asymmetric division of Drosophila neuroblasts (NBs) the scaffold Miranda (Mira) coordinates the subcellular distribution of cell-fate determinants including Staufen (Stau) and segregates them into the ganglion mother cells (GMCs) Here we show the fifth double-stranded RNA (dsRNA)-binding domain (dsRBD5) of Stau is necessary and sufficient for binding to a coiled-coil region of Mira cargo-binding domain (CBD) The crystal structure of Mira514–595/Stau dsRBD5 complex illustrates that Mira forms an elongated parallel coiled-coil dimer and two dsRBD5 symmetrically bind to the Mira dimer through their exposed β-sheet faces revealing a previously unrecognized protein interaction mode for dsRBDs We further demonstrate that the Mira–Stau dsRBD5 interaction is responsible for the asymmetric localization of Stau during Drosophila NB asymmetric divisions we find the CBD-mediated dimer assembly is likely a common requirement for Mira to recognize and translocate other cargos including brain tumour (Brat) the actual contact interfaces on Mira should involve much less residues than those of the identified regions it is not clear whether these cargos competitively or simultaneously interact with the scaffold Mira the exact binding sites of the cargo proteins are yet to be determined (a) Domain organization of Miranda and Staufen (b) SEC-based analyses of the interactions between various Mira and Stau fragments (c) SEC profiles of Trx-Mira514–595 (blue) Trx-Stau951-1,018 (red) and the Trx-Mira514–595/Trx-Stau951-1,018 complex (black) showing pronounced shift in elution volume for the complex compared with either of the two individual proteins The elution volumes of the peaks and the molecular mass standards are indicated at the top of the panel (d) ITC of purified Trx-Mira514–595 and Trx-Stau951-1,018 indicating a complex with a dissociation constant of 1.07±0.25 μM (e) Ribbon diagram representation of the Mira514–595/Stau dsRBD5 complex structure Mira coiled-coil dimer is coloured in navy blue and cyan Stau dsRBD5s are coloured in purple and pink and the grey part indicates the remained fragment after Trx-tag cleavage Here we perform detailed biochemical characterizations of the interaction between Mira and Stau The Mira/Stau binding sites are mapped to residues 514–595 of Mira and Stau dsRBD5 (aa 951–1,018) The crystal structure of Mira514–595/Stau dsRBD5 is solved at 2.5 Å The complex is found to form a 2:2 heterotetramer in which Mira forms an elongated parallel coiled-coil dimer and two dsRBDs bind to two symmetric surfaces at the N-terminal end of the Mira dimer interface this is the first high-resolution complex structure describing the intermolecular dsRBD–protein interaction Stau dsRBD5 uses the exposed face of its β-sheet to interact with Mira revealing a novel target binding mode for dsRBDs We also demonstrate that the direct interaction between Mira and Stau dsRBD5 is crucial for the asymmetric localization of Stau in asymmetrically dividing NBs of Drosophila larval brain We further showed that the dimerization of Mira CBD is essential to coordinate the polarized distribution of its cargos Stau (a) The Mira514–595 forms an asymmetric coiled-coil with one helix curved about 23 degrees (b) The Mira514–595 coiled-coil contains three potential target-binding regions with region I interacting with Stau dsRBD5 (c) The dimerization interface of the Mira coiled-coil The side chains of the residues involved in the dimer interface formation are drawn in the stick model Dotted lines denote hydrogen bonds and salt bridge interactions The target recognition of region I and II might be mainly mediated by hydrophobic packing; whereas target binding of region III might be mainly driven by polar interactions (d) MiraWT could coimmunoprecipitate with StauWT (e) MiraL529E and MiraL557E could not coimmunoprecipitate with StauWT (f) L529EMira or L557EMira mutation abolished or significantly impaired the interaction between Mira and Brat HEK293T cells were cotransfected with full-length Flag-Mira and GFP–Brat Lysates were loaded on the anti-Flag M2 affinity gel and further incubated with or without 0.8 mg purified Trx-Stau dsRBD5 Presence of an excess amount of Trx-Stau dsRBD5 did not interfere with the interaction between Flag-Mira and GFP–Brat GST-tagged Brat NHL domain could only form a complex with wild-type Stau dsRBD5 (but not the H994E mutant) in the presence of Mira CBDL Stau dsRBD5 pulled down by GST-tagged Brat NHL via the bridging of Mira CBDL was indicated with a star demonstrating that the dimer assembly of Mira CBD is crucial for Stau dsRBD5 binding indicating that the compact dimerization of Mira CBD might be a common requirement for cargo recognition through CBD the Mira-binding-deficient H994E mutant could not be pulled down by GST-tagged Brat NHL even in the presence of Mira CBDL suggesting that Mira may use its elongated coiled-coil structure to simultaneously recruit multiple cargos for transport We next employed type I NBs in Drosophila larval central brain as an in vivo model to address whether the Mira–Stau dsRBD5 interaction is of functional significance we generated transgenic flies expressing full-length Flag-tagged WT or mutant forms of Mira and Stau (a,b) Expression of Flag-Stau WT and Flag-Stau H994E transgenes in NB driven by insc-gal4 (a) Flag-Stau WT is localized on the basal cortex in a wild-type NB (b) Flag-Stau H994E shows cytosolic localization in a wild-type NB (c–h) NBs are marked by GFP using mosaic analysis with a repressible cell marker (MARCM) technique (see Methods) (c–f) Staining of various apical and basal proteins (red or blue) and GFP (green) in larval neuroblasts derived from wild type (c) Mira mutant expressing a Flag-Mira WT transgene (e) and Mira mutant expressing a Flag-Mira L529E transgene (f) Pros and aPKC are asymmetrically localized in wild type NBs (d) Mira is not detected in Mira mutant NBs Brat and Pros are cytoplasmic in Mira mutant NBs aPKC is normally localized in a Mira mutant NB (e) Mira (detected by anti-Mira antibody or anti-Flag antibody) Pros and aPKC are normally localized in Mira mutant NBs rescued with Flag-Mira WT in Mira mutant NBs rescued with Flag-Mira L529E majority of NBs exhibit basal localization of Mira L529E (f detected by anti-Mira antibody or anti-Flag antibody) 5.3% of these NBs showing broad cortical localization of Flag-Mira L529E (g) Brat and Pros remain in cytoplasm and aPKC is normally localized in these NBs whereas red arrowheads indicate apical cortex (h) A WT NB lineage contains one Dpn-positive cell while a Mira mutant NB lineage harbours multiple Dpn-positive cells that is reverted by expression of Flag-Mira WT but only partially recused by Flag-Mira L529E variant suggesting that proper cargo-binding/targeting is important for Mira function Together with our in vitro biochemical analysis our data clearly demonstrated that the dimerization of Mira CBD is most likely a prerequisite for cargo recognition and translocation Secondary structures of Drosophila Stau dsRBD5 are shown at the top of the panel The residues of Stau dsRBD5 involved in Mira binding are indicated with black dots Conservation of these Mira interacting residues is highlighted Identical residues or residues with similar polarity or hydrophobicity from invertebrate to vertebrate are coloured in red those only identical in higher order species in green Three dsRNA-binding regions are indicated by dashed frames The key residues for dsRNA-binding are highlighted in cyan (c) Superimposition of Mira514–595/Stau dsRBD5 complex and human STAU1 SSM–dsRBD5 dimer structures showing that the β-sheet face mediated protein binding of dsRBD5 does not conflict with the dimerization through the α1–α2 interface STAU1 SSM–dsRBD5 dimer is coloured in beige and green and Mira dimer is coloured in navy blue and cyan aeolicus RNase III dsRBD with Mira (navy blue and cyan) SSM (dark green) and dsRNA (orange) in surface representations showing that there is no steric hindrance among dsRNA binding and protein binding through β-sheet and α1–α2 interfaces the conformational flexibility of the two loops might facilitate the protein interactions of dsRBDs This suggests that a canonical dsRBD may use its different faces to bind to dsRNA and proteins simultaneously Mira is a key adaptor protein that directs several cell fate determinants to basal cortex during the asymmetric division of Drosophila NB The detailed molecular mechanism and the direct interactions between Mira and its cargos Here we characterize the interaction between Mira and dsRNA-binding protein Stau The crystal structure of the Mira514–595/Stau dsRBD5 complex demonstrates that Mira forms a parallel coiled-coil dimer and two molecules of Stau dsRBD5 symmetrically bind to Mira514–595 through their exposed β-sheet faces Structural based point mutations (H994EStau and L529EMira) verified that the direct interaction between Mira and Stau dsRBD5 Two lines of evidence support the functional importance of this interaction the Mira-binding-deficient Stau mutant H994E exhibited a diffused subcellular distribution during NB ACD endogenous Stau is defectively localized in Mira mutant NB expressing Mira L529E which can simultaneously recognize multiple cargos Whether the conserved β-sheet of STAU1 dsRBD5 is indeed important for mediating protein interactions with physiological relevance is an interesting prospect that warrants further investigation Other multifunctional dsRBDs are expected to exist and these dsRBDs may be capable of simultaneously interacting with dsRNA and proteins Recombinant proteins were expressed in Escherichia coli BL21 (DE3) host cells at 16 °C and were purified by using a Ni2+–NTA agarose affinity chromatography followed by SEC The N-terminal Trx-tagged fragments of Mira and Stau were cleaved by digesting fusion proteins with protease 3C and the proteins were purified by another step of SEC SEC experiments were carried out on an AKTA FPLC system (GE Healthcare) Proteins at concentrations of 10–20 μM in a volume of 100 μl were loaded on a Superose 12 10/300 GL column 20 (GE Healthcare) equilibrated with the buffer containing 50 mM Tris (pH 8.0) Protein elution was detected by absorbance at 280 nm ITC measurements were performed on an ITC200 Micro calorimeter (MicroCal) at 25 °C All protein samples were in 50 mM Tris (pH 8.0) The titrations were carried out by injecting 40 μl aliquots of the Mira fragments into Stau fragments at time intervals of 2 min to ensure that the titration peak returned to the baseline The titration data were analysed using the program Origin7.0 from MicroCal Human HEK293T Cells were transiently cotransfected with the full-length Flag-Mira and HA-Stau or green fluorescent protein (GFP)–Brat WT proteins or various mutants using polyethylenimine transfection reagent MiraL529E or 8 μg MiraL557E were cotransfected with 20 μg StauWT or 13 μg StauH994E MiraL557E were cotransfected with 4 μg Brat Cells were collected 24 h post transfection and lysed in a buffer containing 50 mM Tris (PH 7.4) Each lysate was incubated with anti-Flag M2 affinity gel (Sigma) overnight the lysate containing cotransfected full-length Flag-Mira and GFP–Brat was incubated with anti-flag M2 affinity gel overnight in the presence or absence of 0.8 mg purified Trx-Stau dsRBD5 After extensive wash of the beads with the lysis buffer the captured proteins were boiled in SDS–PAGE loading buffer and subjected to SDS–PAGE Proteins were transferred to 0.45 μM nitrocellulose membrane (Millipore) and the nitrocellulose membrane was blocked using 3% BSA in TBST (20 mM Tris-HCl (pH 7.4) 137 mM NaCl and 0.1% Tween-20) buffer at room temperature for 1 h followed by incubation with the following antibodies: Flag (ABclonal) GFP (ABclonal) at 1/2,000 dilution at 4 °C overnight Membranes were washed three times with TBST buffer incubated with horseradish peroxidase (HRP)-conjugated Goat anti-Rabbit or anti-Mouse antibody (ABclonal) and visualized on a LAS3000 Chemiluminescent Imaging System GST or GST-tagged Brat NHL (8 μM for the final concentration) were first loaded to 40 μl GSH-Sepharose 4B slurry beads in a 500-μl assay buffer containing 50 mM Tris (pH 8.0) The GST fusion protein loaded beads were then mixed with potential binding partners (24 μM each for the final concentration) and the mixtures were incubated for 1 h at 4 °C proteins captured by affinity beads were eluted by boiling Mira (Flag-Mira WT and Flag-Mira L529E) and Stau (Flag-Stau WT and Flag-Stau H994E) variants were subcloned into UASt.attB vector (A gift from Konrad Basler) Drosophila S2 cells were grown at 25 °C in Schneider's medium (Invitrogen) supplemented with 10% fetal bovine serum All transfections were performed using Effectene Reagent (Qiagen) according to the manufacturer’s instructions S2 cells were cotransfected with 0.5 μg plasmids of interest (Mira or Stau) together with act-gal4 plasmid Cells were collected at 48 h later and lysed in Nonidet P-40 lysis buffer containing 50 mM Tris The lysate was collected and cleared by centrifugation at 13,000 r.p.m The samples were separated by 10% polyacrylamide SDS–PAGE gels followed by transferring to PVDF membranes (Millipore) Rabbit anti-Mira antibody (Generated in our lab 1/1,000) and Rabbit anti-Stau antibody (Daniel St Johnston 1/1,000) were diluted in TBST with 5% non-fat dry milk Information about the fly stains used in this study was described in the text or FlyBase (www.flybase.org) Stocks (unless stated below) were obtained from Blooming Stock Center and crosses were maintained at 25 °C on standard medium UAS-Flag-Mira L529E and UAS-Flag-Stau WT and UAS-Flag-Stau H994E were generated in this study Mira (Flag-Mira WT and Flag-Mira L529E) and Stau (Flag-Stau WT and Flag-Stau H994E) variants were subcloned into UASt.attB vector (A gift from Konrad Basler) and transgenic lines were generated by BestGene (ChinoHills,CA) using attP landing site on II chromosome (Best Gene line 9723) To address these Mira variant localization and function in Mira mutant background these transgenes were subsequently crossed into FRT82B or FRT82B.Mira[L44] background to obtain following stocks UASt–Flag-Mira WT/Sm6.cyo; FRT82B.Mira[L44]/Tm3.tb UASt–Flag-Mira L529E/Sm6.cyo; FRT82B/Tm3.tb UASt–Flag-Mira L529E/Sm6.cyo; FRT82B.Mira[L44]/Tm3.tb embryos were collected over a period of 6 h ALH) was subjected to 1-h heat-shocked treatment at 37 °C and larvae with desired genotypes were dissected and examined 1/20; guinea-pig anti-Dpn (generated in our lab) 1/1,000; rabbit anti-Pon (generated in our lab) 1/2,000; rabbit anti-aPKCζ C20 (Santa Cruz Biotechnologies) Secondary antibodies were conjugated to Alexa Fluor 488 TO-PRO-3 (Invitrogen) was used at 1/5,000 for DNA staining and samples were mounted in Vectashield (Vector Laboratories) Images were obtained using Zeiss LSM 510 upright or Leica SP II upright microscope and processed in Adobe Photoshop CS6 and Adobe Illustrator CS6 Accession codes: The atomic coordinates of Mira514–595/Stau dsRBD5 have been deposited to the Protein Data Bank under the accession code 5CFF The structural basis of Miranda-mediated Staufen localization during Drosophila neuroblast asymmetric division Asymmetric and symmetric stem-cell divisions in development and cancer elegans: cortical polarity and spindle positioning Dividing cellular asymmetry: asymmetric cell division and its implications for stem cells and cancer Asymmetric cell division: recent developments and their implications for tumour biology Mechanisms of asymmetric cell division: flies and worms pave the way in The Development of Drosophila melanogaster eds Bate M. 1091–1131Cold Spring Harbor Laboratory Press (1993) Mechanisms of asymmetric stem cell division The prospero gene specifies cell fates in the Drosophila central-nervous-system Cloning of the Drosophila prospero gene and its expression in ganglion mother cells prospero is expressed in neuronal precursors and encodes a nuclear protein that is involved in the control of axonal outgrowth in Drosophila The prospero transcription factor is asymmetrically localized to the cell cortex during neuroblast mitosis in Drosophila Asymmetric segregation of the homeodomain protein prospero during Drosophila development Asymmetric segregation of numb and prospero during cell division Brat is a Miranda cargo protein that promotes neuronal differentiation and inhibits neuroblast self-renewal The brain tumor gene negatively regulates neural progenitor cell proliferation in the larval central brain of Drosophila Asymmetric segregation of the tumor suppressor brat regulates self-renewal in Drosophila neural stem cells a gene required in determination of cell fate during sensory organ formation in Drosophila embryos Staufen-dependent localization of prospero mRNA contributes to neuroblast daughter-cell fate Inscuteable and Staufen mediate asymmetric localization and segregation of prospero RNA during Drosophila neuroblast cell divisions Miranda mediates asymmetric protein and RNA localization in the developing nervous system Miranda directs Prospero to a daughter cell during Drosophila asymmetric divisions Miranda is required for the asymmetric localization of Prospero during mitosis in Drosophila Partner of Numb colocalizes with Numb during mitosis and directs Numb asymmetric localization in Drosophila neural and muscle progenitors Miranda as a multidomain adapter linking apically localized inscuteable and basally localized Staufen and Prospero during asymmetric cell division in Drosophila miranda localizes staufen and prospero asymmetrically in mitotic neuroblasts and epithelial cells in early Drosophila embryogenesis aPKC phosphorylates Miranda to polarize fate determinants during neuroblast asymmetric cell division Identification of Miranda protein domains regulating asymmetric cortical localization Miranda cargo-binding domain forms an elongated coiled-coil homodimer in solution: implications for asymmetric cell division in Drosophila The multifunctional Staufen proteins: conserved roles from neurogenesis to synaptic plasticity An asymmetrically localized Staufen2-dependent RNA complex regulates maintenance of mammalian neural stem cells Asymmetric segregation of the double-stranded RNA binding protein Staufen2 during mammalian neural stem cell divisions promotes lineage progression Staufen 2 regulates mGluR long-term depression and Map1b mRNA distribution in hippocampal neurons Staufen1 regulation of protein synthesis-dependent long-term potentiation and synaptic function in hippocampal pyramidal cells 'Black sheep' that don't leave the double-stranded RNA-binding domain fold RNA recognition by double-stranded RNA binding domains: a matter of shape and sequence X-ray structure of the GCN4 leucine zipper A stepwise model for double-stranded RNA processing by ribonuclease III Interaction of Staufen1 with the 5' end of mRNA facilitates translation of these RNAs Structure of the large ribosomal subunit from human mitochondria PHENIX: a comprehensive Python-based system for macromolecular structure solution Coot: model-building tools for molecular graphics Mosaic analysis with a repressible cell marker for studies of gene function in neuronal morphogenesis Hedgehog signaling acts with the temporal cascade to promote neuroblast cell cycle exit Brain tumor regulates neuromuscular synapse growth and endocytosis in Drosophila by suppressing Mad expression Download references We thank the staff of beamline BL17U and BL17B at SSRF and National Center for Protein Sciences Shanghai for assistance during data collection; and staff members of the Biomedical Core Facility for their help on biochemical analyses; and Konrad Basler This work was supported by grants from the National Major Basic Research Program of China (2011CB808505 National Science Foundation of China (21473034 Program for New Century Excellent Talents in University (NCET-12-0129) Specialized Research Fund for the Doctoral Program of Higher Education (20130071140004) Science & Technology Commission of Shanghai Municipality (08DZ2270500) and Temasek Life Sciences Laboratory and Singapore Millennium Foundation Zelin Shan and Ying Yang: These authors contributed equally to this work Department of Chemistry and Key Laboratory of Molecular Medicine Shanghai Key Laboratory of Molecular Catalysis and Innovative Materials State Key Laboratory of Molecular Neuroscience Hong Kong University of Science and Technology Institute of Neuroscience and State Key Laboratory of Neuroscience Shanghai Institutes for Biological Sciences Department of Systems Biology for Medicine Shanghai Medical College of Fudan University prepared the Se–Met derivative of Mira514–595/Stau dsRBD5 protein complex and grew the crystals and carried out Co-IP experiments in Z.G.L.’s lab helped with structure determination and performed the static light-scattering measurements in M.Z.’s lab All authors contributed to data interpretation The authors declare no competing financial interests Download citation Metrics details little is known about the in vivo structure of full-length mRNAs a biochemical technique for transcriptome-wide identification of RNA secondary structures interacting with RNA-binding proteins (RBPs) Using this technique to investigate RNA structures bound by Staufen 1 (STAU1) in human cells we uncover a dominance of intra-molecular RNA duplexes a depletion of duplexes from coding regions of highly translated mRNAs an unexpected prevalence of long-range duplexes in 3′ untranslated regions (UTRs) and a decreased incidence of single nucleotide polymorphisms in duplex-forming regions We also discover a duplex spanning 858 nucleotides in the 3′ UTR of the X-box binding protein 1 (XBP1) mRNA that regulates its cytoplasmic splicing and stability Our study reveals the fundamental role of mRNA secondary structures in gene expression and introduces hiCLIP as a widely applicable method for discovering new Prices may be subject to local taxes which are calculated during checkout Understanding the transcriptome through RNA structure In vivo genome-wide profiling of RNA secondary structure reveals novel regulatory features Genome-wide probing of RNA structure reveals active unfolding of mRNA structures in vivo Landscape and variation of RNA secondary structure across the human transcriptome Global analysis of RNA secondary structure in two metazoans Metastasis-suppressor transcript destabilization through TARBP2 binding of mRNA hairpins Rbfox proteins regulate alternative mRNA splicing through evolutionarily conserved RNA bridges and sequencing of hybrids reveals RNA–RNA interactions in yeast Unambiguous identification of miRNA:target site interactions by different types of ligation reactions lncRNAs transactivate STAU1-mediated mRNA decay by duplexing with 3′ UTRs via Alu elements Mammalian Staufen1 recruits Upf1 to specific mRNA 3′ UTRs so as to elicit mRNA decay Staufen1 regulates diverse classes of mammalian transcripts Functional signature for the recognition of specific target mRNAs by human Staufen1 protein a double-stranded RNA-binding protein in Caenorhabditis elegans iCLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution Structures of the human and Drosophila 80S ribosome Control of somatic tissue differentiation by the long non-coding RNA TINCR STAU1 binding 3′ UTR IRAlus complements nuclear retention to protect cells from PKR-mediated translational shutdown The intimate relationships of mRNA decay and translation The ribosome uses two active mechanisms to unwind messenger RNA during translation A human sequence homologue of Staufen is an RNA-binding protein that is associated with polysomes and localizes to the rough endoplasmic reticulum Mammalian Staufen is a double-stranded-RNA- and tubulin-binding protein which localizes to the rough endoplasmic reticulum Cell cycle-dependent regulation of the RNA-binding protein Staufen1 Ubiquitination of the peroxisomal targeting signal type 1 receptor suggests the presence of a quality control mechanism during peroxisomal matrix protein import iCLIP: protein–RNA interactions at nucleotide resolution Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling Mammalian microRNAs predominantly act to decrease target mRNA levels Ribosome profiling of mouse embryonic stem cells reveals the complexity and dynamics of mammalian proteomes The ribosome profiling strategy for monitoring translation in vivo by deep sequencing of ribosome-protected mRNA fragments Reversible cross-linking combined with immunoprecipitation to study RNA–protein interactions in vivo Mammalian endoplasmic reticulum stress sensor IRE1 signals by dynamic clustering A language and environment for statistical computing ggplot2: Elegant Graphics For Data Analysis (Springer The Split-Apply-Combine Strategy for Data Analysis BSgenome.Hsapiens.UCSC.hg19: Homo sapiens (human) full genome (UCSC version hg19) GenomicRanges: Representation and manipulation of genomic intervals ShortRead: a bioconductor package for input quality assessment and exploration of high-throughput sequence data Ultrafast and memory-efficient alignment of short DNA sequences to the human genome Fast and effective prediction of microRNA/target duplexes Integrative Genomics Viewer (IGV): high-performance genomics data visualization and exploration Estimating enrichment of repetitive elements from high-throughput sequence data BEDTools: a flexible suite of utilities for comparing genomic features Circos: an information aesthetic for comparative genomics LIN28A is a suppressor of ER-associated translation in embryonic stem cells Differential expression analysis for sequence count data SylArray: a web server for automated detection of miRNA effects from expression data Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources REVIGO summarizes and visualizes long lists of gene ontology terms Direct competition between hnRNP C and U2AF65 protects the transcriptome from the exonization of Alu elements Download references Sibley for discussions on the development of hiCLIP protocol; K Lang for assistance with bioinformatic analyses; D Lukavsky for sharing the STAU1 plasmid and helping in setting up the project; L Maquat for sharing the ARF1 SBS plasmid; the genomic team at the Cancer Research UK Cambridge Institute for Illumina HiSeq sequencing Babu Mohan and Ule group members for support and comments on the manuscript This work was supported by funding from Human Frontier Science Program (RGP0024/2008-C) European Research Council (206726-CLIP and 617837-Translate) and Medical Research Council (U105185858) to J.U. a Wellcome Trust Joint Investigator Award to N.M.L the Nakajima Foundation fellowship and MRC Centenary Early Career Award to Y.S Alessandra Vigilante and Elodie Darbo: These authors contributed equally to this work Cancer Research UK London Research Institute Okinawa Institute of Science & Technology developed the hiCLIP protocol and designed the project; Y.S designed and performed bioinformatic analyses; A.D. All authors contributed to the analysis and interpretation of the data The resulting data comprise hybrid and non-hybrid reads (7) Hybrid reads are selected and adaptors are trimmed to define the sequences of left (L) and right (R) arms which are mapped independently to the transcriptome The left arm of hybrid read locates upstream of adaptor B and the right arm locates downstream of adaptor B Each arm is mapped independently to transcriptome then the duplex is considered to be formed by the same RNA then the duplex is formed by two different RNAs A diagram describing how a hybrid read is used to identify an RNA duplex A diagram describing how the loop (intervening sequence) is defined for each RNA duplex PARS score represents a ratio between reads starts after cutting with dsRNase (positive) / ssRNase (negative) Assuming that the double-stranded RNase fully digests each duplex it is expected that the positive values in PARS-seq will be highest at the last nucleotide of each duplex This might explain why maximum PARS values occur at the positions closer to the 3′ end of duplexes Metaprofiles of the distribution of STAU1 crosslink sites identified by the start sites of non-hybrid hiCLIP reads (blue) standard deviation of the 10 randomizations) around the positions of hiCLIP duplexes Distribution of median probability of RNA bases to be single-stranded from position −50 to +100 nucleotides around the STAU1 crosslink sites and surrounding sequence up to 40 nucleotides on each side Purine content is plotted within each arm of 494 CDS–CDS STAU1-target duplexes and surrounding sequence up to 40 nucleotides on each side Boxplots showing the frequencies of consecutive purine tracks in the 3′ UTR STAU1-target duplexes (boxplot on the left) and in the randomly selected 3′ UTR duplexes that were detected using RNAfold in mRNAs that don’t contain any STAU1-target hiCLIP duplex in their 3′ UTR (boxplot on the right) The plot shows the incremental hypergeometric P value of the 3 most significantly enriched 7mer motifs in the gene list sorted by the downregulation level (the leftmost gene is the most downregulated) The most significantly enriched motif corresponded to the seed sequence of siRNA indicating that most changes in mRNA abundance between UT and KD corresponded to off-target effects we focused our analyses on the comparison between RC and KD which had no significant enrichment of such motifs by the SylArray analysis The cumulative fraction of mRNAs relative to their fold change of mRNA abundance or translation efficiency between STAU1 rescue (RC) and knockdown (KD) cells is plotted The p-values were calculated by the Mann–Whitney U-test (n = 2,269 and 12,122 for the RNAs containing the duplexes in their 3′ UTR or CDS or other mRNAs for the analysis of mRNA abundance and n = 1,986 and 8,199 for the mRNAs containing the duplexes in their 3′ UTR or CDS or other mRNAs for the analysis of translational efficiency) The position of cytoplasmic splicing site is indicated by an arrow The longer RNA duplex overlaps with the region translated in XBP1(s) Thapsigargin induces endoplasmic reticulum stress and cytoplasmic splicing of XBP1 mRNA After the induction of endoplasmic reticulum stress by thapsigargin and the alternative region of XBP1(s) and XBP1(u) mRNAs was amplified by PCR The PCR products of XBP1(s) and XBP1(u) mRNAs were examined by the electropherogram (the expected sizes of PCR products from XBP1(s) and XBP1(u) mRNAs are indicated as s and u respectively while a hybrid PCR product is indicated by 38 We confirmed that after 30 min of the endoplasmic reticulum stress induction Real-time PCR analysis of reporter mRNA levels containing wt in cells treated with siRNA against STAU1 (KD) or in cells where siRNA-resistant STAU1 is induced with doxycycline to rescue expression of STAU1 in spite of knockdown (RC) Differences in expression were compared by the two-tailed Student’s t-test The two independent experiments are marked by black and blue colour and each dot represents replicates performed on separate wells of cells as part of the same experiment Disruption of duplex destabilizes the mRNA and compensatory mutation restores the stability slightly above the wt level This may be because the duplex in the ‘com’ reporter is longer by 2 nucleotides compared to ‘wt’ The mechanism whereby the long-range duplex impacts mRNA stability remains to be determined Node colour indicates the P value (threshold: P value <10−6 and node size indicates the frequency of the GO term in the GOA database Each gene is mapped only to the most specific terms that are applicable to it (in each ontology) Highly similar GO terms are linked by edges in the graph with the edge width depicting the degree of similarity Diagram summarizing the enriched Biological Processes GO terms 3′ UTR-bound mRNAs tend to encode proteins that function in intracellular transport (in red) whereas CDS-bound mRNAs tend to encode proteins that function in the cell cycle M phase (in blue) Diagram summarizing the enriched Cellular Components GO terms in the context of their location in the cell 3′ UTR-bound mRNAs tend to encode membrane proteins that are translated at the endoplasmic reticulum (in red) whereas CDS-bound mRNAs tend to encode nuclear proteins (in blue) Schematic diagram of the functional analyses of CDS and 3′ UTR-bound mRNAs 3′ UTR-bound mRNAs tend to be highly translated and encode membrane proteins that are translated at the endoplasmic reticulum (in grey) CDS-bound mRNAs tend to be lowly translated and encode nuclear proteins that function in the cell cycle M phase Loops formed by RNA duplexes in the 3′ UTR tend to be longer than in the CDS and 3′ UTRs have higher density of bound duplexes This file contains a discussion of challenges of the experimental methods available to study RNA structures in vivo This table contains mapping statistics of high-throughput sequencing data This table contains genomic positions and sequences of STAU1 target mRNA duplexes that are located in CDS or 3'UTR Download citation Cellular and Molecular Life Sciences (2023) The single-stranded nature of cellular RNAs allows them flexibility to adopt different secondary structures that can affect their function current methods of measuring RNA structure in vivo are limited Two papers published in this week's issue of Nature present new techniques to address this gap Howard Chang and colleagues have exploited a click methodology that enables the first global view of RNA secondary structures in living cells for all four bases While some structures are stable and seem to be programmed by sequence reflecting the binding of proteins or modification of the bases This method may allow RNA to be analysed in vivo from a structural genomics perspective Jernej Ule and colleagues have developed a method to specifically measure RNA structures bound by proteins such as a preference for intramolecular interactions and an under-representation of structures in coding regions The results confirm that RNA structure is able to regulate gene expression While the functional significance is not known it is notable that SNPs are not present at the expected frequency in coding regions Die Situation im Wohnquartier von Staufen «gleiche einem Trümmerfeld» das Nachbarhaus ist ebenfalls stark beschädigt In der Brandruine wurde eine Leiche gefunden dass es sich bei den vermissten Personen um die Bewohner des Hauses handle Das Feuer brach am Mittwochabend gegen 20 Uhr aus Es wurden auch mehrere heftige Detonationen gemeldet Die Löscharbeiten dauerten über fünf Stunden und seien schwierig gewesen heisst es in einer Mitteilung der Kantonspolizei Aargau Das betroffene Haus war stark einsturzgefährdet Mehrere umliegende Gebäude wurden vorsorglich evakuiert Die betroffenen Bewohnerinnen und Bewohner wurden betreut – verletzt wurde von ihnen niemand Allerdings zog sich ein Feuerwehrmann bei den Löscharbeiten leichte Verletzungen zu Die nahegelegene SBB-Bahnstrecke Lenzburg-Luzern wurde zunächst vollständig gesperrt konnte jedoch nach einer ersten Sicherheitsprüfung in der Nacht wieder freigegeben werden Für die Registrierung benötigen wir zusätzliche Angaben zu Ihrer Person {| foundExistingAccountText |} {| current_emailAddress |} Geben Sie die E-Mail-Adresse Ihres Benutzerkontos an über den Sie ein neues Passwort erstellen können Sie erhalten in Kürze eine E-Mail mit einem Link Wenn Sie nach 10 Minuten kein E-Mail erhalten haben prüfen Sie bitte Ihren SPAM Ordner und die Angabe Ihrer E-Mail-Adresse Bitte versuchen Sie es später noch ein Mal oder kontaktieren Sie unseren Kundendienst Wir senden Ihnen einen SMS-Code an die Mobilnummer Wir haben Ihnen einen SMS-Code an die Mobilnummer gesendet Bitte geben Sie den SMS-Code in das untenstehende Feld ein Bitte fordern Sie einen neuen Code an oder kontaktieren Sie unseren Kundendienst Die maximale Anzahl an Codes für die angegebene Nummer ist erreicht Es können keine weiteren Codes erstellt werden Wir haben Ihnen ein E-Mail an die Adresse {* emailAddressData *} gesendet Prüfen Sie bitte Ihr E-Mail-Postfach und bestätigen Sie Ihren Account über den erhaltenen Aktivierungslink prüfen Sie bitte Ihren SPAM-Ordner und die Angabe Ihrer E-Mail-Adresse Benutzerdaten anpassen Mit einem SRF-Account erhalten Sie die Möglichkeit Kommentare auf unserer Webseite sowie in der SRF App zu erfassen Bitte prüfen Sie Ihr E-Mail-Postfach. Das Aktivierungs-E-Mail wurde versendet. Vielen Dank für die Verifizierung Ihrer E-Mail-Adresse. In dieser Ansicht können Sie Ihre Benutzerdaten verwalten. Sie können Ihre Daten jederzeit in Ihrem Benutzerkonto einsehen. Definieren Sie ein neues Passwort für Ihren Account {* emailAddressData *}. Definieren Sie ein neues Passwort für Ihren Account. Sie können sich nun im Artikel mit Ihrem neuen Passwort anmelden. Wir haben den Code zum Passwort neusetzen nicht erkannt. Bitte geben Sie Ihre E-Mail-Adresse erneut ein, damit wir Ihnen einen neuen Link zuschicken können. Sie erhalten in Kürze eine E-Mail mit einem Link, um Ihr Passwort zu erneuern. Wenn Sie nach 10 Minuten kein E-Mail erhalten haben, prüfen Sie bitte Ihren SPAM Ordner und die Angabe Ihrer E-Mail-Adresse. Ihr Account wird deaktiviert und kann von Ihnen nicht wieder aktiviert werden. Erfasste Kommentare werden nicht gelöscht. Wollen Sie Ihren Account wirklich deaktivieren? SRF Schweizer Radio und Fernsehen,Zweigniederlassung der Schweizerischen Radio- und Fernsehgesellschaft Alzheimer's Disease and Related Dementias Volume 12 - 2020 | https://doi.org/10.3389/fnagi.2020.578719 Accumulating evidence has indicated that embryonic inflammation could accelerate age-associated cognitive impairment which can be attributed to dysregulation of synaptic plasticity-associated proteins Staufen is a double-stranded RBP that plays a critical role in the modulation of synaptic plasticity and memory relatively few studies have investigated how embryonic inflammation affects cognition and neurobiology during aging or how the adolescent psychosocial environment affects inflammation-induced remote cognitive impairment the aim of this study was to investigate whether these adverse factors can induce changes in Staufen expression and whether these changes are correlated with cognitive impairment CD-1 mice were administered lipopolysaccharides (LPS 50 μg/kg) or an equal amount of saline (control) intraperitoneally during days 15–17 of gestation male offspring were randomly exposed to stress (S) or not treated (CON) and then assigned to five groups: LPS Mice were evaluated at 3-month-old (young) and 15-month-old (middle-aged) Cognitive function was assessed using the Morris water maze test while Staufen expression was examined at both the protein and mRNA level using immunohistochemistry/western blotting and RNAscope technology The results showed that the middle-aged mice had worse cognitive performance and higher Staufen expression than young mice Embryonic inflammation induced cognitive impairment and increased Staufen expression in the middle-aged mice whereas adolescent stress/an enriched environment would accelerated/mitigated these effects Staufen expression was closely correlated with cognitive performance Our findings suggested embryonic inflammation can accelerate age-associated learning and memory impairments and these effects may be related to the Staufen expression Population aging constitutes a significant public health challenge globally. Aging is associated with cognitive decline, such as spatial learning and memory impairments, which are among the earliest and most striking effects of aging (Belblidia et al., 2018) Such age-associated learning and memory impairments can have a strong and negative impact on the quality of life of the affected individuals it is essential to understand the normal aging process as well as the mechanisms underlying cognitive decline the mechanisms that trigger and maintain age-related diseases remain poorly understood and approaches to alleviate brain aging remain ineffective little is known about the in vivo role of RNA-binding proteins (RBPs) in RNA transport to the synapse and subsequent local protein synthesis these observations highlight the importance of Staufen-mediated posttranscriptional regulation in cognition it remains unclear whether an EE can counteract the harmful effects of embryonic inflammation on age-associated learning and memory impairments growing evidence has suggested that exposure to embryonic inflammation can impair spatial learning and memory in the later life; however whether the stress/an EE in adolescence can accelerate/mitigate the age-associated cognitive impairment resulting from the embryonic inflammation remains unknown We speculated that the Staufen protein may be involved in these impairments caused by embryonic inflammation we first explored whether embryonic inflammation could accelerate age-associated cognitive impairment we investigated whether Staufen expression changed with age and under different treatments we examined whether changes in Staufen expression are correlated with deficits in spatial learning and memory All animal experiments were performed in compliance with the guidelines established by the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals The protocol was approved by the Center for Laboratory Animal Sciences at Anhui Medical University Pregnant mice received a daily intraperitoneal injection of LPS or normal saline during gestation days 15–17 the offspring were exposed either to a stressed or an enriched environment following which they were randomly assigned to five groups (LPS+S A Morris water test was applied to 3-month-old and 15-month-old mice The mice were then sacrificed for subsequent Staufen expression analysis group of mice exposed to an enriched environment Young (2 months old) mice in the LPS+S and CON+S were exposed to a variable sequence of chronic, mild, unpredictable stressors (Sun et al., 2018) One of the following four stressors was randomly applied per day over the 4-day cycle (4 days constituted one cycle and the experiment lasted for seven cycles): (1) Restraint: mice were placed in a net pocket made of soft wire (35 × 40 cm2) to restrict their movement Restraining time lasted for 30 min on the first day all the while maintaining normal respiration (2) Suspension: the mice were suspended by the tail from a crossbar (1.2 m high) for 30 min on the first day and the duration was extended by 10 min each time thereby sequentially increasing the stress intensity (3) Illumination: in the normal light–dark cycle repeated cycles of 30 min lights on/30 min lights off were applied from 19:00 to 7: 00 (4) Fasting: the food was removed between 19:00 and 7: 00 while drinking water was provided ad libitum Mice in the LPS+E were reared in large cages to promote social and group activities. Different types of toys, such as pipes, plastic running wheels, and balls, were placed in the cage every week to increase the ability of the mice to avoid difficulties, their adaptation to the novel environment, and the amount of physical exercise until the behavioral examination was completed (Van Loo et al., 2004) United States) was used to record the distance swam Six mice per group were anesthetized with chloral hydrate (360 mg/kg The brains were then rapidly removed from the skull and cut along the midsagittal plane on ice The left hippocampus was stored at -80°C for western blot analysis The right hemisphere was fixed in 4% paraformaldehyde at 4°C for 3 days Continuous coronal sections (3 μm) were prepared using a Leica Microtome (Leica RM 2135 Germany) for subsequent immunohistochemistry and RNAscope assays The streptavidin–biotin–peroxidase complex (SABC) method was used for immunohistochemical staining, as previously described (Nagashima et al., 1992) the sections were treated with periodate-inactivated enzyme for 1 min to deactivate endogenous peroxidases and microwaved in citrate buffer (0.01 mol/L the sections were treated with 5% fetal bovine serum albumin in phosphate-buffered saline at 37°C for 10 min to minimize non-specific staining The tissue sections were treated with an anti-Staufen antibody (1:200; ab73478 United States) and incubated overnight at 4°C the sections were rewarmed at 37°C for 40 min incubated with a secondary antibody (biotin-labeled goat anti-rabbit IgG) and subsequently treated with SABC (SA1022; Wuhan Boster Bioengineering Co. staining was visualized using diaminobenzidine (ZLI-9018; ZsBio Images of the whole hippocampus (4 × 10) and the three subregions [cornu ammonis (CA)1 and dentate gyrus (DG); 20 × 10] were obtained using a digital scanner (Panoramic MIDI) Western blotting was performed as previously reported (Wu T. et al., 2019; Shen et al., 2020) Hippocampal tissue was lysed in RIPA lysis buffer and protein concentrations were determined using the bicinchoninic acid method Protein samples were separated by 15% SDS–PAGE and then transferred onto PVDF immunoblotting membranes The membranes were blocked with 5% dry milk containing 0.1% Tween 20 for 2 h and incubated with a primary monoclonal antibody against Staufen (1:1000; ab73478 The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (1:10,000; ZB2301 ZsBio) for 2 h at room temperature followed by chemiluminescence detection Immunoreactive bands at 63 kDa (Staufen) and 43 kDa (beta-actin internal standard) denoted positive expression Densitometric quantification of the band intensities was performed using Image-J The ratio of the optical density of the anti-Staufen antibody to that of the anti-beta-actin antibody in each sample was calculated as the relative Staufen protein level RNAscope in situ hybridization was performed as previously described (Domi et al., 2019; Gavini et al., 2020) The RNAscope assay was performed on formalin-fixed tissue sections (3 μm) were deparaffinized in xylene and then incubated with 5–8 drops of H2O2 for 10 min The sections were incubated in citrate buffer (10 nmol/L An ImmEdge pen was used to create a barrier around each section The slides were incubated for 30 min at 40°C with the RNAscope protease plus reagent and then with the Staufen mRNA target probes (ACD 322381) for 2 h at 40°C in the HybEZ hybridization oven The sections were then serially incubated with four amplifier probes (30 min each for steps 1 and 2 15 min each for steps 3 and 4) at 40°C the TSAplus fluorescent dye was added to the slide for 30 min at 40°C and then the RNAscope multichannel fluorescent second-generation HRP blocker was also added to the slide for 15 min at 40°C the sections were counterstained with DAPI to visualize the nuclei Fluorescent signals from RNAscope probe hybridization were examined on a laser-scanning confocal microscope (×40 objective; Zeiss LSM 700) tile scan images were obtained using an Olympus IX71 fluorescence microscope (Olympus Japan) equipped with a PXL37 CCD camera (Photometrics Fluorescence images were semi quantitatively analyzed in ImageJ The number of dots in the Staufen mRNA-positive cells relative to the negative control was calculated as the relative levels of Staufen mRNA The negative control was an internal standard and was used to set the light source and exposure time of image acquisition to acceptable background levels The sample size was calculated by G∗Power software (ver Germany) The α error was set at 0.05 and power (1-β) at 0.8 and the essential total sample size for each group in the behavioral assessments and molecular experiments was calculated as 6–8 Parametric data were expressed as means ± standard error of the mean (SEM) repeated-measures analysis of variance (rm-ANOVA) was used to analyze the learning data The memory percentage of distance from the MWM test and data from the western blotting and RNAscope assays were evaluated by one-way ANOVA with age or treatment as independent variables Fisher’s least significant difference test was performed to compare the differences among the groups The correlations between the MWM performance and the relative levels of Staufen protein and mRNA in the hippocampus were analyzed using Pearson’s correlation coefficient The Statistical Package for Social Sciences (SPSS and significance was assumed at P < 0.05 mice in the LPS+S swam significantly longer distances than those in the other four groups (Ps < 0.01) the CON exhibited the shortest swimming distances among the five groups (Ps < 0.01) the LPS swam significantly longer distances than those in the LPS+E and CON-S (Ps < 0.01); however no significant difference in the distance was observed between the latter two groups (P > 0.05) The effect of interaction of ages × days on swimming distance was not significant [F(6,84) = 0.459 Morris water maze performance of CD-1 mice under different treatments The swimming velocity in the learning phase is shown in (A–C); the swimming distances in the learning phase is shown in (D–F); and the memory percentage of distance in the target quadrant is shown in (G–I) The age effects are depicted in (A,D,G) and the treatment effects in (B,E,H) for the 3-month-old mice (3M) mice and (C,F,I) for 15-month-old (15M) mice All values are presented as means ± SEM (n = 8 male mice/group) Different lowercase letters (a/b/c) denote significant differences mice in the LPS+S exhibited a significantly lower percentage of distance than those in the other four groups (Ps < 0.01) mice in the CON showed a significantly greater percentage of distance than those in the other four groups (Ps < 0.01); meanwhile the percentage of distance was significantly lower in the LPS than in the CON+S and LPS+E (Ps < 0.05); no significant difference was observed between the latter two groups (P > 0.05) Staufen protein levels (Ps < 0.05); moreover Staufen protein levels were significantly higher in the LPS than in the CON+S and LPS+E (Ps < 0.05); but no significantly differences was found between the latter two groups (Ps > 0.05) The hippocampal Staufen protein level in CD-1 mice under different treatments (A) Representative images of Staufen immunohistochemical staining (B) Western blotting analysis of Staufen levels; beta-actin was used as a loading control (C) Staufen protein levels in the CON at different ages (D,E) Hippocampal Staufen protein levels in 3-month-old (3M) and 15-month-old (15M) mice in the different treatment groups All values are presented as means ± SEM (n = 6 male mice/group) Representative photomicrographs of Staufen mRNA levels relative to the negative control in the different hippocampal subregions of CD-1 mice of different ages exposed to different treatments The results for the different hippocampal subregions in 3-month and 15-month-old mice are shown in (A,B) The red staining represents a positive Staufen mRNA staining The relative levels of Staufen mRNA in the different hippocampal subregions of CD-1 mice of differing ages exposed to different treatments The age effect is shown in (A) and the treatment effects for 3-month-old (B) and 15-month-old (C) mice The correlations between hippocampal Staufen protein levels and learning and memory performance The correlations between hippocampal Staufen mRNA levels and learning and memory performance In this study we found that the embryonic exposure to LPS could accelerate the cognitive impairments in middle-aged mice and adolescent stress/an EE could exacerbate/relieve this effect avoiding embryonic infection was of great significance to guide healthy borning and fine rearing and avoiding stress and enriching the environment in the adolescence could further mitigate cognitive impairment we also demonstrated that increased Staufen expression in the hippocampus was correlated with the impaired cognition in the different treatment groups our results provide new insights into the mechanisms involved in cognitive decline resulting from embryonic inflammation In the current study, the 15M CON showed a significantly lower swimming velocity than the 3M CON, suggesting a decline in motor ability in middle-aged mice, which is consistent with the previous results (Chen et al., 2015; Zhang et al., 2020; Duan et al., 2020). van der Staay and de Jonge (1993) showed that if the learning ability of two mice was equal the slower mice may need more swimming time to reach the escape platform (escape latency) than the faster mice; however the swimming distance had no significantly difference we assessed the swimming distance as an indicator of spatial learning and memory in our study Our results indicated that embryonic inflammation or adolescent stress alone does not affect spatial learning and memory in young adults; however deteriorative effect on cognitive behavior the current results also suggested that embryonic inflammation can impair spatial learning and memory abilities in the middle-aged mice to a greater extent than the effect of adolescent stress we did not observe any difference between the LPS+E and CON+S suggesting that an EE in adolescence can mitigate the age-associated cognitive impairment resulting from embryonic inflammation Staufen, a dsRBP, was initially identified in a screen for anterior–posterior patterning mutants in Drosophila embryos. Staufen is recruited to stress granules during the stress response, and can play an important role in mRNA localization, translation, and/or stability (Lebeau et al., 2008; Zimyanin et al., 2008; Paul et al., 2018) no study has investigated whether aging and embryonic inflammation can affect Staufen expression we explored the changes in Staufen expression (protein and mRNA) that occur as a result of aging and exposure to embryonic inflammation or the effect of adolescent psychosocial environment (stress or an EE) on hippocampal Staufen expression following exposure to embryonic inflammation our data indicated a significant effect of age and this age-related increase in hippocampal Staufen expression (protein and mRNA) was in accordance with the behavioral change observed impaired spatial learning and memory abilities treatment also exerted a significant effect on Staufen expression we found that the LPS+S showed the highest levels of hippocampal Staufen protein no significant difference in the Staufen protein levels was found between the LPS+E and the CON+S these observations support that the more detrimental the factor indicating that the effect of embryonic inflammation was significantly stronger than the effect of adolescent stress while both factors combined exerted the strongest effect an adolescent EE could partially reverse the changes in Staufen expression further illustrating that stress upregulated Staufen protein levels the levels of Staufen mRNA was significantly higher in the CA3 subregion of the LPS+E than in the CON+S indicating that an EE could partially reverse the change of Staufen mRNA expression resulting from the embryonic inflammation and the effect was more obvious in the CA1 and DG subregions than in the CA3 subregion no studies have investigated the correlation between Staufen expression and memory Our results suggest that the cognitive impairment induced by the embryonic inflammation may be related to changes in Staufen protein levels our correlation analysis indicated that Staufen expression (protein and mRNA) was significantly correlated with cognitive ability in all the treatment groups These findings provided the first evidence that Staufen expression is associated with impaired spatial learning and memory this correlation was also age-dependent and treatment-related A positive correlation was found between Staufen protein levels and the learning swimming distance while a negative correlation was recorded between Staufen protein levels and the memory percentage of distance These results suggested that the increase in hippocampal Staufen protein levels was associated with the observed age-associated learning and memory impairments following exposure to embryonic inflammation the pattern of correlation between cognitive performance and Staufen mRNA or protein levels was similar further supporting that the changes in Staufen levels occured at the level of transcription a positive correlation was found between the learning swimming distance and Staufen mRNA levels in the CA1 CA3 subregions in all the treatment groups (LPS+S a negative correlation was recorded between the memory percentage of distance and Staufen mRNA levels in the CA1 and DG subregions in the LPS+S and LPS; and in the CA1 subregion of the CON+S These results further suggest that the impaired cognitive performance induced by exposure to embryonic inflammation may be attributable to increased Staufen transcription This occurs preferentially in the CA1 subregion an effect that was dependent on the intensity of the adverse stimulus Staufen mRNA levels might be more related to impaired learning ability our study is the first to report that exposure to embryonic inflammation and adolescent stress could and accumulatively aggravate the age-associated learning and memory impairments while an adolescent EE could ameliorate the changes resulting from embryonic inflammation this study is the first to report that age and embryonic inflammation can enhance the hippocampal Staufen expression at both the protein and mRNA levels while adolescent stress/an EE can partially increase/reverse this effect our results also indicated that the changes in hippocampal Staufen expression were closely correlated with impaired spatial learning and memory abilities especially during “pathological” aging This suggests that the learning and memory impairment resulting from embryonic inflammation may be related to changes in Staufen protein levels Our study also had several limitations. Firstly, it has recently been revealed that left–right anatomical and functional differences exist in the rodent hippocampus (Sakaguchi and Sakurai, 2020) a considerable amount of brain tissue was needed for the experiment and because the unilateral hippocampal tissue available to us may not have been enough to meet the needs of the experiment all brain tissue was prepared in the same manner because we designed the experiment with a focus on the effects of embryonic inflammation and the factors that would aggravate or alleviate these effects so we did not set up an LPS+S+E group to investigate the compound effect We will further enrich our groups in our subsequent study we must admit that we can only find this phenomenon but we didn’t invest the mechanism underlying age-associated learning and memory impairments but we have provided a new insight into the mechanism of cognitive impairment caused by embryonic infection and we still need to further clarify it in our future research All datasets presented in this study are included in the article/Supplementary Material The animal study was reviewed and approved by the Center for Laboratory Animal Sciences at Anhui Medical University C-YR and Z-ZZ performed the behavioral test and collected the data LC and FW designed the study and performed the statistical analysis G-HC designed the study and revised the manuscript All authors read and approved the final manuscript This work was financially supported by the National Natural Science Foundation of China (81370444 and 81671316) the Natural Science Foundation for the Youth of China (81301094) and the Natural Science Foundation for the Youth of Anhui Province (1708085QH182) This funding played important roles in the design of the study and collection The 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/fnagi.2020.578719/full#supplementary-material Lipopolysaccharide-induced spatial memory and synaptic plasticity impairment is preventable by captopril Consequences of brucellosis infection during pregnancy: a systematic review of the literature Exercise reverses behavioral and synaptic abnormalities after maternal inflammation Osmotin attenuates LPS-induced neuroinflammation and memory impairments via the TLR4/NFκB signaling pathway Characterizing age-related decline of recognition memory and brain activation profile in mice Cation-chloride cotransporters in the nervous system: general features and clinical correlations conditional silencing of Staufen 2 alters synaptic plasticity Short-term exposure to enriched environment rescues chronic stress-induced impaired hippocampal synaptic plasticity Effects of prenatal infection on brain development and behavior: a review of findings from animal models Bonnet-Magnaval Chronic adjunction of 1-deoxynojirimycin protects from age-related behavioral and biochemical changes in the SAMP8 mice Activation of PPARγ attenuates the expression of physical and affective nicotine withdrawal symptoms through mechanisms involving amygdala and hippocampus neurotransmission ACE2 activator diminazene aceturate ameliorates Alzheimer’s disease-like neuropathology and rescues cognitive impairment in SAMP8 mice The staufen/pumilio pathway is involved in Drosophila long-term memory Role of physical exercise in the regulation of epigenetic mechanisms in inflammation Differential effects of prenatal stress on the morphological maturation of hippocampal neurons Staufen-1 amplifies proapoptotic activation of the unfolded protein response Hypothalamic C2-domain protein involved in MC4R trafficking and control of energy balance The brain-specific double-stranded RNA-binding protein Staufen2 is required for dendritic spine morphogenesis The curvilinear relationship of early-life adversity and successful aging: the mediating role of mental health Effect of zerumbone on scopolamine-induced memory impairment and anxiety-like behaviours in rats Outdoor ambient air pollution and neurodegenerative diseases: the neuroinflammation hypothesis The effect of Porphyromonas gingivalis lipopolysaccharide on pregnancy in the rat Staufen-1 regulation of protein synthesis-dependent long-term potentiation and synaptic function in hippocampal pyramidal cells Early life stress impairs fear memory and synaptic plasticity; a potential role for GluN2B Maternal inflammation linearly exacerbates offspring age-related changes of spatial learning and memory Inflammatory insult during pregnancy accelerates age-related behavioral and neurobiochemical changes in CD-1 mice Epigenetic-mediated decline in synaptic plasticity during aging Pathological changes in hippocampal neuronal circuits underlie age-associated neurodegeneration and memory loss: positive clue toward SAD Virus-induced obesity in mice: association with a hypothalamic lesion Staufen-2 deficiency leads to impaired response to novelty in mice Systemic LPS causes chronic neuroinflammation and progressive neurodegeneration Unraveling the pathways to neuronal homeostasis and disease: mechanistic insights into the role of RNA-binding proteins and associated factors Left-right functional difference of the rat dorsal hippocampus 751 for short-term memory and long-term memory Hydrogen gas protects against delayed encephalopathy after acute carbon monoxide poisoning in a rat model Recent life stress exposure is associated with poorer long-term memory Environmental enrichment restores neurogenesis and rapid acquisition in aged rats Long-term memory consolidation: the role of RNA-binding proteins with prion-like domains Clostridium butyricum attenuates chronic unpredictable mild stress-induced depressive-like behavior in mice via the gut-brain axis Prenatal immune challenge induces behavioral deficits and increases brain nitric oxide and zinc levels in the male rat offspring The Drosophila transcription factor Adf-1(nalyot) regulates dendrite growth by controlling Fas II and Staufen expression downstream of CaMKII and neural activity Chronic acarbose treatment alleviates age-related behavioral and biochemical changes in SAMP8 mice Long-term moderate exercise rescues age-related decline in hippocampal neuronal complexity and memory RNA binding proteins and the regulation of neuronal synaptic plasticity Effects of age on water escape behavior and on repeated acquisition in rats CrossRef Full Text | Google Scholar Preference for social contact versus environmental enrichment in male laboratory mice Morris water maze: procedures for assessing spatial and related forms of learning and memory Can outbred mice be used as a mouse model of mild cognitive impairment Google Scholar Age- and gender-dependent impairments of neurobehaviors in mice whose mothers were exposed to lipopolysaccharide during pregnancy Lipopolysaccharide exposure during late embryogenesis triggers and drives Alzheimer-like behavioral and neuropathological changes in CD-1 mice Effects of mind-body exercises on cognitive function in older adults: a meta-analysis Histone H3K9 trimethylation downregulates the expression of brain-derived neurotrophic factor in the dorsal hippocampus and impairs memory formation during anaesthesia and surgery Accelerated deficits of spatial learning and memory resulting from prenatal inflammatory insult are correlated with abnormal pPhosphorylation and methylation of histone 3 in CD-1 mice Author response: gram-negative bacterial molecules associate with Alzheimer disease pathology PubMed Abstract | CrossRef Full Text | Google Scholar Effects of prenatal exposure to inflammation coupled with stress exposure during adolescence on cognition and synaptic protein levels in aged CD-1 mice In vivo imaging of oskar mRNA transport reveals the mechanism of posterior localization Wang F and Chen G-H (2020) Effects of Embryonic Inflammation and Adolescent Psychosocial Environment on Cognition and Hippocampal Staufen in Middle-Aged Mice Copyright © 2020 Wu, Zhang, Ge, Ren, Zhang, Cao, Wang and Chen. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) distribution or reproduction in other forums is permitted provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited in accordance with accepted academic practice distribution or reproduction is permitted which does not comply with these terms *Correspondence: Gui-Hai Chen, ZG9jdG9yY2doQDE2My5jb20= Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher 94% of researchers rate our articles as excellent or goodLearn more about the work of our research integrity team to safeguard the quality of each article we publish Metrics details Staufen (Stau) proteins are core factors of mRNA localization particles They consist of three to four double-stranded RNA binding domains (dsRBDs) and a C-terminal dsRBD-like domain Mouse Staufen2 (mStau2)-like Drosophila Stau (dmStau) contains four dsRBDs Existing data suggest that only dsRBDs 3–4 are necessary and sufficient for mRNA binding we show that dsRBDs 1 and 2 of mStau2 bind RNA with similar affinities and kinetics as dsRBDs 3 and 4 While RNA binding by these tandem domains is transient all four dsRBDs recognize their target RNAs with high stability Rescue experiments in Drosophila oocytes demonstrate that mStau2 partially rescues dmStau-dependent mRNA localization a rescue with mStau2 bearing RNA-binding mutations in dsRBD1–2 fails confirming the physiological relevance of our findings our data show that the dsRBDs 1–2 play essential roles in the mRNA recognition and function of Stau-family proteins of different species dendritically localized RNAs produce proteins with synaptic functions such as Ca2+/calmodulin kinase II (CaMKII) the cytoskeletal protein Arc or microtubule-associated protein 2 (MAP2) one of the unresolved questions is how specific RNA binding can be achieved by dsRBD 3–4 the noncanonical dsRBDs 1 and 2 exhibit RNA-binding activity of equal affinity and kinetic properties as the known RNA-binding dsRBDs 3–4 Mutational analyses and biophysical characterization of RNA binding revealed that dsRBD 1–2 have to act in concert with dsRBD 3–4 to allow for stable we demonstrate the importance of RNA binding by dsRBDs 1–2 for Stau function in vivo and show that mStau2 can partially substitute for dmStau function during early Drosophila development The requirement of a combination of two dsRBD-tandem domains and thus the possibility of binding to two stem loops allows recognition of combinations of secondary structure and thus a much more complex readout for specific binding This observation might help to explain how Stau proteins can bind selectively to their RNA targets in vivo The dsRBD1–2 tandem domain of mStau2 binds RNA a EMSAs with full-length mStau2 (mStau2 FL) and different SRS RNAs from the 3′UTR of the Rgs4 mRNA b EMSAs with mStau2 tandem domains dsRBD1–2 or dsRBD3–4 and SRS2 c NMR titration experiments of mStau2 dsRBD1–2 with SRS2 RNA d Crystal structure of SRS2 at 1.73 Å resolution Iminos from c showing significant line broadening are indicated by colored (orange to red) spheres e EMSAs with mStau2 tandem domains dsRBD 1–2 or dsRBD 3–4 and SRS2 RNA extended by five basepairs (SRS2 + 5) f NMR titration experiments of mStau2 dsRBD 1–2 with SRS2 + 5 RNA Left: overlay of 1H,15N-HSQC spectra of dsRBD1–2 in absence and presence of 2x excess SRS2 + 5 RNA Resonance shifts and line broadening of several signals are observed Right: comparison of 1D imino traces of SRS2 + 5 RNA at different stoichiometric ratios with dsRBD 1–2 Strong line broadening of imino signals is observed in presence of protein Source data are provided as a Source Data file Our NMR data and the crystal structure thus confirm that SRS2 folds into a canonical stem-loop structure all imino signals are observable at equimolar RNA:protein ratio Line broadening for the imino signals in the basepairs at the bottom of the stem suggests this as a main interaction region Because the protein may not slide off the hairpin end but rather gets stopped there protein binding to a hairpin RNA is expected to introduce some asymmetry to binding and thus differential line broadening the 18 bp stem of SRS2 + 5 allows for significant sliding as reflected by the severe line broadening observed for all imino signals in the basepairs of the stem upon protein binding It is therefore well possible that our observed binding to a minimal stem-loop RNA only reflects a partial recognition and that for a full binding a longer stem is required This interpretation is consistent with our general observation that longer RNAs are bound stronger than shorter ones SPR shows mStau2 binding to SRS2 + 5 RNA and SRS* RNA and b mStau2 dsRBD 3–4 binding to surface-coupled SRS2 + 5 and SRS* RNAs The tandem domains dsRBD 1–2 and dsRBD 3–4 bind transiently with fast kinetics The steady-state binding curves do not saturate up to 1 µM protein concentration but can be described by a two-site binding fit with KD1 of 18 nM and 130 nM c mStau2 dsRBD 1–4 binding to surface-coupled SRS2 + 5 and SRS* RNAs is stable with slower kinetics The steady-state binding curve saturates at approximately 1 µM and is described by a Hill fit with an apparent overall KD of 357 nM and a Hill coefficient n = 1.7 for SRS2 + 5 and an apparent overall KD of 330 nM and a Hill coefficient n = 1.8 for SRS* d SRS2 + 5 and SRS* bind to surface-coupled mStau2 FL stably and with high affinity The steady-state binding curves can be described by a two-site binding fit with KD1 of 1.3 nM and 10.6 nM and KD2 of 185 nM and 195 nM Also, dsRBD 3–4 bound with similar properties, yielding a KD1 of 18 nM for SRS2 + 5 RNA and of 9 nM for the SRS* RNA (Fig. 2b; Table 2) KD2 was in the micromolar range and could not be determined Together these findings confirm the RNA-binding activities of dsRBD 1–2 and of dsRBD 3–4 The observed fast kinetics for KD1 explain why in EMSAs no high-affinity band shifts were observed In order to obtain structural insights into RNA-binding preferences of dsRBDs 1 and 2, 1H,15N-HSQC NMR spectra of the individual dsRBDs and of the tandem dsRBD 1–2 were measured. The spectra of the two individual domains show that they are well-folded (Supplementary Fig. 7) and also nicely match with the NMR spectrum of the tandem domain dsRBD 1–2 This indicates that in the context of the tandem domains the structures of the individual dsRBDs 1 and 2 are not altered and do not significantly interact with each other NMR titrations of mStau2 dsRBD1 and dsRBD2 with SRS2 + 5 RNA c Overlay of 1H,15N-HSQC spectra of dsRBD1 (a) or dsRBD2 (c) in absence and presence of SRS2 + 5 RNA Resonance shifts and line broadening of several signals are observed for both domains that there are two sets of signals observed for dsRBD1 where only one set is affected by RNA binding The second set of signals may reflect the presence of an alternate conformation of a region of dsRBD1 d Comparison of 1D imino NMR spectra of SRS2 + 5 RNA at different stoichiometric ratios with dsRBD1 (b) or dsRBD2 (d) Strong line broadening of imino signals is observed in presence of dsRBD2 but not dsRBD1 pointing at reduced RNA binding affinity for dsRBD1 This indicates that the two dsRBDs bind RNA with different kinetics which is suggestive of a lower binding affinity of dsRBD1 compared with dsRBD2 SPR experiments with mStau2 dsRBD1 or dsRBD2 mStau2 dsRBD1 alone binds neither to (a) SRS2 + 5 nor to (b) SRS* dsRBD2 binds to (c) SRS2 + 5 and to (d) SRS* with fast kinetics in a non-cooperative fashion with KDs of 650 nM or 829 nM Steady-state binding curves are described by a Hill fit Together these results indicate that the two domains act in concert to bind dsRNA with better affinities and that this requires the presence of the linker which itself does not appear to contribute to the RNA recognition The dsRBD1–2 tandem domain with a range of mutations in dsRBD1 were tested for binding to SRS2 + 5 (Supplementary Fig. 10; Table 3) the steady-state binding curves are best described by Hill-fits with Hill coefficients n ≈ 1 Whereas the observed KDs of dsRBD 1–2 H36A and K59A are similar to that of dsRBD2 alone and K59A K60A bind with even lower affinity than dsRBD2 alone These results indicate that binding activity of dsRBD1 was abolished by these mutations where steady-state binding to SRS2 + 5 was fitted with a first-order binding reaction and a KD of 132 nM indicating that RNA-binding activity of dsRBD1 might be compromised but not completely abolished This mutation possibly has a less drastic effect indicating that the effects of these mutations are less dramatic Binding of dsRBD 1–2 F157A was again strongly impaired and no KD could be determined thus confirming the results obtained for SRS2 + 5 SPR with mutated dsRBD 1–4 confirms a contribution of dsRBD1–2 a Schematic drawing of mStau2 with its two mutations in dsRBD 1–2 c SPR experiments with mStau2 dsRBD 1–4 double-mutants binding to SRS2 + 5 RNA and d Binding to (b) SRS2 + 5 and (d) SRS* is strongly decreased for the F40A F157A mutant as compared to mStau2 dsRBD 1–4 wild-type Binding of the F40A H169A mutant shows dramatically altered kinetics (c e) as compared with dsRBD 1–4 wild-type and resembles binding by the tandem domain dsRBD 3–4 alone this indicates that in dsRBD 1–4 F40A H169A RNA binding is mediated by dsRBD 3–4 alone Since mutations in dsRBD 1–2 impair the affinity of dsRBD 1–4 and because the interactions become much more transient our data indicate that for efficient and stable RNA binding of mStau2 all four dsRBDs have to act in concert Functional interaction of mStau2 with oskar mRNA in Drosophila GFP-mStau2[F40A H169A] and GFP-mStau2[F40A F157A] in germline of stauR9/stauD3 mutant females In dmStau-expressing oocytes oskar (cyan) localizes almost exclusively to the posterior pole (right) and bicoid (green) to the anterior pole (left) during stage 9 of oogenesis Transgenic GFP-Stau protein is shown in red Insets show magnified regions of the upper anterior corner b Typical localization of oskar mRNA in oocytes as function of expressed Stau protein RNA signal was redistributed into a 100 × 100 square matrix and statistically evaluated to obtain average (green) and variability (magenta) of RNA distribution In wild-type oocytes (top left) most signal is found close to the posterior pole (right of the panels) by stage 9 oskar mRNA accumulates at the anterior pole Scale bar: 20% length of anteroposterior axis d Center of mass (relative to geometric center at 0 c) and fraction at posterior pole of oskar mRNA (d) during stage 9 P-values show result of pairwise Mann–Whitney U tests vs the stau null condition (Bonferroni corrected alpha value: 0.01) Center line: median; box limits: 25th and 75th percentile; whiskers: 10th and 90th percentile e–h Interaction of GFP-tagged Stau molecules with oskar (e mRNPs are sorted by their mRNA content using quantitative smFISH g) and normalized GFP-Stau signal intensity (f h) were plotted as function of mRNA content of the mRNPs The normalized GFP-Stau signal intensities are fitted linear models In pairwise comparisons of oskar mRNPs (f) all slopes are significantly different (p < 0.0001) except for GFP-mStau2[F40A H169A] vs GFP-mStau2[F40A F157A] (p = 0.016 the slope of GFP-mStau2[F40A F157A] differs from the other three (p < 0.01) these rescue experiments confirm the importance of RNA binding by dsRBD 1–2 for the in vivo function of Stau proteins suggesting that dsRBDs 1 and 2 fulfill other functions we have demonstrated that the mStau2 dsRBDs 1 and 2 also possess RNA-binding activity we could show that the two dsRBDs 1 and 2 work together as a tandem domain to achieve their full functionality Our data further confirm that also dsRBD 3–4 act as a tandem domain Our comparison of RNA-binding affinities of single domains and tandem domains bearing mutations suggests that the first binding event with moderate affinity is achieved by the second dsRBD in each tandem domain Model of the molecular recognition of dsRNA by mStau2 a dsRNA recognition by the mStau2 tandem domain dsRBD1–2 dsRBD2 binds dsRNA promiscuously with moderate affinity and slides along the stem dsRBD2 positions dsRBD1 close to the dsRNA When a suitable dsRNA structure is reached thereby strongly increasing the affinity of the tandem domain to dsRNA dsRBD4 does the first promiscuous binding with moderate affinity When dsRBD2 and dsRBD4 position dsRBD1 and dsRBD3 the respective domains also bind the dsRNA Only when suitable dsRNA binding sites for both tandem domains are in sufficient spatial proximity can all four dsRBDs be bound and form a stable complex with the RNA target To confirm in vivo that RNA binding by the dsRBDs 1–2 is important for the function of the full-length protein we utilized the Drosophila oocyte as model system In the germline of otherwise stau null flies different variants of Stau were expressed and the rescue of the mutant phenotype assessed by analyzing oskar mRNA localization to the posterior pole a moderate rescue of oskar mRNA localization was observed mStau2-rescue constructs bearing RNA-binding mutations in dsRBD 1–2 failed to rescue oskar localization This observation confirms the importance of dsRBD 1–2 for RNA binding and RNA localization in vivo It will be interesting to see the basis of these functional differences in future experiments mStau2 involves two tandem domains with four dsRBDs for its sequential RNA-target recognition Future work will have to answer whether a defined spatial arrangement of two stem-loops is recognized by each tandem dsRBD or if all four domains act as a molecular ruler for a single stem loop of defined length RNA-binding proteins in higher eukaryotes very often contain multiple RNA-binding domains34 It is thought that these act in a combinatorial fashion such as we have shown for the dsRBDs of mStau2 for most of these multidomain proteins the manner in which they act cooperatively for function and specificity is not well understood each of which can bind to secondary structures It is likely that the combination of secondary structure elements as well as their spatial arrangement determine the specificity of Stau binding for transport of selected mRNAs in vivo according to the manufacturer’s instructions the primers pBSKS-rsEGFP2 FW and rsEGFP + 3 C RV were used to create an rsEGFP2 sequence with a pBlueScript-KS 5′ overhang for In-Fusion cloning and a PreScission protease cleavage site as 3′ overhang and the primers 3 C + Stau2 FW and pBSKS-mStau2 RV to create a mStau2 sequence with a PreScission cleavage site 5′ overhang and a pBlueScript-KS 3′ overhang for In-Fusion cloning The PCR products from these reactions served as templates for a third PCR with the primers pBSKS-rsEGFP2 FW and pBSKS-mStau2 RV to create rsEGFP2-mStau2 sequences with 5′ and 3′ overhangs for In-Fusion cloning into BamHI/XbaI-linearized pBlueScript-KS The resulting plasmids served as templates for PCR with primers pUASp-rsEGFP2 FW and pUASp-mStau2 RV or pUASp-dmStau RV to amplify rsEGFP2-mStau2/dmStau sequences with 5′ and 3′ overhangs for In-Fusion cloning into the BamHI/XbaI-linearized pUASp-attB plasmid mStau2 FL was expressed as a HisSUMO-tagged fusion protein in High Five insect cells recombinant baculovirus was produced with the Bac-to-Bac Expression System (Invitrogen) in Sf21 insect cells as described by the manufacturer’s protocol Uniformly 15N- or 15N,13C-labeled proteins for NMR experiments were expressed in 15N-M9 minimal medium (1 × 15N-labeled M9 salt solution 1× trace metals) supplemented with antibiotics Hundred milliliters precultures were grown overnight at 37 °C shaking at 150 rpm and used to inoculate 1 L prewarmed M9 minimal medium Protein expression was induced with 0.25 mM IPTG and cultures were cooled for protein expression overnight at 18 °C High Five cell pellets containing HisSUMO-tagged mStau2 FL were lysed by sonication in lysis buffer (1× PBS and the soluble protein fraction was purified by Ni-IMAC on HisTrap FF (GE) Bound protein was eluted with 200 mM imidazole after extensive washing with 15 CV lysis buffer The protein was dialyzed in low salt buffer (40 mM Bis-Tris pH 7 2 mM DTT) overnight before further purification on a HiTrap Heparin HP column (GE) and size exclusion chromatography (SEC) on Superdex200 Increase (GE) coli Rosetta cell pellets containing HisSUMO-tagged fusion proteins were lysed by sonication in lysis buffer (1× PBS The lysate was cleared by the centrifugation the protein was digested overnight with PreScission protease upon dialysis in low-salt buffer (40 mM Bis-Tris pH 7 subtractive Ni-IMAC affinity chromatography on a HiTrap Heparin HP column (GE) and size exclusion chromatography (SEC) on Superdex 75 (GE) Size-exclusion chromatography was performed in minimal buffer (40 mM Bis-Tris pH 7 2 mM DTT) or the indicated buffer required for downstream applications In order to produce partially double-stranded template DNA FW (T7prom) and RV primers were annealed after unfolding at 60 °C for 5 min by slow cooling to RT This DNA template mixture was used for a 5 mL in vitro transcription reaction containing a template specifically optimized concentration of MgCl2 (see below) 80 mg per mL PEG8000 and 0.5 mg per mL T7 RNA polymerase in 1× TRX buffer (40 mM Tris/HCl pHRT 8.0 The reaction was incubated for 2 h at 37 °C The reaction was stopped by removal of precipitants by centrifugation at 48,384 g for 5 min and subsequent RNA precipitation with 0.1 V 3 M NaOAc and 3 V absolute ethanol at −20 °C overnight The optimal MgCl2 concentration for each RNA was determined beforehand by MgCl2 screening in 50 µL reactions containing 4–60 mM MgCl2 Quality and quantity of RNA in each MgCl2 concentration were examined by 8% urea PAGE RNA was pelleted by centrifugation at 48,384 g air-dried and subsequently dissolved in 1× denaturing RNA loading dye The RNA was purified by 8% 1x TBE- 8 M urea PAGE in an Owl sequencing chamber (Thermo Fisher Scientific) in 1x TBE running buffer at constant 300 V for 17–20 h RNA bands were visualized by UV shadowing and the desired band was excised from the gel and extracted by electroelution in a Whatman Elutrap electroelution system (GE Healthcare) at constant 200 V in 1x TBE for 8 h Eluted RNA was dialyzed against 5 M NaCl at 4 °C overnight and subsequently twice against RNase-free water at 4 °C overnight before drying in a Concentrator Plus SpeedVac (Eppendorf) RNAs for electrophoretic mobility shift assays (EMSA) were labeled radioactively for sensitive detection of protein–RNA interactions In vitro transcribed RNA was 5′ dephosphorylated in 20 µL reactions containing 10 pmol RNA 2 U FastAP thermosensitive alkaline phosphatase (Thermo Fisher) and 20 U of the RNase inhibitor SUPERaseIn (Thermo Fisher) the dephosphorylated RNA was phenol/chloroform extracted and precipitated with 0.1 V 3 M NaOAc 3 V absolute ethanol and subsequent chilling at −20 °C for ≥15 min 10 pmol dephosphorylated RNA or chemically synthesized RNA were 5′-phosphorylated with 32P from γ-32P ATP (Hartmann Analytic) in a 20 µL reaction with T4 polynucleotide kinase (New England Biolabs) in 1× buffer A The labeling reaction was incubated at 37 °C for 30 min and subsequently stopped at 72 °C for 10 min Remaining free nucleotides were removed by purification on a NucAway™ Spin column (Ambion) according to the manufacturer’s instructions Eluted radiolabeled RNA was diluted to a final concentration of 100 nM in RNase-free H2O and stored at −20 °C protein at the indicated final concentration was mixed with 5 nM radiolabeled RNA in RNase-free protein buffer supplemented with 4% glycerol and 30 µg per mL yeast tRNA as a competitor in a final volume of 20 µL In order to allow protein–RNA complexes to form the mixtures were incubated for >20 min at RT Separation of protein–RNA complexes was performed by native PAGE on 6% polyacrylamide 1x TBE gels in 40 min at constant 110 V in 1x TBE running buffer 10% (v/v) acetic acid for 10 min before drying in a vacuum gel drier (BioRad) Visualization of radioactivity occurred after exposure of radiograph films (Kodak) in a Protec Optimax developer (Hohmann) or by PhosphorImaging with a Fujifilm FLA-3000 Each experiment was performed as a triplicate on different days and separation of protein–RNA complexes was performed by 1–1.5% agarose gel electrophoresis Visualization of RNA was achieved by GelRed (Biotium) staining Fluorescence was visualized with a Fusion SL imaging system (Vilber Lourmat) by UV at 254 nm Ligand RNA for binding studies by Surface Plasmon Resonance was biotinylated to allow immobilization on a streptavidin-coated surface the PierceTM RNA 3′ End Biotinylation Kit (Thermo Fisher Scientific) was used according to the manufacturer’s protocol Fifty pmol of RNAs were used per 30 µL reaction the RNA was redissolved in 100 µL RNase-free water Surface Plasmon Resonance (SPR) experiments were performed with a BIACORE 3000 system (GE Healthcare) biotinylated RNA in a volume of 60 µL was streptavidin-captured on a SA-Chip (GE Healthcare) surface at a flow rate of 10 µL per min after three consecutive 1 min conditioning injections of 50 mM NaOH Full-length mStau interacted strongly with the blank SA-Chip surface to assess the interaction of RNA with mStau FL the protein was diluted in HBS-EP buffer (10 mM HEPES pH 7.4 0.005% surfactant P20) and covalently amine-coupled to a CM5-Chip (GE Healthcare) according to the manufacturer’s instructions Kinetic analysis of protein–RNA interactions was performed at a flow rate of 30 µL per min in HBS-EP buffer RNA in HBS-EP buffer at the indicated concentrations was injected for 4–5 min to allow for association subsequent dissociation was allowed for 10 to 15 min in HBS-EP buffer two 1 min regeneration injections with 1 M NaCl were performed Data were analyzed in the BIAevaluation software (GE Healthcare) After double-referencing of obtained binding curves against the signal in a ligand-free reference channel or against a HisSUMO-coupled reference channel average values for the analyte response at equilibrium were calculated Steady-state binding curves were obtained by plotting the response at equilibrium against analyte concentration and curve fitting with the Origin Pro 9.0 (OriginLab) software using the two-site binding or Hill1 fits available in the software All experiments were performed at least in triplicate on different days and the results of the n ≥ 3 experiments were averaged were performed with the BIAevaluation software (GE Healthcare) using the bivalent analyte model available in the software Circular dichroism (CD) spectra were collected with a Jasco J-715 spectropolarimeter from 260 nm to 190 nm in a continuous scanning mode with a scanning speed of 50 nm per min at a bandwidth of 1 nm Five scans were collected per measurement and response time was 8 s Spectra were analyzed with SpectraManager (Jasco) Static light scattering (SLS) experiments were performed with a 270 Dual Detector and a VE3580 RI Detector (Malvern) after SEC on a 10/300 Superdex200 Increase column (GE Healthcare) at 7 °C and a flow rate of 0.5 mL per min Data were analyzed with the OmniSEC 5.02 software (Malvern) RNA assignment of imino groups was based on 1H,1H-NOESY spectra; an initial protein backbone assignment was made with HNCACB spectra Titration experiments with the single mStau2 dsRBDs 1 and 2 as well as the tandem domain dsRBD1–2 were performed at 50 µM protein concentration the RNA ligand was added in molar ratios of 0.5 To determine the binding interface of Stau2 dsRBD1–2 to RNA imino signals of the unbound RNA were compared with the respective resonances of a Stau2 dsRBD1–2-RNA (1:1) complex The bound spectrum was scaled so that imino signals which do not show additional exchange-mediated line broadening in the complex have the same peak height as in the free spectrum (still with larger line broadening) The UASp-rsEGFP2-mStau2 vectors were injected into embryos of y[1] M{vas-int.Dm}ZH-2A w[*]; PBac{y[ + ]-attP-3B}VK00033 (FBti0076453) females to facilitate psiC31 mediated insertion into the same locus on the 3 L chromosome arm All stocks were raised on normal cornmeal agar at 25 °C Further information on experimental design is available in the Nature Research Reporting Summary linked to this article The data that support the findings of this study are available from the corresponding author upon reasonable request In the right place at the right time: visualizing and understanding mRNA localization Molecular insights into intracellular RNA localization The central dogma decentralized: new perspectives on RNA function and local translation in neurons Germ plasm biogenesis--an oskar-centric perspective Identification of a novel homolog of the Drosophila staufen protein in the chromosome 8q13-q21.1 region A genome-wide approach identifies distinct but overlapping subsets of cellular mRNAs associated with Staufen1- and Staufen2-containing ribonucleoprotein complexes Staufen2 isoforms localize to the somatodendritic domain of neurons and interact with different organelles The mammalian staufen protein localizes to the somatodendritic domain of cultured hippocampal neurons: implications for its involvement in mRNA transport Two rat brain staufen isoforms differentially bind RNA ‘Black sheep’ that don’t leave the double-stranded RNA-binding domain fold The brain-specific double-stranded RNA-binding protein Staufen2: nucleolar accumulation and isoform-specific exportin-5-dependent export NMR approaches for structural analysis of multidomain proteins and complexes in solution LR-HSQMBC: a sensitive NMR technique to probe very long-range heteronuclear coupling pathways ATP-independent diffusion of double-stranded RNA binding proteins Molecular basis for asymmetry sensing of siRNAs by the Drosophila Loqs-PD/Dcr-2 complex in RNA interference Identification of small-molecule inhibitors of the HuR/RNA interaction using a fluorescence polarization screening assay followed by NMR validation Structural basis of siRNA recognition by TRBP double-stranded RNA binding domains RNA-binding proteins: modular design for efficient function Site-directed mutagenesis by overlap extension using the polymerase chain reaction Protein production by auto-induction in high density shaking cultures Overview of the CCP4 suite and current developments Auto-rickshaw: an automated crystal structure determination platform as an efficient tool for the validation of an X-ray diffraction experiment Continuous-flow optical pumping NMR in a closed circuit system The CCPN data model for NMR spectroscopy: development of a software pipeline Multiple steps in the localization of bicoid RNA to the anterior pole of the Drosophila oocyte Aster migration determines the length scale of nuclear separation in the Drosophila syncytial embryo A versatile platform for creating a comprehensive UAS-ORFeome library in Drosophila Enzymatic production of single-molecule FISH and RNA capture probes Ex vivo ooplasmic extract from developing Drosophila oocytes for quantitative TIRF microscopy Terminal deoxynucleotidyl transferase mediated production of labeled probes for single-molecule FISH or RNA capture Klar ensures thermal robustness of oskar localization by restraining RNP motility An RNA-binding atypical tropomyosin recruits kinesin-1 dynamically to oskar mRNPs Independent and coordinate trafficking of single Drosophila germ plasm mRNAs Lenth R. V. Least-Squares Means: The R Package lsmeans. J. Stat. Softw. 69, https://doi.org/10.18637/jss.v069.i01 (2016) R: A language and environment for statistical computing ggplot2: Elegant Graphics for Data Analysis Download references Gregor Witte for support with static light-scattering experiments Kiebler for fruitful discussions and support We acknowledge the support by the Bavarian NMR center and the X-ray crystallography platform at the Helmholtz Zentrum München X-ray data were collected at the Swiss Light Source (SLS) synchrotron beamline PXIII and the European Synchrotron radiation Facility (ESRF) at beamline ID 23–2 This work was supported by the Deutsche Forschungsgemeinschaft (FOR2333 to S.H. Present address: Institute of Molecular Biotechnology Center for Integrated Protein Science Munich at Chair of Biomolecular NMR Spectroscopy All authors reviewed and approved the paper Journal peer review information: Nature Communications thanks the anonymous reviewer(s) for their contribution to the peer review of this work Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations Download citation DOI: https://doi.org/10.1038/s41467-019-09655-3 Germany) are now offering their combined services in North America The two companies have joined forces to provide consulting services and process optimization for press shops untapped potentials are identified and addressed as well as Schuler experts together improve the whole value stream of the press shop like improvements of Overall Equipment Efficiency (OEE) die change optimizations and the implementation of Total Productive Maintenance (TPM) as well as setup time and output and logistics optimizations Schuler (Canton, MI Auch in diesem Jahr haben wir wieder die Schelmenzunft unterstützt Am Sonntag sicherten wir den Kinderumzug mit mehreren Fahrzeugen ab Heute Nacht begleiteten wir den Umzug und die Verbrennung auf dem Marktplatz this isn't some weird idea for a Pixar movie -- this is actually happening Youtuber Tom Scott visits the town of Staufen in south-west Germany Staufen has a very unique problem: it is splitting apart It turns out that the government bored in the area for geothermic energy but unfortunately the groundwater combined with a layer of anhydrite to form gypsum which expanded causing the ground to bulge and the buildings to form these huge cracks The German town of Staufen is falling apart at the seams decided to invest in geothermal energy back in 2007 the decision backfired when the underground drilling went wrong causing hundreds of buildings to begin cracking apart The town rests on a layer of soft anhydrite below which is a layer of groundwater confined to an aquifer It was this combination which proved to be fatal for the Baden Württenburg hamlet which soon formed gypsum and expanded by about 50 percent Over 270 buildings have suffered fractures in the ten years since and things don’t appear to be getting any better Photo: Ekem/Wikimedia Commons “We’ve been in crisis mode for ten years,” Mayor Michael Benitz told news agency DPA expanding the layers and forcing the earth upwards.” In some places the town of Staufen has risen nearly 62 centimeters and moved sideways by 45 centimeters and the town fears that several more buildings will need to be torn down to avoid accidental collapsing Demolitions are necessary because structures structures are not shifting uniformly but instead different parts of a building are shifting by slightly different amounts The financial fallout for the residents is immense with over 400 claims filed so far with a mediation body established to manage the crisis Photo: Baden de/Panoramio “Fractures have become our daily companions,” resident Csaba-Peter Gaspar told The Local His apartment is located in the town’s historic core and has suffered significant damage Pumps in Staufen are working around the clock to dredge groundwater and minimize imminent damage but while they have helped reduce the buildup of cement and slow down the rise of the ground to millimeters from the initial phase of several centimeters it is uncertain how long they will need to be in operation before things go back to normal “I’m working on the assumption that we’ll be grappling with this problem for several more years probably even decades,” Staufen’s mayor said Staufen is not the only community suffering as a result of the botched geothermal drilling Geologists from nearby Freiburg have reported similar issues unfolding in the nearby towns of Böblingen and Rudersberg the Berlin-based German Geothermal Association insists that the benefits outweighs the risks and that geothermal energy remains a smart and environmentally sound option Staufen is in for decades of devastation and ballooning repair costs Metrics details Stau is required for localization of pros RNA but not of Pros protein Loss of localization of Stau or of pros RNA alters GMC development but only in embryos with reduced levels of Pros protein suggesting that pros RNA and Pros protein act redundantly to specify GMC fate We also find that GMCs do not transcribe the pros gene showing that inheritance of pros RNA and/or Pros protein from the neuroblast is essential for GMC specification Constructing the cerebral cortex: neurogenesis and fate determination elegans HAM-1 protein in neuroblasts enables daughter cells to adopt distinct fates Maggot's hair and bug's eye: role of cell interactions and intrinsic factors in cell fate specification Neuroblasts: a model for the asymmetric division of stem cells Asymmetric segregation of the homeodomain protein Prospero during Drosophila development Asymmetric segregation of Numb and Prospero during cell division The prospero gene specifies cell fates in the Drosophila central nervous system Staufen protein associates with the 3′ UTR of bicoid mRNA to form particles that move in a microtubule-dependent manner a gene required to localize maternal RNA in the Drosophila egg and microfilaments regulate asymmetric protein localization in Drosophila neuroblasts Mating type switching in yeast controlled by asymmetric localization of ASH1 mRNA Actin-dependent localization of an RNA encoding a cell-fate determinant in yeast Asymmetric accumulation of Ash1p in postanaphase nuclei depends on a myosin and restricts yeast mating-type switching to mother cells Neurogenesis of the peripheral nervous system in Drosophila embryos: DNA replication patterns and cell lineages Anew visible light fluorochrome for confocal microscopy Evolution of neuroblast identity: seven-up and prospero expression reveal homologous and divergent cell fates in Drosophila and Schistocerca The role of segment polarity genes during Drosophila neurogenesis Miranda directs Propsero to a daughter cell during Drosophila asymmetric divisions Download references Spana for pros intron DNA; and C.Thummel and F This work was supported by an NIH postdoctoral fellowship to S.F Doe: These authors contributed equally to this work Department of Cell & Structural Biology Download citation