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• W3092662710 abstract "Recently, the US Food and Drug Administration (FDA) approved the first small interfering RNA (siRNA) drug, marking a significant milestone in the therapeutic use of RNA interference (RNAi) technology. However, off-target gene silencing by siRNA remains one of the major obstacles in siRNA therapy. Although siRNA off-target effects caused by a mechanism known for microRNA (miRNA)-mediated gene repression have been extensively discussed, whether RNAi can cause unintended cleavage through the effector protein AGO2 at sites harboring partially complementary sequences to the siRNA remains unknown. Here, we report a strategy to establish a comprehensive picture of siRNA cleaved and noncleaved off-targets by performing SpyCLIP using wild-type and catalytically inactive AGO2 mutants in parallel. Additionally, we investigated naturally occurring cleavage events mediated by endogenous miRNAs using the same strategy. Our results demonstrated that AGO2 SpyCLIP is a powerful method to identify both the cleaved and noncleaved targets of siRNAs, providing valuable information for improving siRNA design rules. Recently, the US Food and Drug Administration (FDA) approved the first small interfering RNA (siRNA) drug, marking a significant milestone in the therapeutic use of RNA interference (RNAi) technology. However, off-target gene silencing by siRNA remains one of the major obstacles in siRNA therapy. Although siRNA off-target effects caused by a mechanism known for microRNA (miRNA)-mediated gene repression have been extensively discussed, whether RNAi can cause unintended cleavage through the effector protein AGO2 at sites harboring partially complementary sequences to the siRNA remains unknown. Here, we report a strategy to establish a comprehensive picture of siRNA cleaved and noncleaved off-targets by performing SpyCLIP using wild-type and catalytically inactive AGO2 mutants in parallel. Additionally, we investigated naturally occurring cleavage events mediated by endogenous miRNAs using the same strategy. Our results demonstrated that AGO2 SpyCLIP is a powerful method to identify both the cleaved and noncleaved targets of siRNAs, providing valuable information for improving siRNA design rules. In mammalian cells, Argonaute (AGO) family proteins associate with small RNAs, including small interfering RNAs (siRNAs) and microRNAs (miRNAs), and assemble with other core components to form the RNA-induced silencing complex (RISC), regulating the expression of target RNAs that contain partially or fully complementary sites to the siRNAs and miRNAs.1Bartel D.P. Metazoan MicroRNAs.Cell. 2018; 173: 20-51Abstract Full Text Full Text PDF PubMed Scopus (1517) Google Scholar, 2Daugaard I. Hansen T.B. Biogenesis and Function of Ago-Associated RNAs.Trends Genet. 2017; 33: 208-219Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 3Wu L. Belasco J.G. Let me count the ways: mechanisms of gene regulation by miRNAs and siRNAs.Mol. Cell. 2008; 29: 1-7Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar, 4Carthew R.W. Sontheimer E.J. Origins and Mechanisms of miRNAs and siRNAs.Cell. 2009; 136: 642-655Abstract Full Text Full Text PDF PubMed Scopus (3633) Google Scholar Among the four members of the AGO family of proteins expressed in mammals, AGO2 is the only one that exhibits endonuclease activity and cleaves target RNAs that contain fully complementary sites to small RNAs, resulting in RNA interference (RNAi).5Liu J. Carmell M.A. Rivas F.V. Marsden C.G. Thomson J.M. Song J.J. Hammond S.M. Joshua-Tor L. Hannon G.J. Argonaute2 is the catalytic engine of mammalian RNAi.Science. 2004; 305: 1437-1441Crossref PubMed Scopus (1980) Google Scholar,6Meister G. Landthaler M. Patkaniowska A. Dorsett Y. Teng G. Tuschl T. Human Argonaute2 mediates RNA cleavage targeted by miRNAs and siRNAs.Mol. Cell. 2004; 15: 185-197Abstract Full Text Full Text PDF PubMed Scopus (1407) Google Scholar In contrast, the ability to mediate translational repression and accelerated deadenylation of the target RNAs harboring partially complementary sequences to the miRNAs and siRNAs is shared by all four mammalian AGOs,3Wu L. Belasco J.G. Let me count the ways: mechanisms of gene regulation by miRNAs and siRNAs.Mol. Cell. 2008; 29: 1-7Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar,7Fabian M.R. Sonenberg N. Filipowicz W. Regulation of mRNA translation and stability by microRNAs.Annu. Rev. Biochem. 2010; 79: 351-379Crossref PubMed Scopus (2175) Google Scholar a mechanism known for miRNA-mediated gene repression and siRNA off-target effects. siRNA has become a powerful tool to silence target genes that has therapeutic potential.8Pecot C.V. Calin G.A. Coleman R.L. Lopez-Berestein G. Sood A.K. RNA interference in the clinic: challenges and future directions.Nat. Rev. Cancer. 2011; 11: 59-67Crossref PubMed Scopus (640) Google Scholar,9Castanotto D. Rossi J.J. The promises and pitfalls of RNA-interference-based therapeutics.Nature. 2009; 457: 426-433Crossref PubMed Scopus (1018) Google Scholar Many RNAi therapeutics using chemically synthesized siRNAs have been undergoing clinical trials for the treatment of various diseases. Patisiran is the first FDA (US Food and Drug Administration)-approved siRNA drug to treat hereditary transthyretin-mediated amyloidosis (hATTR) by silencing transthyretin.10Adams D. Gonzalez-Duarte A. O’Riordan W.D. Yang C.C. Ueda M. Kristen A.V. Tournev I. Schmidt H.H. Coelho T. Berk J.L. et al.Patisiran, an RNAi Therapeutic, for Hereditary Transthyretin Amyloidosis.N. Engl. J. Med. 2018; 379: 11-21Crossref PubMed Scopus (1178) Google Scholar,11Wood H. FDA approves patisiran to treat hereditary transthyretin amyloidosis.Nat. Rev. Neurol. 2018; 14: 570Google Scholar Later, givosiran is approved by the FDA to treat adults with acute hepatic porphyria by silencing aminolevulinate synthase 1 (ALAS1).12Balwani M. Sardh E. Ventura P. Peiró P.A. Rees D.C. Stölzel U. Bissell D.M. Bonkovsky H.L. Windyga J. Anderson K.E. et al.ENVISION InvestigatorsPhase 3 Trial of RNAi Therapeutic Givosiran for Acute Intermittent Porphyria.N. Engl. J. Med. 2020; 382: 2289-2301Crossref PubMed Scopus (165) Google Scholar,13Scott L.J. Givosiran: First Approval.Drugs. 2020; 80: 335-339Crossref PubMed Scopus (146) Google Scholar More siRNA drugs, such as inclisiran14Fitzgerald K. Frank-Kamenetsky M. Shulga-Morskaya S. Liebow A. Bettencourt B.R. Sutherland J.E. Hutabarat R.M. Clausen V.A. Karsten V. Cehelsky J. et al.Effect of an RNA interference drug on the synthesis of proprotein convertase subtilisin/kexin type 9 (PCSK9) and the concentration of serum LDL cholesterol in healthy volunteers: a randomised, single-blind, placebo-controlled, phase 1 trial.Lancet. 2014; 383: 60-68Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar and ARC-52015Yuen M.F. Chan H.L.Y. Given B. Hamilton J. Schluep T. Lewis D.L. Lai C.L. Locarnini S. Lau J.Y. Gish R.G. Phase II, dose ranging study of ARC-520, a siRNA-based therapeutic, in patients with chronic hepatitis B virus infection.Hepatology. 2014; 60: 1280AGoogle Scholar to treat hypercholesterolemia and chronic hepatitis B virus infection, respectively, are at different developmental phases. For stable expression in cells, siRNAs can be generated from short hairpin RNA (shRNA) precursors that are transcribed from RNA polymerase III promoters such as U6 and H1.16Brummelkamp T.R. Bernards R. Agami R. A system for stable expression of short interfering RNAs in mammalian cells.Science. 2002; 296: 550-553Crossref PubMed Scopus (3926) Google Scholar,17Paddison P.J. Caudy A.A. Bernstein E. Hannon G.J. Conklin D.S. Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells.Genes Dev. 2002; 16: 948-958Crossref PubMed Scopus (1294) Google Scholar Alternatively, siRNAs can be produced from a miRNA gene by substituting the mature miRNA sequence with the siRNA sequence via RNA polymerase II promoters.18Stegmeier F. Hu G. Rickles R.J. Hannon G.J. Elledge S.J. A lentiviral microRNA-based system for single-copy polymerase II-regulated RNA interference in mammalian cells.Proc. Natl. Acad. Sci. USA. 2005; 102: 13212-13217Crossref PubMed Scopus (441) Google Scholar,19Dickins R.A. Hemann M.T. Zilfou J.T. Simpson D.R. Ibarra I. Hannon G.J. Lowe S.W. Probing tumor phenotypes using stable and regulated synthetic microRNA precursors.Nat. Genet. 2005; 37: 1289-1295Crossref PubMed Scopus (428) Google Scholar Despite the difference in their origins, all siRNAs have to incorporate into the four AGO family proteins to execute their functions, thus inevitably causing off-target effects resulting from imperfect base pairing of the siRNAs with the target RNAs; through this mechanism, they can repress nontarget genes via the miRNA-mediated repression pathway,20Jackson A.L. Bartz S.R. Schelter J. Kobayashi S.V. Burchard J. Mao M. Li B. Cavet G. Linsley P.S. Expression profiling reveals off-target gene regulation by RNAi.Nat. Biotechnol. 2003; 21: 635-637Crossref PubMed Scopus (1857) Google Scholar,21Jackson A.L. Burchard J. Schelter J. Chau B.N. Cleary M. Lim L. Linsley P.S. Widespread siRNA “off-target” transcript silencing mediated by seed region sequence complementarity.RNA. 2006; 12: 1179-1187Crossref PubMed Scopus (711) Google Scholar posing a major challenge for RNAi application in clinical practice. However, whether AGO2-siRNA can also cause unintended cleavage of RNA targets in mammalian cells guided by partially complementary target sites remains unknown. Although cleaving target mRNAs is known to be the major mechanism by which miRNAs silence gene expression in plants,22Reinhart B.J. Weinstein E.G. Rhoades M.W. Bartel B. Bartel D.P. MicroRNAs in plants.Genes Dev. 2002; 16: 1616-1626Crossref PubMed Scopus (1654) Google Scholar,23Tang G. Reinhart B.J. Bartel D.P. Zamore P.D. A biochemical framework for RNA silencing in plants.Genes Dev. 2003; 17: 49-63Crossref PubMed Scopus (780) Google Scholar only a few cases of cleavage have been reported for endogenous miRNAs in mammals.24Yekta S. Shih I.H. Bartel D.P. MicroRNA-directed cleavage of HOXB8 mRNA.Science. 2004; 304: 594-596Crossref PubMed Scopus (1381) Google Scholar,25Hansen T.B. Wiklund E.D. Bramsen J.B. Villadsen S.B. Statham A.L. Clark S.J. Kjems J. miRNA-dependent gene silencing involving Ago2-mediated cleavage of a circular antisense RNA.EMBO J. 2011; 30: 4414-4422Crossref PubMed Scopus (663) Google Scholar Whether AGO2-miRNA-mediated cleavage should be considered an important mechanism for gene regulation in mammalian cells is still being debated. The cleavage product by AGO2 usually undergoes fast decay, which renders the detection of the cleavage intermediates very difficult. To date, global surveys of AGO2-mediated cleavage events in mammalian cells are rare6Meister G. Landthaler M. Patkaniowska A. Dorsett Y. Teng G. Tuschl T. Human Argonaute2 mediates RNA cleavage targeted by miRNAs and siRNAs.Mol. Cell. 2004; 15: 185-197Abstract Full Text Full Text PDF PubMed Scopus (1407) Google Scholar,26Bracken C.P. Szubert J.M. Mercer T.R. Dinger M.E. Thomson D.W. Mattick J.S. Michael M.Z. Goodall G.J. Global analysis of the mammalian RNA degradome reveals widespread miRNA-dependent and miRNA-independent endonucleolytic cleavage.Nucleic Acids Res. 2011; 39: 5658-5668Crossref PubMed Scopus (62) Google Scholar due to the lack of an accurate experimental approach to determine AGO2-small RNA-target RNA interaction maps. In this study, we used our recently developed SpyCLIP method,27Zhao Y. Zhang Y. Teng Y. Liu K. Liu Y. Li W. Wu L. SpyCLIP: an easy-to-use and high-throughput compatible CLIP platform for the characterization of protein-RNA interactions with high accuracy.Nucleic Acids Res. 2019; 47: e33Crossref PubMed Scopus (9) Google Scholar a covalent link-based crosslinking and immunoprecipitation (CLIP) method with high efficiency and accuracy, along with catalytically inactive AGO2 mutants to systematically examine the siRNA off-targets and the AGO2-mediated target RNA cleavage events within human cells when associated with siRNAs that are produced from shRNA precursors. We also investigated the AGO2-mediated cleavage activities mediated by endogenous miRNAs and revealed the widespread potential miRNA-induced cleavage events in the cells. Our results demonstrated that SpyCLIP using wild-type and cleavage-incompetent AGO2 is a valuable tool for the global study of AGO2-mediated cleavage activities, regardless of whether it associates with exogenous siRNAs or endogenous miRNAs. In mammalian cells, the AGO2-programmed RISC complex uses siRNA to cleave target RNAs at a discrete position, usually between the 10th and 11th nucleotides downstream of the first nucleotide that was complementary to the siRNA guide strand, and the resultant RNA fragment is then rapidly degraded by exoribonucleases.28Martinez J. Patkaniowska A. Urlaub H. Lührmann R. Tuschl T. Single-stranded antisense siRNAs guide target RNA cleavage in RNAi.Cell. 2002; 110: 563-574Abstract Full Text Full Text PDF PubMed Scopus (1162) Google Scholar This AGO2-siRNA-mediated target RNA cleavage and degradation process is very quick; thus, identification of AGO2-siRNA cleavage sites is a major technical challenge. We hypothesized that a catalytically inactive AGO2-programmed RISC would accomplish siRNA-target RNA recognition and binding without the cleavage event, which would retain the RISC at the binding site and, therefore, permit accurate detection of all binding sites and could distinguish on- and off-target sites compared with those of the wild-type AGO2-programmed RISC counterpart (Figure 1). The UV CLIP method is a highly advanced high-throughput tool that can capture all the RNAs that bound to a specific RNA-binding protein with precise location information of the binding sites and has been successfully used in the identification of miRNA-mRNA interaction maps.29Chi S.W. Zang J.B. Mele A. Darnell R.B. Argonaute HITS-CLIP decodes microRNA-mRNA interaction maps.Nature. 2009; 460: 479-486Crossref PubMed Scopus (1377) Google Scholar Therefore, we adopted our recently developed SpyCLIP to systematically detect the siRNA on- and off-target effects. We first constructed Lenti-X 293T cell lines that stably expressed FLAG- and Spy-tagged AGO2 or its catalytically inactive mutant AGO2-D597A.5Liu J. Carmell M.A. Rivas F.V. Marsden C.G. Thomson J.M. Song J.J. Hammond S.M. Joshua-Tor L. Hannon G.J. Argonaute2 is the catalytic engine of mammalian RNAi.Science. 2004; 305: 1437-1441Crossref PubMed Scopus (1980) Google Scholar The expression of these ectopic AGO2 proteins was induced to near-endogenous levels by adjusting the doxycycline (Dox) concentration (Figure 2A). We then transiently transfected wild-type or mutant AGO2-expressing stable cells with shRNA vectors that produce siRNA sequences targeting the TP53 or LMNA gene (Figure S1). Luciferase reporter mRNAs harboring fully complementary sequences to the siRNAs within the 3′ untranslated region (3′ UTR) demonstrated the strong silencing effects of these siRNAs in wild-type AGO2-expressing cells but the impaired silencing effects in mutant AGO2-expressing cells, which confirmed the effectiveness of these siRNAs and the dominant-negative function of the catalytically inactive AGO2 protein (Figure 2B). Next, we performed SpyCLIP of both the wild-type and catalytically inactive AGO2 proteins in the cells transfected with TP53- or LMNA-specific shRNA. We found that the siRNA cleavage site on the TP53 or LMNA mRNA can be precisely located by comparing the signal strength of the potential sites identified using wild-type AGO2 and its catalytically inactive counterparts (Figure 2C); efficient cleavage by AGO2-siRNA can dramatically reduce the target mRNA concentration and, therefore, decrease the signal strength of wild-type AGO2 SpyCLIP. The siRNA on-target binding site identified by our strategy exhibited a binding strength that is similar to typical miRNA binding sites in the cells expressing catalytically inactive AGO2 (Figure S2). Thus, we concluded that SpyCLIP using the wild-type and catalytically inactive AGO2 proteins in parallel can faithfully capture the cleavage sites of a given siRNA. Measuring the signal strength of each AGO2-siRNA binding site is critical for identifying potential siRNA-mediated off-target sites with high confidence. We first calculated the signal strength of the wild-type AGO2 SpyCLIP clusters that either depend on or do not depend on siRNA expression by comparing the SpyCLIP clusters in the cells transfected with or without shRNAs (Figure 3A). The signal strength of the AGO2-bound sites independent of shRNA expression did not show obvious differences after introducing shRNA, which indicates that introducing siRNA from shRNA-expressing vectors did not cause significant alterations in the AGO2 binding events guided by endogenous miRNAs. To identify the siRNA-related AGO2-bound sites without introducing an artificial bias, we calculated the fold enrichment of the read counts with siRNA expression versus without siRNA expression in each wild-type AGO2 SpyCLIP cluster. Only the clusters exhibiting a fold enrichment higher than the k value30Jung I. Park J.C. Kim S. piClust: a density based piRNA clustering algorithm.Comput. Biol. Chem. 2014; 50: 60-67Crossref PubMed Scopus (24) Google Scholar were defined as siRNA-enriched clusters (Figure S3). Following this data-processing pipeline, we identified all siRNA-enriched clusters targeted by the TP53 and LMNA siRNAs and compared the abundance of the siRNA-enriched clusters in the wild-type and mutant AGO2 SpyCLIP. Clusters that were significantly enriched by AGO2 mutant SpyCLIP in the cells transfected with shRNAs were defined as off-target sites. The on-target sites (indicated by red circles in Figure 3B) for both the TP53 and LMNA siRNAs are the most significantly enriched ones in mutant AGO2 SpyCLIP compared with the wild-type AGO2 counterparts, which was consistent with the model (Figure 1) that RNAs bearing siRNA on-target sites are cleaved by AGO2 and degraded rapidly, whereas RNAs bearing on-target sites bound with catalytically inactive AGO2 remain stable. The majority of the identified siRNA-dependent clusters showed similar signal strength in the wild-type and mutant AGO2 SpyCLIP data (indicated by black dots in Figure 3B). Notably, a portion of the off-target sites had relatively low signal strength in wild-type AGO2 SpyCLIP but significantly higher signal strength in mutant AGO2 SpyCLIP (>2-fold, indicated by red dots in Figure 3B), an interesting feature reminiscent of on-target sites by cleavage mechanism. We proposed that these off-target sites might also undergo AGO2-mediated cleavage events as the on-target site did and defined them as AGO2-cleavage-activity-dependent off-target sites (cleaved off-target sites). The remaining SpyCLIP-identified off-target sites that exhibited 1.4- to 2-fold enrichment over their counterparts in wild-type AGO2 SpyCLIP were defined as AGO2-cleavage-activity-independent off-target sites (noncleaved off-target sites, indicated by blue dots in Figure 3B). Intriguingly, analysis of the genomic distribution of the TP53 and LMNA siRNA off-target sites showed a significant portion of SpyCLIP-defined cleaved off-target sites located within the coding regions (CDSs) of mRNAs, a region usually occupied by cleavage-competent siRNAs (Figure 3C), which further implies that these cleaved off-targets might be AGO2 cleavage activity dependent. By contrast, the noncleaved off-targets had a strong preference for the 3′ UTRs of protein-coding genes, an action mode similar to that of miRNAs (Figure 3C). Analysis of the base-pairing tendency of these off-target sites with the guide strand of the TP53 and LMNA siRNAs demonstrated a preference for the 5′ half of the siRNA sequence, which corresponded to the seed region of siRNAs (Figure S4). These observations agreed with previous reports showing that the off-target effects of siRNAs are mediated by a miRNA-like mechanism.31Anderson E.M. Birmingham A. Baskerville S. Reynolds A. Maksimova E. Leake D. Fedorov Y. Karpilow J. Khvorova A. Experimental validation of the importance of seed complement frequency to siRNA specificity.RNA. 2008; 14: 853-861Crossref PubMed Scopus (103) Google Scholar,32van Dongen S. Abreu-Goodger C. Enright A.J. Detecting microRNA binding and siRNA off-target effects from expression data.Nat. Methods. 2008; 5: 1023-1025Crossref PubMed Scopus (209) Google Scholar In addition, we performed RNA sequencing of cells transfected with different siRNAs and found that endogenous TP53 and LMNA were, indeed, downregulated upon corresponding siRNA treatment (Figures 3D and 3E), suggesting effective siRNA function within the cells. By comparing the fold changes of the expression levels of genes bearing SpyCLIP-identified cleaved and noncleaved off-target sites upon corresponding siRNA transfection, we found that the genes bearing cleaved off-target sites, indeed, exhibited notably higher levels of downregulation than those of the genes bearing noncleaved off-target sites (Figure 3F). In total, we identified 16 and 19 potential cleaved off-target sites for TP53 and LMNA siRNAs, respectively (Figures S5 and S6). To experimentally verify these potential cleaved off-target sites, six candidates from AGO2-siTP53 or AGO2-siLMNA SpyCLIP-defined cleaved off-target sites were randomly picked for validation. We inserted one copy of the candidate site into the 3′ UTR of a luciferase reporter and compared its expression levels in wild-type AGO2-expressing cells and catalytically inactive AGO2-expressing cells. To prevent endogenous AGO2 from masking the dominant-negative effect of mutant AGO2, we transiently knocked down endogenous AGO2 (Figure S7) by transfecting the cells with a siRNA targeting the 3′ UTR of endogenous AGO2 mRNA, which would not interfere with the expression of AGO2 mRNA bearing a 3′ UTR composed of the herpes simplex virus thymidine kinase (HSV TK) poly(A) signal sequence transcribed from the lentiviral vector. Consistent with our bioinformatic-defined cleaved off-target sites, the majority of these candidates showed marked repression by wild-type AGO2 (indicated by blue bars in Figures 3I and 3J). Three of the siTP53 candidates and two of the siLMNA candidates exhibited significant de-repression upon AGO2 mutant expression (compare blue and orange bars indicated with three asterisks in Figures 3I and 3J), indicating that these sites are responsive to AGO2 cleavage activity. These validated cleaved off-target sites appeared to require more extensive base paring, exhibiting stronger base pairing to the mRNAs in the middle and/or in the 3′ region of the siRNAs (Figures 3G and 3H), consistent with previous studies.33Bartel D.P. MicroRNAs: target recognition and regulatory functions.Cell. 2009; 136: 215-233Abstract Full Text Full Text PDF PubMed Scopus (15317) Google Scholar Strikingly, we identified cleaved off-target sites that break the “2–8 seed match” rule for both siRNAs we tested (NUP155 for siTP53 and CTNNAL1 for siLMNA; Figures 3G and 3H), which had higher levels of overall base pairing and almost perfect base pairing in the 3′ region, an observation that expands our current understanding of AGO2-small RNA target recognition and repression mechanisms. Also, it should be noted that both the top 1 cleaved off-target candidate for siTP53 and the siLMNA identified by SpyCLIP showed elevated expression levels in RNA-sequencing data (Figures S5 and S6), which provided a typical example that the secondary effects often complicated gene expression alteration-based transcriptome or proteome analysis strategies and highlighted the unique value of developing a straightforward biochemical strategy to capture off-target sites. To further decipher the AGO2-mediated cleavage events guided by endogenous miRNAs in mammalian cells, we performed SpyCLIP against wild-type AGO2; its two catalytically inactive counterparts, AGO2-D597A and AGO2-D669A; and naturally cleavage-incompetent AGO1 and analyzed the clusters enriched within the catalytically inactive AGOs according to the strategy described in Figure 1A. The clusters identified from AGO2-D597A and AGO2-D669A were almost identical (Figure 4A), which confirmed that SpyCLIP was a highly reproducible tool to generate RNA-binding maps for AGOs. We thus combined the SpyCLIP data of AGO2-D597A and AGO2-D669A, hereinafter referred to as data from the AGO2 mutant, for further analyses. The majority of clusters identified by SpyCLIP are shared between AGO2 and the AGO2 mutant (defined as AGO2 noncleaved sites), which is consistent with the notion that the miRNA-mediated noncleavage repressive pathway is the dominant function of AGO2 in mammalian cells. Intriguingly, clusters identified by AGO1 SpyCLIP showed a strong correlation with those identified by the AGO2 mutant (Figure 4B). Further analysis demonstrated that the AGO2 mutant-enriched clusters, when compared with those of wild-type AGO2, also exhibited a high enrichment ratio in AGO1 SpyCLIP clusters (Figure 4C). These observations suggest that naturally cleavage-incompetent AGO1 functionally mimics the cleavage-incompetent AGO2 mutant. Next, we focused on the clusters from AGO1 and AGO2 mutant SpyCLIP that exhibited at least 2-fold enrichment over their counterparts detected in wild-type AGO2 SpyCLIP data and defined them as endogenous AGO2 cleavage sites. The distribution of the clusters enriched in wild-type AGO1 and AGO2 mutant showed a preference for 3′ UTRs, whereas clusters enriched in AGO2-mediated cleaved sites showed a significant preference for the coding regions (Figure 4D), and the distribution pattern did not change when corrected by region size. These results were consistent with the notion that, unlike miRNA-mediated repression that required persistent association of the RISC with the target RNA,34Zhao Y. Lin J. Xu B. Hu S. Zhang X. Wu L. MicroRNA-mediated repression of nonsense mRNAs.eLife. 2014; 3: e03032Crossref PubMed Scopus (22) Google Scholar AGO2-mediated cleavage is a process with a fast turnover (cut and run) and, therefore, has an increased ability to target mRNA coding regions despite ribosome trafficking. We further investigated the base-pairing rules of AGO2-mediated cleavage sites and noncleaved sites. The AGO2-mediated noncleaved sites generally exhibited the highest base-pairing tendency within nucleotides 1–8 of the miRNAs, followed by nucleotides 12–19, consistent with previous results.27Zhao Y. Zhang Y. Teng Y. Liu K. Liu Y. Li W. Wu L. SpyCLIP: an easy-to-use and high-throughput compatible CLIP platform for the characterization of protein-RNA interactions with high accuracy.Nucleic Acids Res. 2019; 47: e33Crossref PubMed Scopus (9) Google Scholar However, the AGO2-mediated cleavage sites have less requirement for base pairing within nucleotides 1–8 of the miRNAs but required additional base pairing of the 9th, 10th, and 11th nucleotides of the miRNAs and extra base pairing in the middle and 3′ regions of the miRNAs (Figure 4E; Figure S8). The duplexes of miR-346 and miR-339-5p and their SpyCLIP-identified potential cleavage target CD2BP2 and TOP3A mRNAs are shown as examples (Figure 4F). The exact base-pairing rule of endogenous AGO2-miRNA-mediated cleavage warrants further investigations. Gene Ontology (GO) analysis of these potential AGO2-miRNA cleaved target RNAs showed enrichment in the processes of DNA topological change, acrosome assembly, and several other critical cellular processes (Figure S9). In summary, AGO2-mediated cleavage of RNAs by endogenous miRNAs might not be as rare as previously estimated. Performing SpyCLIP against wild-type and catalytically inactive AGO2 proteins overcomes the limitation of rapid degradation of cleaved RNA intermediates and provides a convenient option for genome-wide identification of endogenous AGO2 cleavage targets. The off-target effect of RNAi is one of the major obstacles for its therapeutic applications. miRNA-like off-target effects (cleavage-independent RNA degradation and translational repression) resulting from imperfect base pairing of the siRNAs with nontarget binding sites have been investigated extensively. In addition, unexpected cleavage events are dangerous, because the cleavage mechanism can reduce the abundance of recognized mRNA more significantly by leading to much faster decay of the targets than miRNA-mediated repressive mechanisms. Although the number of AGO2-cleaved off-target sites we identified in this study is lower than that of the miRNA-like noncleaved off-target sites, it should be noted that among the ∼22,000 genes encoded in the human genome, only fewer than 8,000 genes are expressed in each type of cell, and more cleavage-dependent off-target sites would be detected if more cell lines were analyzed. This phenomenon should not be overlooked in future siRNA applications in vivo. To date, no reliable experimental method or computational algorithm has been available to effectively identify off-target sites in cells. Although transcriptome or proteome analyses can provide useful information on gene expression alterations in cells transfected with siRNA, genome-wide changes represent a mixture of both primary and secondary effects that are indistinguishable with our current knowledge and, therefore, could not provide sufficient information to improve the design rule for siRNAs. In this study, we reported a straightforward strategy to establish a comprehensive picture of siRNA off-targets by performing SpyCLIP using wild-type AGO2 and catalytically inactive AGO2 mutants in parallel. Using this strategy, we successfully identified many active off-target sites for the siRNA we tested and revealed unexpected base-pairing rules (seed independent) for AGO2-siRNA-guided target cleavage. This strategy" @default.
• W3092662710 created "2020-10-22" @default.
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• W3092662710 date "2020-12-01" @default.
• W3092662710 modified "2023-10-02" @default.
• W3092662710 title "Identifying Cleaved and Noncleaved Targets of Small Interfering RNAs and MicroRNAs in Mammalian Cells by SpyCLIP" @default.
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