Wilms’ tumor 1-associating protein complex regulates alternative splicing and polyadenylation at potential G-quadruplex-forming splice site sequences
2021; Elsevier BV; Volume: 297; Issue: 5 Linguagem: Inglês
10.1016/j.jbc.2021.101248
ISSN1083-351X
AutoresKeiko Horiuchi, Takeshi Kawamura, Takao Hamakubo,
Tópico(s)RNA and protein synthesis mechanisms
ResumoWilms' tumor 1-associating protein (WTAP) is a core component of the N6-methyladenosine (m6A)-methyltransferase complex, along with VIRMA, CBLL1, ZC3H13 (KIAA0853), RBM15/15B, and METTL3/14, which generate m6A, a key RNA modification that affects various processes of RNA metabolism. WTAP also interacts with splicing factors; however, despite strong evidence suggesting a role of Drosophila WTAP homolog fl(2)d in alternative splicing (AS), its role in splicing regulation in mammalian cells remains elusive. Here we demonstrate using RNAi coupled with RNA-seq that WTAP, VIRMA, CBLL1, and ZC3H13 modulate AS, promoting exon skipping and intron retention in AS events that involve short introns/exons with higher GC content and introns with weaker polypyrimidine-tract and branch points. Further analysis of GC-rich sequences involved in AS events regulated by WTAP, together with minigene assay analysis, revealed potential G-quadruplex formation at splice sites where WTAP has an inhibitory effect. We also found that several AS events occur in the last exon of one isoform of MSL1 and WTAP, leading to competition for polyadenylation. Proteomic analysis also suggested that WTAP/CBLL1 interaction promotes recruitment of the 3′-end processing complex. Taken together, our results indicate that the WTAP complex regulates AS and alternative polyadenylation via inhibitory mechanisms in GC-rich sequences. Wilms' tumor 1-associating protein (WTAP) is a core component of the N6-methyladenosine (m6A)-methyltransferase complex, along with VIRMA, CBLL1, ZC3H13 (KIAA0853), RBM15/15B, and METTL3/14, which generate m6A, a key RNA modification that affects various processes of RNA metabolism. WTAP also interacts with splicing factors; however, despite strong evidence suggesting a role of Drosophila WTAP homolog fl(2)d in alternative splicing (AS), its role in splicing regulation in mammalian cells remains elusive. Here we demonstrate using RNAi coupled with RNA-seq that WTAP, VIRMA, CBLL1, and ZC3H13 modulate AS, promoting exon skipping and intron retention in AS events that involve short introns/exons with higher GC content and introns with weaker polypyrimidine-tract and branch points. Further analysis of GC-rich sequences involved in AS events regulated by WTAP, together with minigene assay analysis, revealed potential G-quadruplex formation at splice sites where WTAP has an inhibitory effect. We also found that several AS events occur in the last exon of one isoform of MSL1 and WTAP, leading to competition for polyadenylation. Proteomic analysis also suggested that WTAP/CBLL1 interaction promotes recruitment of the 3′-end processing complex. Taken together, our results indicate that the WTAP complex regulates AS and alternative polyadenylation via inhibitory mechanisms in GC-rich sequences. Alternative splicing (AS) is the process by which a single gene produces multiple mRNAs, thereby substantially increasing both the protein diversity and complexity. Ninety-five percent of human multiexon genes are alternatively spliced, and the majority of these transcripts vary across tissues, developmental stage, or physiological state (1Wang E.T. Sandberg R. Luo S. Khrebtukova I. Zhang L. Mayr C. Kingsmore S.F. Schroth G.P. Burge C.B. Alternative isoform regulation in human tissue transcriptomes.Nature. 2008; 456: 470-476Crossref PubMed Scopus (3776) Google Scholar, 2Pan Q. Shai O. Lee L.J. Frey B.J. Blencowe B.J. Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing.Nat. Genet. 2008; 40: 1413-1415Crossref PubMed Scopus (2733) Google Scholar). 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The Drosophila homologs of WTAP and VIRMA, Fl(2)d and Virilizer (Vir), respectively, are required for alternative splicing of Sex lethal (SXL), a master regulator of Drosophila sex determination pathway (26Granadino B. Campuzano S. Sánchez L. The Drosophila melanogaster fl(2)d gene is needed for the female-specific splicing of sex-lethal RNA.EMBO J. 1990; 9: 2597-2602Crossref PubMed Scopus (76) Google Scholar, 27Hilfiker A. Amrein H. Dübendorfer A. Schneiter R. Nöthiger R. The gene virilizer is required for female-specific splicing controlled by Sxl, the master gene for sexual development in Drosophila.Development. 1995; 121: 4017-4026Crossref PubMed Google Scholar). The female-specific expression of the RNA-binding protein SXL regulates the AS and/or translation of SXL, transformer(tra), and male-specific-lethal 2 (msl-2) pre-mRNA, which controls sex determination, sexual behavior, and dosage compensation. Recent studies have shown that this process is affected by m6A modifications, catalyzed by Mettl14 and Ime4 (the Drosophila homologs of METTL3) (28Lence T. Akhtar J. Bayer M. Schmid K. Spindler L. Ho C.H. Kreim N. Andrade-Navarro M.A. Poeck B. Helm M. Roignant J.Y. m 6 A modulates neuronal functions and sex determination in Drosophila.Nature. 2016; 540: 242-247Crossref PubMed Scopus (378) Google Scholar, 29Haussmann I.U. Bodi Z. Sanchez-Moran E. Mongan N.P. Archer N. Fray R.G. Soller M. m 6 A potentiates Sxl alternative pre-mRNA splicing for robust Drosophila sex determination.Nature. 2016; 540: 301-304Crossref PubMed Scopus (415) Google Scholar). Moreover, biochemical analyses have revealed the physical interactions between FL(2)d, Ime4, and Mettl14 (28Lence T. Akhtar J. Bayer M. Schmid K. Spindler L. Ho C.H. Kreim N. Andrade-Navarro M.A. Poeck B. Helm M. 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In contrast, Ime4-null mutants, lacking m6A marks in mRNA, remain viable and fertile (29Haussmann I.U. Bodi Z. Sanchez-Moran E. Mongan N.P. Archer N. Fray R.G. Soller M. m 6 A potentiates Sxl alternative pre-mRNA splicing for robust Drosophila sex determination.Nature. 2016; 540: 301-304Crossref PubMed Scopus (415) Google Scholar), indicating that Fl(2)d and Vir have distinct functions, independent of the m6A pathway. Since our previous proteomic data revealed an evolutionarily conserved formation of the WTAP complex and its interaction with general splicing factors (15Horiuchi K. Kawamura T. Iwanari H. Ohashi R. Naito M. Kodama T. Hamakubo T. Identification of Wilms' tumor 1-associating protein complex and its role in alternative splicing and the cell cycle.J. Biol. Chem. 2013; 288: 33292-33302Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar), here, we investigate whether the WTAP complex regulates AS in mammalian cells, independent or dependent on the m6A modifications. In this study, we performed comprehensive analysis of AS events regulated by WTAP complex via RNAi coupled with RNA-seq analysis using HUVECs, in which we identified interacting proteins of WTAP using shotgun proteomics (15Horiuchi K. Kawamura T. Iwanari H. Ohashi R. Naito M. Kodama T. Hamakubo T. Identification of Wilms' tumor 1-associating protein complex and its role in alternative splicing and the cell cycle.J. Biol. Chem. 2013; 288: 33292-33302Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar). We demonstrate that the WTAP complex major components WTAP, VIRMA, ZC3H13, and CBLL1 function as splicing regulators to promote exon skipping and intron retention, several of which occur in the last exon with competition between splicing and polyadenylation. Proteomic analysis of CBLL1-interacting proteins identified 3′-end processing factors: cleavage and polyadenylation specific factors CPSF1 and CPSF2, FIP1L1 and symplekin, suggesting a role for WTAP/CBLL1 in facilitating polyadenylation and determining the last exon. Further analysis of the sequences in the regulated introns/exons, as well as minigene analysis, revealed the involvement of G-quadruplex formation near 5′ and 3′ splice sites in AS regulation by WTAP, suggesting possible participation of the G-quadruplex-interacting/WTAP-interacting proteins, such as FMR1, FXR1, and FXR2. To determine the implications of the WTAP complex in gene expression and splicing regulation, we selected knockdown (KD) of WTAP complex major components (15Horiuchi K. Kawamura T. Iwanari H. Ohashi R. Naito M. Kodama T. Hamakubo T. Identification of Wilms' tumor 1-associating protein complex and its role in alternative splicing and the cell cycle.J. Biol. Chem. 2013; 288: 33292-33302Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar) (WTAP, VIRMA, ZC3H13, and CBLL1, hereinafter referred to as the "major components") in duplicates to obtain the overlapped events with higher confidence. RNA-seq on HUVECs treated with siRNA against the major components yielded on average ∼39 million uniquely mapped 150 nt pair-end sequencing reads of each library (Fig. 1A). KD efficiency was evaluated by immunoblot analysis (Fig. S1). Transcriptomic analysis with R software DESeq2 revealed 900, 420, 1090, 554 differentially expressed genes (DEGs) by KD of WTAP, VIRMA, ZC3H13, and CBLL1, respectively (FDR ≤0.01 and fold change ≥1.5, Fig. 1, B and C, left panel. TPM (transcripts per million) values are in Table S1). Gene ontology (GO) analysis of the DEGs showed enrichment of cellular processes linked to cell cycle, immune response, morphogenesis, and cell migration by WTAP, cell cycle and chemokine secretion by VIRMA, cell migration and the metabolic process by ZC3H13, and cell cycle, cell adhesion, and cell migration by CBLL1 (Fig. S2). Heatmap of the WTAP-regulated genes showed that majority of genes are similarly regulated by KD of VIRMA (74.7%), ZC3H13 (86.2%), and CBLL1 (73.0%) as well as KD of other WTAP-interacting proteins BCLAF1/THRAP3 (70.8%), RBM15/RBM15B (54.9%), METTL3 (57.4%), and METTL14 (59.0%) (one replicate each, as reference) (Fig. 1D and Fig. S3A). (Note, a similar trend was inferred when the change in gene expression was in the same direction as that of WTAP-KD and |Log2FC| ≥ 0.1). Differentially expressed isoforms (DEIs) were evaluated with EBSeq software using transcript estimated counts obtained with the RSEM software (https://deweylab.github.io/RSEM/), which is capable of detecting DEIs regulated by alternative splicing and/or alternative polyadenylation, as well as DEIs regulated by transcription and/or RNA stability. As expected, DEI analysis identified larger number of genes as differentially expressed, showing 1518, 1298, 2086, and 1255 isoforms (corresponding to 1261, 1129, 1660, and 1061 genes) that were upregulated by KD of WTAP, VIRMA, ZC3H13, and CBLL1, respectively, whereas the expressions of 1293, 1042, 1688, and 983 isoforms (corresponding to 1103, 952, 1459, and 880 genes) were downregulated by KD (FDR ≤0.01 and fold change ≥1.5, Fig. 1, B and C, right panel and TPM values are in Table S2). Regulation of the WTAP transcript by the WTAP complex (VIRMA, ZC3H13, and CBLL1) via competition between splicing and polyadenylation was also observed by DEI analysis, consistent with our previous study (Fig. S3B). Approximately 72% of the DEGs were also detected by DEI analysis. GO analysis of the DEIs showed similar enrichment of GO terms compared with the DEGs (Fig. S2). We next examined AS using the vast-tools software, which detects splicing events of five categories based on the database: alternative 5′ splice site (Alt 5′ss), alternative 3′ splice site (Alt 3′ss), alternative (cassette) exon skipping/inclusion (Alt Ex), microexons (MIC), and intron retention (IR). In total, 1016, 624, 748, and 517 AS events were identified as affected with a minimum difference in percent spliced-in (ΔPSI) of 15% by KD of WTAP, VIRMA, ZC3H13, and CBLL1, compared with control siRNA-treated samples, respectively (Fig. 2A and Table S3). All categories of AS events were affected (Fig. 2A); most of the events corresponded to cassette exon skipping/inclusion and IR in all KD samples. However, once normalized according to the overall distribution of AS events (identified as AS when at least one of the compared samples shows 10 < PSI < 90, Fig. 2A left), cassette exon skipping/inclusion was most enriched (Fig. 2B). KD of WTAP and ZC3H13 showed higher ΔPSI than that of VIRMA and CBLL1 in AltEx and IR (Fig. 2C). Comparing AS events among the major components KD samples, AS events were generally found to change in same direction in most cases (∼73%, 5% > ΔPSI in the same direction) as shown in the heatmap of ΔPSI of AS events in which at least one KD sample showed >15% of ΔPSI (1970 events, Fig. 2D and Fig. S4A). Similar trends were also observed by KD of other WTAP-interacting proteins, BCALF1/THRAP3, RBM15/RBM15B, METTL3, and METTL14 (∼65%, one replicate each, as reference). Seventy events were commonly affected by KD of all four major components (Fig. 2E) with ΔPSI ≥15%. Interestingly, reduced IR (37%) and cassette exon inclusion (43%) are the most affected AS categories by KD, indicating that WTAP complex inhibits splicing (Fig. 2F). There were 229 commonly regulated events with ΔPSI ≥15% in at least three of the major components exhibiting the similar distribution of the AS categories (Fig. S4B). As shown in Figure 3, the RT-PCR assays validated several AS events predicted as commonly regulated by the four major components. Several intron retention events (ABCD4, PTAR1, CCNT2, and IRF7 genes) were reduced by KD of the major components as well as the other WTAP-interacting proteins, BCLAF1/THRAP3, RBM15/15B, METTL3, and METTL14, while the opposite was also observed in the case of the YTHDC2 gene (Fig. 3A). The regulated intron retentions were often observed in the last exon (Fig. 3A, described below). Given that most of the affected IR is reduced by KD of WTAP complex, IR observed in YTHDC2 gene might be regulated indirectly, such as via other protein regulated by WTAP complex. In the case of MSL1 and WTAP genes, splicing was increased instead of alternative polyadenylation, leading to the formation of longer isoforms by KD of WTAP complex major components (Fig. 3B). We performed 3′ rapid amplification of cDNA ends (RACE) to isolate the alternative polyadenylation isoforms of MSL1 and WTAP. Sequencing the 3′ end of the mRNAs revealed an alternative polyadenylation event involving a poly(A) signal located in intron 3 and intron 5, respectively. Significant increase in ratio of exon inclusion and polyadenylated products were detected in MSL1 and WTAP transcripts by KD of the major components, suggesting that WTAP complex might promote polyadenylation (Fig. 3C). Increased exon inclusion was also validated in EPB49, EGFL7, EXD3, SUV420H2, HDAC7, and SLC2A6 genes (Fig. 3D). To examine sequence elements associated with the regulated AS events by the WTAP complex, common AS events that were affected by KD of at least three of the major components were analyzed using Matt software (http://matt.crg.eu/). Analysis of sequence feature of decreased retained introns (91 introns) compared with nonaffected retained introns (i.e., 3247 introns of 10 < percent intron retention (PIR) of control sample < 90, but not affected by WTAP complex KD) revealed that the introns more spliced by KD of WTAP complex are significantly short, more GC contents, and enriched in the last intron (Fig. 4A). The 3′ss displayed weaker branch points, as determined by SVM-BPfinder and SF1 binding site scores (34Corioni M. Antih N. Tanackovic G. Zavolan M. Krämer A. Analysis of in situ pre-mRNA targets of human splicing factor SF1 reveals a function in alternative splicing.Nucleic Acids Res. 2011; 39: 1868-1879Crossref PubMed Scopus (44) Google Scholar, 35Corvelo A. Hallegger M. Smith C.W. 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Analysis of sequence feature of the increased exon inclusion (75 exons) compared with the nonaffected exon skipping/inclusion events (i.e., 978 exons of 10 < PSI of control sample < 90, but not affected by WTAP complex KD) revealed that the exons that were more included by KD of WTAP complex were more GC-rich and marginally longer compared with nonaffected exons (Fig. 4B). The flanking upstream and downstream introns display significantly higher GC contents, weaker branch points, and are significantly shorter compared with those of nonaffected exons (Fig. 4B). Taken together, these results indicate that the WTAP complex represses splicing of the introns with a short length and higher GC contents. The relation of GC content and intron length has been described. Bioinformatic study on gene structure across various eukaryotes revealed that the mammalian genomes exhibit two groups of exons: GC-rich exons flanked by short introns and GC-poor exons flanked by long introns with lower GC content than the exons, which may be recognized by the splicing machinery under the different mechanism: intron and exon definition (36Amit M. Donyo M. Hollander D. Goren A. Kim E. Gelfman S. Lev-Maor G. Burstein D. Schwartz S. Postolsky B. Pupko T. Ast G. Differential GC content between exons and introns establishes distinct strategies of splice-site recognition.Cell Rep. 2012; 1: 543-556Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar). It, therefore, appears that the WTAP complex is involved in the regulation of the former group of the exons. The observed enrichment of GC-rich sequences in WTAP-regulated exons/introns prompted us to further examine the involvement of G-quadruplexes in AS regulation, as WTAP interacts with G-quadruplex interacting proteins, such as FMR1, FXR1, and FXR2 (15Horiuchi K. Kawamura T. Iwanari H. Ohashi R. Naito M. Kodama T. Hamakubo T. Identification of Wilms' tumor 1-associating protein complex and its rol
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