Splicing inhibition of U2AF65 leads to alternative exon skipping Sunghee Choa, Heegyum Moona, Tiing Jen Loha, Ha Na Janga, Yongchao Liua, Jianhua Zhoub, Takbum Ohnc, Xuexiu Zhenga, and Haihong Shena,1 aSchool of Life Sciences, Gwangju Institute of Science and Technology, Gwangju 500-712, Korea; bJiangSu Key Laboratory of Neuroregeneration, Nantong University, Nantong 226001, China; and cDepartment of Cellular & Molecular Medicine, College of Medicine, Chosun University, Gwangju 501-759, Korea Edited by Michael R. Green, University of Massachusetts Medical School, Worcester, MA, and approved June 11, 2015 (received for review January 11, 2015) U2 snRNP auxiliary factor 65 kDa (U2AF65) is a general splicing factor mutations in SMN2 pre-mRNA cause predominantly skipping of that contacts polypyrimidine (Py) tract and promotes prespliceo- exon 7, which produces SMNΔ7, a truncated and less stable pro- some assembly. In this report, we show that U2AF65 stimulates al- tein with reduced self-oligomerization activity. Alternative exon 7 ternative exon skipping in spinal muscular atrophy (SMA)-related splicing of SMN pre-mRNA was modulated by orchestrated RNA– survival motor neuron (SMN) pre-mRNA. A stronger 5′ splice-site protein and protein–protein interactions, secondary structures of mutation of alternative exon abolishes the stimulatory effects of RNA, and RNA sequences (25–27). Among the mutations on U2AF65. U2AF65 overexpression promotes its own binding only on SMN2 pre-mRNA, the most functionally understood one is the the weaker, not the stronger, Py tract. We further demonstrate C-to-U point mutation on exon 7, which plays an important role in that U2AF65 inhibits splicing of flanking introns of alternative exon alternative splicing of exon 7 (25–27). In vitro analysis using HeLa 65 in both three-exon and two-exon contexts. Similar U2AF effects nuclear extract and S100 extract demonstrates that SRSF1 pro- were observed in Fas (Apo-1/CD95) pre-mRNA. Strikingly, we dem- motes exon 7 inclusion through contacting the enhancer sequence 65 onstrate that U2AF even inhibits general splicing of adenovirus on exon 7 of SMN1 pre-mRNA, and that C-to-U mutation on major late (Ad ML) or β-globin pre-mRNA. Thus, we conclude that SMN2 pre-mRNA disrupts SRSF1 binding and then conse- 65 U2AF possesses a splicing Inhibitory function that leads to alter- quently disrupts the enhancer function of SRSF1 (28). However, native exon skipping. cell-based analysis shows a different result, indicating that SRSF1 CELL BIOLOGY does not play an essential role in SMN exon 7 splicing (29). In 65 U2AF | pre-mRNA splicing | splicing inhibition | exon exclusion | SMN contrast, cell transfection analysis demonstrates that heteroge- neous nuclear ribonucleoprotein (hnRNP) A1 interacts with the re-mRNA splicing is a process in which noncoding intron C-to-U mutation on SMN2 pre-mRNA to inhibit exon 7 splicing Psequences are removed and exon sequences are then ligated (29). A possible explanation for these different results is that different together (1, 2). Pre-mRNA splicing is carried out by spliceosome, analysis systems could provide different conclusions. a large RNA–protein complex that contains five small nuclear Although the roles of U2AF65 in alternative splicing are ver- ribonucleoproteins (U snRNPs) and more than 100 additional pro- ified to some extent, the function and mechanism are unclear. teins (3). Pre-mRNA splicing occurs in the consensus sequences at The previous reports have shown that U2AF65 roles in alterna- the 5′ splice-site, 3′ splice-site, and branch point that are necessary tive splicing are the target of alternative splicing regulatory fac- for splicing. The sequence between 3′ AG dinucleotide and branch tors, as demonstrated with increased U2AF65 binding by other point is the polypyrimidine (Py) tract that directs spliceosome as- splicing regulatory proteins (30, 31). More recently, genome- sembly on the 3′ splice-site. Alternative splicing provides an im- wide analysis has demonstrated that upstream intronic binding of portant regulatory mechanism in higher eukaryotes for multiple U2AF65 interferes with the immediate downstream 3′ splice-site proteins produced from a single gene (4, 5). 65 The U2 snRNP auxiliary factor 65 kDa (U2AF ) exists as a Significance heterodimer with U2AF35 (6). U2AF65 contains three C-terminal RNA recognition motifs (RRMs) and an N-terminal arginine/ Transcription is a biological procedure in which DNA is tran- serine-rich (RS) domain (7, 8). Using U2AF65 depletion/adding scribed to an RNA molecule. However, only fragments of this back technology with in vitro HeLa nuclear extract, it was dem- RNA are needed for protein synthesis. These fragments are onstrated that U2AF65 is an essential splicing factor (9). Whereas exons that are interrupted by introns. Introns are removed by U2AF65 binds to Py tract to promote prespliceosome assembly 35 so-called RNA splicing process. Some exons could be alterna- and branchpoint/U2 snRNA base pairing, U2AF plays a role in tively included or excluded from the final RNA molecule. In this the 3′ splice-site (10, 11). As U2AF65 prefers high C/U-rich se- 65 study, we have found that U2 snRNP auxiliary factor 65 kDa quences in the Py tract, a stronger interaction between U2AF 65 65 (U2AF ), a general splicing regulator, can significantly pro- and Py tract promotes prespliceosome assembly (12). U2AF is mote the exclusion of alternative exons. Strikingly, U2AF65 also essential in vertebrate development (13, 14). Its expression suppresses flanking intron splicing of alternative exons, and level is related to myotonic dystrophy, cystic fibrosis, and cancers even constitutive intron splicing. We deduce that the stimula- (15, 16). tory effects of U2AF65 on alternative exon exclusion are in- Proximal spinal muscular atrophy (SMA) is an autosomal re- duced by the splicing inhibitory effects of U2AF65. cessive genetic disease (17) and a leading cause of infant mor- tality. The motor neurons in the anterior horn of spinal cord are Author contributions: S.C. and H.S. designed research; S.C., H.M., T.J.L., H.N.J., and Y.L. severely damaged in patients with type 1 SMA, usually leading to performed research; S.C., Y.L., J.Z., T.O., X.Z., and H.S. analyzed data; and X.Z. and H.S. death before age 2 y as a result of a lack of respiratory support wrote the paper. (18, 19). In patients with SMA, the SMN1 gene is deleted or The authors declare no conflict of interest. mutated, whereas the SMN2 gene, a duplicate of the SMN1 gene, This article is a PNAS Direct Submission. is included (20). SMN2 genomic DNA contains a few nucleotide Freely available online through the PNAS open access option. mutations compared with SMN1 (21, 22). Full-length SMN protein 1To whom correspondence should be addressed. Email: [email protected]. functions in the U snRNP assembly/disassembly, as well as in This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. the β-actin mRNA transport in neurons (23, 24). However, the 1073/pnas.1500639112/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1500639112 PNAS Early Edition | 1of6 Downloaded by guest on September 25, 2021 of alternative or constitutive exons to cause exon skipping or A 293A C33A SH-SY5Y GM03813 inclusion (32). In the SMN pre-mRNA, it was demonstrated that 65 siRNA − − − − U2AF interacts more strongly with the SMN1 Py tract than the 6 7 8 65 6 8 SMN1 SMN2 Py tract (33). However, it is unclear how U2AF itself 6 7 8 PCR - SMN2 regulates alternative splicing. 6 8 65 RT Here we identified the function of U2AF in the alternative U2AF65 65 GAPDH splicing. Through siRNA-knockdown and overexpression of U2AF , MC3 65 IB α-tubulin we show that U2AF promotes alternative exon exclusion of both SMN2 inclusion/total (%) 12.8 13.5 37.9 14.2 15.6 45.9 27.0 28.3 61.5 46.1 48.0 65.1 ′ SD 0.2 0.3 0.5 0.4 0.3 0.6 0.3 0.1 0.3 2.0 2.0 3.0 SMN2 and SMN1 pre-mRNA. Mutations of 5 splice-site in exon 7 1 2 3 4 5 6 7 8 9 10 11 12 to a higher score sequence abolished the U2AF65 effects. Highly 65 B 293A C33A SH-SY5Y GM03813 expressed U2AF also represses splicing of exon 7, flanking introns uucuuuuuuuuuuuuuuuuuuuuuugag 65 in three or two exon contexts. Strikingly, U2AF also inhibits intron − − − − 6 7 8 β 6 8 splicing of adenovirus major late (Ad ML) and -globin pre-mRNA. 6 7 8 1kb 65 515nt RT-PCR GAPDH In addition, U2AF selectively increases its own binding on the SMN2 Flag IB α-tubulin weaker Py tract sequence, but not the stronger Py tract. Our Skipping/total (%) 48.8 46.8 95.6 47.9 50.3 95.1 47.4 47.9 95.6 58.8 60.9 96.5 65 SD 0.5 1.0 1.0 1.5 0.3 0.3 3.0 2.2 0.9 0.9 1.5 0.8 results support the conclusion that the U2AF activity in pro- 1 2 3 4 5 6 7 8 9 10 11 12 moting alternative exon skipping comes from its own splicing inhibitory activity. C 293A C33A SH-SY5Y GM03813 uucuuuuuuuuuuuuuuuuuuuuuugag − − − − Results 6 7 8 515nt 6 8 6 7 8 65 RT-PCR 1kb GAPDH U2AF Stimulates Alternative Exon Exclusion. To determine the role Flag of U2AF65 in alternative exon spicing, we first applied siRNA- SMN1 IB α-tubulin 65 65 Skipping/total (%) 4.1 3.8 44.6 3.1 2.8 31.5 4.9 2.9 30.8 13.9 17.8 53.4 directed knockdown of U2AF .