{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,23]],"date-time":"2026-08-23T18:38:32Z","timestamp":1787510312541,"version":"build-2736575974"},"reference-count":59,"publisher":"MDPI AG","issue":"12","license":[{"start":{"date-parts":[[2020,12,9]],"date-time":"2020-12-09T00:00:00Z","timestamp":1607472000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Ministry of Education and the National Research Foundation of Korea","award":["NRF-2020R1A2C2004682"],"award-info":[{"award-number":["NRF-2020R1A2C2004682"]}]},{"name":"Ministry of Education and the National Research Foundation of Korea","award":["2019R1I1A1A01057372"],"award-info":[{"award-number":["2019R1I1A1A01057372"]}]},{"name":"Ministry of Education and the National Research Foundation of Korea","award":["2016R1A5A1007318"],"award-info":[{"award-number":["2016R1A5A1007318"]}]},{"name":"GIST Research Institute (GRI) IIBR\u201d grant funded by the GIST","award":["2020"],"award-info":[{"award-number":["2020"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cells"],"abstract":"<jats:p>Splicing factor 3b subunit 1 (SF3B1) is an essential protein in spliceosomes and mutated frequently in many cancers. While roles of SF3B1 in single intron splicing and roles of its cancer-linked mutant in aberrant splicing have been identified to some extent, regulatory functions of wild-type SF3B1 in alternative splicing (AS) are not well-understood yet. Here, we applied RNA sequencing (RNA-seq) to analyze genome-wide AS in SF3B1 knockdown (KD) cells and to identify a large number of skipped exons (SEs), with a considerable number of alternative 5\u2032 splice-site selection, alternative 3\u2032 splice-site selection, mutually exclusive exons (MXE), and retention of introns (RI). Among altered SEs by SF3B1 KD, survival motor neuron 2 (SMN2) pre-mRNA exon 7 splicing was a regulatory target of SF3B1. RT-PCR analysis of SMN exon 7 splicing in SF3B1 KD or overexpressed HCT116, SH-SY5Y, HEK293T, and spinal muscular atrophy (SMA) patient cells validated the results. A deletion mutation demonstrated that the U2 snRNP auxiliary factor 65 kDa (U2AF65) interaction domain of SF3B1 was required for its function in SMN exon 7 splicing. In addition, mutations to lower the score of the polypyrimidine tract (PPT) of exon 7, resulting in lower affinity for U2AF65, were not able to support SF3B1 function, suggesting the importance of U2AF65 in SF3B1 function. Furthermore, the PPT of exon 7 with higher affinity to U2AF65 than exon 8 showed significantly stronger interactions with SF3B1. Collectively, our results revealed SF3B1 function in SMN alternative splicing.<\/jats:p>","DOI":"10.3390\/cells9122647","type":"journal-article","created":{"date-parts":[[2020,12,10]],"date-time":"2020-12-10T20:18:22Z","timestamp":1607631502000},"page":"2647","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":10,"title":["U2AF65-Dependent SF3B1 Function in SMN Alternative Splicing"],"prefix":"10.3390","volume":"9","author":[{"given":"Namjeong","family":"Choi","sequence":"first","affiliation":[{"name":"School of Life Sciences, Gwangju Institute of Science and Technology, Gwangju 500-712, Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yongchao","family":"Liu","sequence":"additional","affiliation":[{"name":"School of Life Sciences, Gwangju Institute of Science and Technology, Gwangju 500-712, Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jagyeong","family":"Oh","sequence":"additional","affiliation":[{"name":"School of Life Sciences, Gwangju Institute of Science and Technology, Gwangju 500-712, Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jiyeon","family":"Ha","sequence":"additional","affiliation":[{"name":"School of Life Sciences, Gwangju Institute of Science and Technology, Gwangju 500-712, Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xuexiu","family":"Zheng","sequence":"additional","affiliation":[{"name":"School of Life Sciences, Gwangju Institute of Science and Technology, Gwangju 500-712, Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Haihong","family":"Shen","sequence":"additional","affiliation":[{"name":"School of Life Sciences, Gwangju Institute of Science and Technology, Gwangju 500-712, Korea"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2020,12,9]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"671","DOI":"10.1146\/annurev.ge.20.120186.003323","article-title":"Pre-mRNA splicing","volume":"20","author":"Green","year":"1986","journal-title":"Annu. Rev. Genet."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"701","DOI":"10.1016\/j.cell.2009.02.009","article-title":"The spliceosome: Design principles of a dynamic RNP machine","volume":"136","author":"Wahl","year":"2009","journal-title":"Cell"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"3008","DOI":"10.1101\/gad.8.24.3008","article-title":"Dynamic association of proteins with the pre-mRNA branch region","volume":"8","author":"MacMillan","year":"1994","journal-title":"Genes Dev."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1706","DOI":"10.1126\/science.273.5282.1706","article-title":"Interaction of U2AF65 RS region with pre-mRNA branch point and promotion of base pairing with U2 snRNA","volume":"273","author":"Valcarcel","year":"1996","journal-title":"Science"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"609","DOI":"10.1038\/355609a0","article-title":"Cloning and domain structure of the mammalian splicing factor U2AF","volume":"355","author":"Zamore","year":"1992","journal-title":"Nature"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"233","DOI":"10.1101\/gad.10.2.233","article-title":"Evidence that sequence-independent binding of highly conserved U2 snRNP proteins upstream of the branch site is required for assembly of spliceosomal complex A","volume":"10","author":"Gozani","year":"1996","journal-title":"Genes Dev."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"4536","DOI":"10.1093\/emboj\/20.16.4536","article-title":"A novel U2 and U11\/U12 snRNP protein that associates with the pre-mRNA branch site","volume":"20","author":"Will","year":"2001","journal-title":"EMBO J."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"410","DOI":"10.1261\/rna.2271406","article-title":"Biochemical and NMR analyses of an SF3b155-p14-U2AF-RNA interaction network involved in branch point definition during pre-mRNA splicing","volume":"12","author":"Spadaccini","year":"2006","journal-title":"RNA"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"664","DOI":"10.1016\/j.jmb.2005.11.067","article-title":"Multiple U2AF65 binding sites within SF3b155: Thermodynamic and spectroscopic characterization of protein-protein interactions among pre-mRNA splicing factors","volume":"356","author":"Thickman","year":"2006","journal-title":"J. Mol. Biol."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"10092","DOI":"10.1021\/bi060429o","article-title":"The SF3b155 N-terminal domain is a scaffold important for splicing","volume":"45","author":"Cass","year":"2006","journal-title":"Biochemistry"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"1409","DOI":"10.1101\/gad.12.10.1409","article-title":"Phosphorylation of spliceosomal protein SAP 155 coupled with splicing catalysis","volume":"12","author":"Wang","year":"1998","journal-title":"Genes Dev."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1392","DOI":"10.1002\/j.1460-2075.1996.tb00481.x","article-title":"Three recognition events at the branch-site adenine","volume":"15","author":"Query","year":"1996","journal-title":"EMBO J."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"4752","DOI":"10.1128\/MCB.18.8.4752","article-title":"A potential role for U2AF-SAP 155 interactions in recruiting U2 snRNP to the branch site","volume":"18","author":"Gozani","year":"1998","journal-title":"Mol. Cell Biol."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"291","DOI":"10.1146\/annurev.biochem.72.121801.161720","article-title":"Mechanisms of alternative pre-messenger RNA splicing","volume":"72","author":"Black","year":"2003","journal-title":"Annu. Rev. Biochem."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"105","DOI":"10.1016\/j.molcel.2015.09.005","article-title":"SRSF1-Regulated Alternative Splicing in Breast Cancer","volume":"60","author":"Anczukow","year":"2015","journal-title":"Mol. Cell"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"30","DOI":"10.1016\/j.semcdb.2014.03.016","article-title":"The alternative splicing side of cancer","volume":"32","author":"Biamonti","year":"2014","journal-title":"Semin. Cell Dev. Biol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"556","DOI":"10.2174\/138920208786847971","article-title":"Alternative splicing and tumor progression","volume":"9","author":"Ghigna","year":"2008","journal-title":"Curr. Genom."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"9926","DOI":"10.1073\/pnas.1500639112","article-title":"Splicing inhibition of U2AF65 leads to alternative exon skipping","volume":"112","author":"Cho","year":"2015","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"457","DOI":"10.1038\/s41571-020-0350-x","article-title":"Roles and mechanisms of alternative splicing in cancer\u2014Implications for care","volume":"17","author":"Bonnal","year":"2020","journal-title":"Nat. Rev. Clin. Oncol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1033","DOI":"10.1016\/j.celrep.2015.09.053","article-title":"Cancer-Associated SF3B1 Hotspot Mutations Induce Cryptic 3\u2032 Splice Site Selection through Use of a Different Branch Point","volume":"13","author":"Darman","year":"2015","journal-title":"Cell Rep."},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"DeBoever, C., Ghia, E.M., Shepard, P.J., Rassenti, L., Barrett, C.L., Jepsen, K., Jamieson, C.H., Carson, D., Kipps, T.J., and Frazer, K.A. (2015). Transcriptome sequencing reveals potential mechanism of cryptic 3\u2032 splice site selection in SF3B1-mutated cancers. PLoS Comput. Biol., 11.","DOI":"10.1371\/journal.pcbi.1004105"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"399","DOI":"10.1038\/nature11547","article-title":"Pancreatic cancer genomes reveal aberrations in axon guidance pathway genes","volume":"491","author":"Biankin","year":"2012","journal-title":"Nature"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"353","DOI":"10.1038\/nature11143","article-title":"Whole-genome analysis informs breast cancer response to aromatase inhibition","volume":"486","author":"Ellis","year":"2012","journal-title":"Nature"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"133","DOI":"10.1038\/ng.2523","article-title":"Recurrent mutations at codon 625 of the splicing factor SF3B1 in uveal melanoma","volume":"45","author":"Harbour","year":"2013","journal-title":"Nat. Genet."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"332","DOI":"10.1097\/CMR.0000000000000071","article-title":"Rare SF3B1 R625 mutations in cutaneous melanoma","volume":"24","author":"Kong","year":"2014","journal-title":"Melanoma Res."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"714","DOI":"10.1016\/j.cell.2013.01.019","article-title":"Evolution and impact of subclonal mutations in chronic lymphocytic leukemia","volume":"152","author":"Landau","year":"2013","journal-title":"Cell"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"233","DOI":"10.1182\/blood-2015-03-633537","article-title":"SF3B1 mutation identifies a distinct subset of myelodysplastic syndrome with ring sideroblasts","volume":"126","author":"Malcovati","year":"2015","journal-title":"Blood"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"445","DOI":"10.1002\/wrna.1222","article-title":"Splicing factor mutations and cancer","volume":"5","author":"Yoshida","year":"2014","journal-title":"Wiley Interdiscip. Rev. RNA"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"10615","DOI":"10.1038\/ncomms10615","article-title":"Cancer-associated SF3B1 mutations affect alternative splicing by promoting alternative branchpoint usage","volume":"7","author":"Alsafadi","year":"2016","journal-title":"Nat. Commun."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"1123","DOI":"10.1038\/onc.2016.279","article-title":"Cancer-associated SF3B1 mutants recognize otherwise inaccessible cryptic 3\u2032 splice sites within RNA secondary structures","volume":"36","author":"Kesarwani","year":"2017","journal-title":"Oncogene"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"82","DOI":"10.1016\/j.molcel.2019.07.017","article-title":"Disease-Causing Mutations in SF3B1 Alter Splicing by Disrupting Interaction with SUGP1","volume":"76","author":"Zhang","year":"2019","journal-title":"Mol. Cell"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"2100","DOI":"10.1038\/s41467-017-02007-z","article-title":"Molecular basis of differential 3\u2032 splice site sensitivity to anti-tumor drugs targeting U2 snRNP","volume":"8","author":"Vigevani","year":"2017","journal-title":"Nat. Commun."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"919","DOI":"10.1016\/S0140-6736(80)90847-8","article-title":"Classification of spinal muscular atrophies","volume":"1","author":"Pearn","year":"1980","journal-title":"Lancet"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"97","DOI":"10.1006\/nbdi.1996.0010","article-title":"The neurobiology of childhood spinal muscular atrophy","volume":"3","author":"Crawford","year":"1996","journal-title":"Neurobiol. Dis."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"1023","DOI":"10.1016\/S0092-8674(00)80368-2","article-title":"The SMN-SIP1 complex has an essential role in spliceosomal snRNP biogenesis","volume":"90","author":"Fischer","year":"1997","journal-title":"Cell"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"261","DOI":"10.1016\/S0896-6273(01)00357-9","article-title":"Neurotrophin-induced transport of a beta-actin mRNP complex increases beta-actin levels and stimulates growth cone motility","volume":"31","author":"Zhang","year":"2001","journal-title":"Neuron"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"299","DOI":"10.1016\/j.bbagrm.2016.12.008","article-title":"Diverse role of survival motor neuron protein","volume":"1860","author":"Singh","year":"2017","journal-title":"Biochimica Biophysica Acta Gene Regul. Mech."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"348","DOI":"10.1002\/ana.23995","article-title":"Spinal muscular atrophy: Development and implementation of potential treatments","volume":"74","author":"Arnold","year":"2013","journal-title":"Ann. Neurol."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"2768","DOI":"10.1093\/hmg\/ddx166","article-title":"A-44G transition in SMN2 intron 6 protects patients with spinal muscular atrophy","volume":"26","author":"Wu","year":"2017","journal-title":"Hum. Mol. Genet."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"6307","DOI":"10.1073\/pnas.96.11.6307","article-title":"A single nucleotide in the SMN gene regulates splicing and is responsible for spinal muscular atrophy","volume":"96","author":"Lorson","year":"1999","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"731","DOI":"10.1080\/14728222.2020.1783241","article-title":"RNA in spinal muscular atrophy: Therapeutic implications of targeting","volume":"24","author":"Singh","year":"2020","journal-title":"Expert Opin. Ther. Targets"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"3017","DOI":"10.1016\/S0140-6736(16)31408-8","article-title":"Treatment of infantile-onset spinal muscular atrophy with nusinersen: A phase 2, open-label, dose-escalation study","volume":"388","author":"Finkel","year":"2016","journal-title":"Lancet"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"271","DOI":"10.1038\/nbt.1610","article-title":"Rescue of the spinal muscular atrophy phenotype in a mouse model by early postnatal delivery of SMN","volume":"28","author":"Foust","year":"2010","journal-title":"Nat. Biotechnol."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"520","DOI":"10.1038\/gt.2017.34","article-title":"How the discovery of ISS-N1 led to the first medical therapy for spinal muscular atrophy","volume":"24","author":"Singh","year":"2017","journal-title":"Gene Ther."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"3","DOI":"10.1016\/j.pediatrneurol.2019.06.007","article-title":"From Clinical Trials to Clinical Practice: Practical Considerations for Gene Replacement Therapy in SMA Type 1","volume":"100","author":"Mendell","year":"2019","journal-title":"Pediatr. Neurol."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"706","DOI":"10.1038\/s41582-020-00413-4","article-title":"Spinal muscular atrophy\u2014Insights and challenges in the treatment era","volume":"16","author":"Mercuri","year":"2020","journal-title":"Nat. Rev. Neurol."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"2873","DOI":"10.4155\/fmc-2019-0239","article-title":"More is needed to complement the available therapies of spinal muscular atrophy","volume":"11","author":"Singh","year":"2019","journal-title":"Future Med. Chem."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"194440","DOI":"10.1016\/j.bbagrm.2019.194440","article-title":"Structures of SF3b1 reveal a dynamic Achilles heel of spliceosome assembly: Implications for cancer-associated abnormalities and drug discovery","volume":"1862","author":"Maji","year":"2019","journal-title":"Biochim. Biophys. Acta Gene Regul. Mech."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"743","DOI":"10.1016\/j.bbagrm.2015.01.004","article-title":"hnRNP L inhibits CD44 V10 exon splicing through interacting with its upstream intron","volume":"1849","author":"Loh","year":"2015","journal-title":"Biochim. Biophys. Acta"},{"key":"ref_50","doi-asserted-by":"crossref","unstructured":"Singh, N.N., Seo, J., Rahn, S.J., and Singh, R.N. (2012). A multi-exon-skipping detection assay reveals surprising diversity of splice isoforms of spinal muscular atrophy genes. PLoS ONE, 7.","DOI":"10.1371\/journal.pone.0049595"},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"E5593","DOI":"10.1073\/pnas.1419161111","article-title":"rMATS: Robust and flexible detection of differential alternative splicing from replicate RNA-Seq data","volume":"111","author":"Shen","year":"2014","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"R183","DOI":"10.1186\/gb-2007-8-9-r183","article-title":"The DAVID Gene Functional Classification Tool: A novel biological module-centric algorithm to functionally analyze large gene lists","volume":"8","author":"Huang","year":"2007","journal-title":"Genome Biol."},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"1476","DOI":"10.1038\/s41467-017-01559-4","article-title":"Binding to SMN2 pre-mRNA-protein complex elicits specificity for small molecule splicing modifiers","volume":"8","author":"Sivaramakrishnan","year":"2017","journal-title":"Nat. Commun."},{"key":"ref_54","doi-asserted-by":"crossref","unstructured":"Corvelo, A., Hallegger, M., Smith, C.W., and Eyras, E. (2010). Genome-wide association between branch point properties and alternative splicing. PLoS Comput. Biol., 6.","DOI":"10.1371\/journal.pcbi.1001016"},{"key":"ref_55","doi-asserted-by":"crossref","first-page":"31","DOI":"10.1007\/978-3-319-89689-2_2","article-title":"Mechanism of Splicing Regulation of Spinal Muscular Atrophy Genes","volume":"20","author":"Singh","year":"2018","journal-title":"Adv. Neurobiol."},{"key":"ref_56","doi-asserted-by":"crossref","first-page":"377","DOI":"10.1038\/ng854","article-title":"Disruption of an SF2\/ASF-dependent exonic splicing enhancer in SMN2 causes spinal muscular atrophy in the absence of SMN1","volume":"30","author":"Cartegni","year":"2002","journal-title":"Nat. Genet."},{"key":"ref_57","doi-asserted-by":"crossref","first-page":"460","DOI":"10.1038\/ng1207","article-title":"A negative element in SMN2 exon 7 inhibits splicing in spinal muscular atrophy","volume":"34","author":"Kashima","year":"2003","journal-title":"Nat. Genet."},{"key":"ref_58","doi-asserted-by":"crossref","first-page":"1235","DOI":"10.1038\/emboj.2010.19","article-title":"The splicing regulator Sam68 binds to a novel exonic splicing silencer and functions in SMN2 alternative splicing in spinal muscular atrophy","volume":"29","author":"Pedrotti","year":"2010","journal-title":"EMBO J."},{"key":"ref_59","doi-asserted-by":"crossref","unstructured":"Moon, H., Jang, H.N., Liu, Y., Choi, N., Oh, J., Ha, J., Zheng, X., and Shen, H. (2019). Activation of Cryptic 3\u2032 Splice-Sites by SRSF2 Contributes to Cassette Exon Skipping. Cells, 8.","DOI":"10.3390\/cells8070696"}],"container-title":["Cells"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2073-4409\/9\/12\/2647\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T10:42:47Z","timestamp":1760179367000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2073-4409\/9\/12\/2647"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2020,12,9]]},"references-count":59,"journal-issue":{"issue":"12","published-online":{"date-parts":[[2020,12]]}},"alternative-id":["cells9122647"],"URL":"https:\/\/doi.org\/10.3390\/cells9122647","relation":{},"ISSN":["2073-4409"],"issn-type":[{"value":"2073-4409","type":"electronic"}],"subject":[],"published":{"date-parts":[[2020,12,9]]}}}