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U1-70K deficiency caused extensive exon skipping consistent with widespread exon definition. Surprisingly, exons with the strongest 5\u2019 splice sites (SSs) are skipped. Furthermore, strengthening of non-canonical 5\u2019 splice sites with a complementary mutant U1 snRNA or a splice modifying drug induced exon skipping in response to U1-70K depletion. We suggest that U1-70K plays a previously unappreciated role in destabilizing the U1-5\u2019SS interaction to facilitate the transition to the U6-5\u2019SS interaction required for productive splicing following exon definition. Consistent with this idea, knock-down of Prp28\/DDX23 that exchanges U1 for U6 at the 5\u2019SS also promotes exon skipping mimicking U1-70K depletion. Replacing WT U1-70K in degron-containing cells with a mutant in the RNAPII interface caused widespread exon skipping. We propose that frequent exon definition occurs at the transcription elongation complex and is enabled by U1-70K contact with transcribing RNAPII.<\/jats:p>","DOI":"10.64898\/2026.09.07.749963","type":"posted-content","created":{"date-parts":[[2026,9,10]],"date-time":"2026-09-10T10:20:12Z","timestamp":1789035612000},"source":"Crossref","is-referenced-by-count":0,"title":["Widespread exon definition is promoted by the U1 snRNP 70K subunit and requires sites of contact with RNA Polymerase II"],"prefix":"10.64898","author":[{"given":"Nova","family":"Fong","sequence":"first","affiliation":[{"name":"RNA Bioscience Initiative, Dept. Biochemistry and Molecular Genetics, University of Colorado School of Medicine, PO Box 6511, Aurora CO. 80045, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ryan","family":"Sheridan","sequence":"additional","affiliation":[{"name":"RNA Bioscience Initiative, Dept. 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