{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,8,13]],"date-time":"2026-08-13T01:27:38Z","timestamp":1786584458481,"version":"build-2736575974"},"posted":{"date-parts":[[2026,8,10]]},"group-title":"Molecular Biology","reference-count":53,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,8,10]],"date-time":"2026-08-10T00:00:00Z","timestamp":1786320000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/www.biorxiv.org\/about\/FAQ#license"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,8,10]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  Pseudouridines (\u03a8s) are highly enriched in conserved, functionally critical regions of spliceosomal snRNAs, particularly within the U2 branch site recognition region (BSRR), which contains six \u03a8s in humans and three in\n                  <jats:italic>S. cerevisiae<\/jats:italic>\n                  . To investigate how U2 BSRR pseudouridylation influences branch site sequence (BSS) recognition, we developed a large-scale, high-throughput screening system in\n                  <jats:italic>S. cerevisiae<\/jats:italic>\n                  that allows a library of pre-mRNAs with randomized BSSs to be spliced in distinct U2 BSRR pseudouridylation backgrounds. Screening and next-generation sequencing (NGS) revealed that different U2 pseudouridylation backgrounds exhibit distinct recognition patterns and efficiencies for specific BSSs. Notably, \u03a842 and \u03a844 generally enhanced splicing, whereas \u03a838 alone, and in some contexts together with \u03a835, consistently impaired BSS recognition. The differential effects were validated using endogenous\n                  <jats:italic>S. cerevisiae<\/jats:italic>\n                  genes. In addition, splicing assays with engineered pre-mRNA constructs guided by the screening results demonstrated that BSRR \u03a8s directly influence BSS selection, supporting a model in which U2 pseudouridylation modulates BSS recognition and could contribute to alternative splicing in more complex eukaryotes. Finally, synthetic-lethality analyses with a Prp5 mutant, together with Prp5-U2-pre-mRNA binding assays, indicate that U2 BSRR \u03a8s are critical for Prp5 recruitment and, consequently, for proper U2-BSS interactions during spliceosome assembly. Collectively, these findings establish U2 pseudouridylation as a key determinant of branch site recognition and spliceosome function.\n                <\/jats:p>","DOI":"10.64898\/2026.08.09.743773","type":"posted-content","created":{"date-parts":[[2026,8,11]],"date-time":"2026-08-11T03:40:14Z","timestamp":1786419614000},"source":"Crossref","is-referenced-by-count":0,"title":["Pseudouridines in the U2 Branch Site Recognition Region Differentially Contribute to Branch Site Recognition during Pre-mRNA Splicing"],"prefix":"10.64898","author":[{"given":"Rui","family":"Zhang","sequence":"first","affiliation":[{"name":"Department of Biochemistry and Biophysics, Center for RNA Biology, University of Rochester Medical Center, Rochester, NY 14642, USA"},{"name":"Department of Biology, University of Rochester, Rochester, NY 16427, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Meemanage D.","family":"De Zoysa","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Biophysics, Center for RNA Biology, University of Rochester Medical Center, Rochester, NY 14642, USA"},{"name":"Relay Therapeutics, 60 Hampshire Street, Cambridge, MA 02139"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jonathan L.","family":"Chen","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Biophysics, Center for RNA Biology, University of Rochester Medical Center, Rochester, NY 14642, USA"},{"name":"Department of Orthopedics and Therapy Services, Penn State Health Milton S. Hershey Medical Center, 500 University Drive, Hershey, PA 17033-0850"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hironori","family":"Adachi","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Biophysics, Center for RNA Biology, University of Rochester Medical Center, Rochester, NY 14642, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0333-2898","authenticated-orcid":false,"given":"Yu","family":"Sun","sequence":"additional","affiliation":[{"name":"Department of Biology, University of Rochester, Rochester, NY 16427, USA"},{"name":"Biogen, 225 Binney Street, Cambridge, MA 02142"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2368-2799","authenticated-orcid":false,"given":"Yi-Tao","family":"Yu","sequence":"additional","affiliation":[{"name":"Department of Biochemistry and Biophysics, Center for RNA Biology, University of Rochester Medical Center, Rochester, NY 14642, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"54368","reference":[{"key":"2026081217504438000_2026.08.09.743773v1.1","doi-asserted-by":"crossref","first-page":"177","DOI":"10.1007\/978-1-0716-3191-1_14","article-title":"In Vitro Reconstitution of Pseudouridylation Catalyzed by Human Box H\/ACA Ribonucleoprotein Particles","volume":"2666","year":"2023","journal-title":"Methods Mol Biol"},{"key":"2026081217504438000_2026.08.09.743773v1.2","doi-asserted-by":"publisher","DOI":"10.1021\/bi00190a008"},{"key":"2026081217504438000_2026.08.09.743773v1.3","doi-asserted-by":"publisher","DOI":"10.1146\/annurev.biochem.72.121801.161720"},{"key":"2026081217504438000_2026.08.09.743773v1.4","doi-asserted-by":"publisher","DOI":"10.1146\/annurev-genet-112618-043830"},{"key":"2026081217504438000_2026.08.09.743773v1.5","doi-asserted-by":"publisher","DOI":"10.1038\/nature13802"},{"key":"2026081217504438000_2026.08.09.743773v1.6","doi-asserted-by":"publisher","DOI":"10.1080\/152165400300089295"},{"key":"2026081217504438000_2026.08.09.743773v1.7","doi-asserted-by":"publisher","DOI":"10.1261\/rna.066837.118"},{"key":"2026081217504438000_2026.08.09.743773v1.8","doi-asserted-by":"publisher","DOI":"10.1016\/bs.enz.2017.02.001"},{"key":"2026081217504438000_2026.08.09.743773v1.9","doi-asserted-by":"publisher","DOI":"10.1261\/rna.061226.117"},{"key":"2026081217504438000_2026.08.09.743773v1.10","doi-asserted-by":"publisher","DOI":"10.1016\/j.molcel.2009.09.040"},{"key":"2026081217504438000_2026.08.09.743773v1.11","first-page":"909","article-title":"Localization of modified nucleotides in Schizosaccharomyces pombe spliceosomal small nuclear RNAs: modified nucleotides are clustered in functionally important regions","volume":"2","year":"1996","journal-title":"Rna"},{"key":"2026081217504438000_2026.08.09.743773v1.12","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.R109.076893"},{"key":"2026081217504438000_2026.08.09.743773v1.13","doi-asserted-by":"publisher","DOI":"10.1101\/gad.1536107"},{"key":"2026081217504438000_2026.08.09.743773v1.14","doi-asserted-by":"publisher","DOI":"10.1016\/j.tibs.2012.02.009"},{"key":"2026081217504438000_2026.08.09.743773v1.15","doi-asserted-by":"publisher","DOI":"10.1016\/S1097-2765(03)00270-3"},{"key":"2026081217504438000_2026.08.09.743773v1.16","doi-asserted-by":"publisher","DOI":"10.4161\/rna.7.2.11207"},{"key":"2026081217504438000_2026.08.09.743773v1.17","doi-asserted-by":"crossref","unstructured":"Kastner B , Will CL , Stark H , Luhrmann R . 2019. Structural Insights into Nuclear pre-mRNA Splicing in Higher Eukaryotes. Cold Spring Harb Perspect Biol 11.","DOI":"10.1101\/cshperspect.a032417"},{"key":"2026081217504438000_2026.08.09.743773v1.18","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkt1330"},{"key":"2026081217504438000_2026.08.09.743773v1.19","doi-asserted-by":"publisher","DOI":"10.1016\/j.molcel.2010.01.032"},{"key":"2026081217504438000_2026.08.09.743773v1.20","doi-asserted-by":"publisher","DOI":"10.1146\/annurev-biochem-060614-034316"},{"key":"2026081217504438000_2026.08.09.743773v1.21","doi-asserted-by":"publisher","DOI":"10.1093\/genetics\/133.4.851"},{"key":"2026081217504438000_2026.08.09.743773v1.22","doi-asserted-by":"publisher","DOI":"10.1038\/nchembio.1836"},{"key":"2026081217504438000_2026.08.09.743773v1.23","doi-asserted-by":"publisher","DOI":"10.1101\/gad.253708.114"},{"key":"2026081217504438000_2026.08.09.743773v1.24","doi-asserted-by":"publisher","DOI":"10.1038\/sj.emboj.7600718"},{"key":"2026081217504438000_2026.08.09.743773v1.25","doi-asserted-by":"publisher","DOI":"10.1093\/emboj\/cdg191"},{"key":"2026081217504438000_2026.08.09.743773v1.26","doi-asserted-by":"publisher","DOI":"10.1128\/MCB.19.3.2142"},{"key":"2026081217504438000_2026.08.09.743773v1.27","unstructured":"Moore MJ , Query CC , and Sharp PA . 1993. Splicing of precursors to mRNA by the spliceosome.. in The RNA World (ed. RFaA Gesteland, J.F. , Ed.), pp. 303-357. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York."},{"key":"2026081217504438000_2026.08.09.743773v1.28","doi-asserted-by":"publisher","DOI":"10.1038\/nsb873"},{"key":"2026081217504438000_2026.08.09.743773v1.29","doi-asserted-by":"publisher","DOI":"10.1006\/meth.2001.1249"},{"key":"2026081217504438000_2026.08.09.743773v1.30","doi-asserted-by":"crossref","first-page":"983","DOI":"10.1038\/s41589-025-02025-9","article-title":"A Psi-Psi codon-anticodon pairing in nonsense suppression and translational recoding","volume":"22","year":"2026","journal-title":"Nat Chem Biol"},{"key":"2026081217504438000_2026.08.09.743773v1.31","doi-asserted-by":"publisher","DOI":"10.1016\/0092-8674(87)90564-2"},{"key":"2026081217504438000_2026.08.09.743773v1.32","doi-asserted-by":"publisher","DOI":"10.1016\/j.molcel.2010.02.036"},{"key":"2026081217504438000_2026.08.09.743773v1.33","doi-asserted-by":"publisher","DOI":"10.1101\/gad.1524307"},{"key":"2026081217504438000_2026.08.09.743773v1.34","doi-asserted-by":"crossref","unstructured":"Plaschka C , Newman AJ , Nagai K . 2019. Structural Basis of Nuclear pre-mRNA Splicing: Lessons from Yeast. Cold Spring Harb Perspect Biol 11.","DOI":"10.1101\/cshperspect.a032391"},{"key":"2026081217504438000_2026.08.09.743773v1.35","doi-asserted-by":"crossref","unstructured":"Reddy R , Busch H . 1988. Small Nuclear RNAs: RNA Sequences, Structure, and Modifications. in Structure and Function of Major and Minor Small Nuclear Ribonucleoprotein Particles (ed. ML Birnstiel ), pp. 1\u201337. Springer-Verlag, Berlin.","DOI":"10.1007\/978-3-642-73020-7_1"},{"key":"2026081217504438000_2026.08.09.743773v1.36","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2014.08.028"},{"key":"2026081217504438000_2026.08.09.743773v1.37","doi-asserted-by":"publisher","DOI":"10.1016\/0092-8674(89)90426-1"},{"key":"2026081217504438000_2026.08.09.743773v1.38","doi-asserted-by":"publisher","DOI":"10.1016\/S0092-8674(00)80925-3"},{"key":"2026081217504438000_2026.08.09.743773v1.39","doi-asserted-by":"publisher","DOI":"10.1016\/0092-8674(93)90298-5"},{"key":"2026081217504438000_2026.08.09.743773v1.40","doi-asserted-by":"publisher","DOI":"10.1038\/362171a0"},{"key":"2026081217504438000_2026.08.09.743773v1.41","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2009.02.009"},{"key":"2026081217504438000_2026.08.09.743773v1.42","doi-asserted-by":"publisher","DOI":"10.1146\/annurev-biochem-091719-064225"},{"key":"2026081217504438000_2026.08.09.743773v1.43","unstructured":"Will CL , L\u00fchrmann R . 2011. Spliceosome structure and function. Cold Spring Harb Perspect Biol 3."},{"key":"2026081217504438000_2026.08.09.743773v1.44","doi-asserted-by":"publisher","DOI":"10.15252\/embj.201593113"},{"key":"2026081217504438000_2026.08.09.743773v1.45","doi-asserted-by":"publisher","DOI":"10.1016\/j.molcel.2007.09.022"},{"key":"2026081217504438000_2026.08.09.743773v1.46","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M413288200"},{"key":"2026081217504438000_2026.08.09.743773v1.47","doi-asserted-by":"publisher","DOI":"10.1007\/s13238-011-1087-1"},{"key":"2026081217504438000_2026.08.09.743773v1.48","unstructured":"Yu YT , Scharl EC , Smith CM , Steitz JA . 1999. The growing world of small nuclear ribonucleoproteins. in The RNA World (ed. RF Gesteland , Cech, T. R ., Atkins, J. F. ), pp. 487\u2013524. Cold Spring Harbor Laboratory Press, Cold Spring Harbor."},{"key":"2026081217504438000_2026.08.09.743773v1.49","doi-asserted-by":"publisher","DOI":"10.1093\/emboj\/17.19.5783"},{"key":"2026081217504438000_2026.08.09.743773v1.50","doi-asserted-by":"publisher","DOI":"10.1261\/rna.7110804"},{"key":"2026081217504438000_2026.08.09.743773v1.51","doi-asserted-by":"publisher","DOI":"10.1261\/rna.5159504"},{"key":"2026081217504438000_2026.08.09.743773v1.52","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkl1084"},{"key":"2026081217504438000_2026.08.09.743773v1.53","doi-asserted-by":"publisher","DOI":"10.1101\/gad.3.10.1545"}],"container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.64898\/2026.08.09.743773","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,13]],"date-time":"2026-08-13T00:51:05Z","timestamp":1786582265000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.64898\/2026.08.09.743773"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,8,10]]},"references-count":53,"URL":"https:\/\/doi.org\/10.64898\/2026.08.09.743773","relation":{},"subject":[],"published":{"date-parts":[[2026,8,10]]},"subtype":"preprint"}}