Tandem donor splice sites (5'ss) are unique regions with at least two GU dinucleotides serving as splicing cleavage sites. The Δ3 tandem 5'ss are a specific subclass of 5'ss separated by 3 nucleotides which can affect protein function by inserting/deleting a single amino acid. One 5'ss is typically preferred, yet factors governing particular 5'ss choice are not fully understood. A highly conserved exon 21 of the STAT3 gene was chosen as a model to study Δ3 tandem 5'ss splicing mechanisms. Based on multiple lines of experimental evidence, endogenous U1 snRNA most likely binds only to the upstream 5'ss. However, the downstream 5'ss is used preferentially, and the splice site choice is not dependent on the exact U1 snRNA binding position. Downstream 5'ss usage was sensitive to exact nucleotide composition and dependent on the presence of downstream regulatory region. The downstream 5'ss usage could be best explained by two novel interactions with endogenous U6 snRNA. U6 snRNA enables the downstream 5'ss usage in STAT3 exon 21 by two mechanisms: (i) binding in a novel non-canonical register and (ii) establishing extended Watson-Crick base pairing with the downstream regulatory region. This study suggests that U6:5'ss interaction is more flexible than previously thought.

Centre for Cardiovascular Surgery and Transplantation 656 91 Brno Czech Republic

Centre of Molecular Biology and Genetics University Hospital and Masaryk University Brno Czech Republic

Faculty of Medicine Masaryk University 625 00 Brno Czech Republic

National Centre for Biomolecular Research Faculty of Science Masaryk University 62500 Brno Czech Republic

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Pan  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; 40:1413–1415. PubMed

Wang  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; 456:470–476. PubMed PMC

Sheth  N., Roca  X., Hastings  M.L., Roeder  T., Krainer  A.R., Sachidanandam  R.  Comprehensive splice-site analysis using comparative genomics. Nucleic Acids Res.  2006; 34:3955–3967. PubMed PMC

Ule  J., Blencowe  B.J.  Alternative splicing regulatory networks: functions, mechanisms, and evolution. Mol. Cell. 2019; 76:329–345. PubMed

Lee  Y., Rio  D.C.  Mechanisms and regulation of alternative pre-mRNA splicing. Annu. Rev. Biochem.  2015; 84:291–323. PubMed PMC

Zhou  Z., Fu  X.D.  Regulation of splicing by SR proteins and SR protein-specific kinases. Chromosoma. 2013; 122:191–207. PubMed PMC

Geuens  T., Bouhy  D., Timmerman  V.  The hnRNP family: insights into their role in health and disease. Hum. Genet.  2016; 135:851–867. PubMed PMC

Sammeth  M., Foissac  S., Guigó  R.  A general definition and nomenclature for alternative splicing events. PLoS Comput. Biol.  2008; 4:e1000147. PubMed PMC

Chern  T.M., Van Nimwegen  E., Kai  C., Kawai  J., Carninci  P., Hayashizaki  Y., Zavolan  M.  A simple physical model predicts small exon length variations. PLoS Genet.  2006; 2:e45. PubMed PMC

Séraphin  B., Kretzner  L., Rosbash  M.  A U1 snRNA:pre-mRNA base pairing interaction is required early in yeast spliceosome assembly but does not uniquely define the 5′ cleavage site. EMBO J.  1988; 7:2533–2538. PubMed PMC

Siliciano  P.G., Guthrie  C.  5′ splice site selection in yeast: genetic alterations in base-pairing with U1 reveal additional requirements. Genes Dev.  1988; 2:1258–1267. PubMed

Freund  M., Hicks  M.J., Konermann  C., Otte  M., Hertel  K.J., Schaal  H.  Extended base pair complementarity between U1 snRNA and the 5′ splice site does not inhibit splicing in higher eukaryotes, but rather increases 5′ splice site recognition. Nucleic Acids Res.  2005; 33:5112–5119. PubMed PMC

Roca  X., Krainer  A.R.  Recognition of atypical 5′ splice sites by shifted base-pairing to U1 snRNA. Nat. Struct. Mol. Biol.  2009; 16:176–182. PubMed PMC

Roca  X., Akerman  M., Gaus  H., Berdeja  A., Bennett  C.F., Krainer  A.R.  Widespread recognition of 5′ splice sites by noncanonical base-pairing to U1 snRNA involving bulged nucleotides. Genes Dev.  2012; 26:1098–1109. PubMed PMC

Tan  J., Ho  J.X.J., Zhong  Z., Luo  S., Chen  G., Roca  X.  Noncanonical registers and base pairs in human 5′ splice-site selection. Nucleic Acids Res.  2016; 44:3908–3921. PubMed PMC

Dewilde  S., Vercelli  A., Chiarle  R., Poli  V.  Of alphas and betas: distinct and overlapping functions of STAT3 isoforms. Front. Biosci.  2008; 13:6501–6514. PubMed

Hu  X., li  J., Fu  M., Zhao  X., Wang  W.  The JAK/STAT signaling pathway: from bench to clinic. Signal Transduct. Target Ther.  2021; 6:402. PubMed PMC

Aigner  P., Just  V., Stoiber  D.  STAT3 isoforms: alternative fates in cancer?. Cytokine. 2019; 118:27–34. PubMed

Zheng  M., Turton  K.B., Zhu  F., Li  Y., Grindle  K.M., Annis  D.S., Lu  L., Drennan  A.C., Tweardy  D.J., Bharadwaj  U.  et al. .  A mix of s and δs variants of stat3 enable survival of activated b-cell-like diffuse large b-cell lymphoma cells in culture. Oncogenesis. 2016; 5:e184. PubMed PMC

Dawes  R., Bournazos  A.M., Bryen  S.J., Bommireddipalli  S., Marchant  R.G., Joshi  H., Cooper  S.T.  SpliceVault predicts the precise nature of variant-associated mis-splicing. Nat. Genet.  2023; 55:324–332. PubMed PMC

Zuker  M.  Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res.  2003; 31:3406–3415. PubMed PMC

Erkelenz  S., Theiss  S., Otte  M., Widera  M., Peter  J.O., Schaal  H.  Genomic HEXploring allows landscaping of novel potential splicing regulatory elements. Nucleic Acids Res.  2014; 42:10681–10697. PubMed PMC

Ke  S., Shang  S., Kalachikov  S.M., Morozova  I., Yu  L., Russo  J.J., Ju  J., Chasin  L.A.  Quantitative evaluation of all hexamers as exonic splicing elements. Genome Res.  2011; 21:13060–13074. PubMed PMC

Souček  P., Réblová  K., Kramárek  M., Radová  L., Grymová  T., Hujová  P., Kováčová  T., Lexa  M., Grodecká  L., Freiberger  T.  High-throughput analysis revealed mutations’ diverging effects on SMN1 exon 7 splicing. RNA Biol. 2019; 16:13064–13076. PubMed PMC

Case  D.A.D.A., Betz  R.M.R., Cerutti  D.D.S., T.E.  C.I., Darden  T.A., Duke  R.E., Giese  T.J., Gohlke  H., Goetz  A.W., Homeyer  N.  et al. .  Amber 2016. 2016; San Francisco: University of California.

Zgarbová  M., Otyepka  M., Šponer  J., Mládek  A., Banáš  P., Cheatham  T.E., Jurečka  P.  Refinement of the Cornell et al. Nucleic acids force field based on reference quantum chemical calculations of glycosidic torsion profiles. J. Chem. Theory Comput.  2011; 7:2886–2902. PubMed PMC

Aduri  R., Psciuk  B.T., Saro  P., Taniga  H., Schlegel  H.B., SantaLucia  J.  AMBER force field parameters for the naturally occurring modified nucleosides in RNA. J. Chem. Theory Comput.  2007; 3:1464–1475. PubMed

Lavery  R., Zakrzewska  K., Beveridge  D., Bishop  T.C., Case  D.A., Cheatham  T., Dixit  S., Jayaram  B., Lankas  F., Laughton  C.  et al. .  A systematic molecular dynamics study of nearest-neighbor effects on base pair and base pair step conformations and fluctuations in B-DNA. Nucleic Acids Res.  2009; 38:299–313. PubMed PMC

Humphrey  W., Dalke  A., Schulten  K.  VMD: visual molecular dynamics. J. Mol. Graph.  1996; 14:33–38. PubMed

Yeo  G., Burge  C.B.  Maximum entropy modeling of short sequence motifs with applications to RNA splicing signals. J. Comput. Biol.  2004; 11:377–394. PubMed

Kondo  Y., Oubridge  C., van Roon  A.M.M., Nagai  K.  Crystal structure of human U1 snRNP, a small nuclear ribonucleoprotein particle, reveals the mechanism of 5′ splice site recognition. eLife. 2015; 4:e04986. PubMed PMC

Hansen  S.R., White  D.S., Scalf  M., Corrêa  I.R., Smith  L.M., Hoskins  A.A.  Multi-step recognition of potential 5′ splice sites by the Saccharomyces cerevisiae U1 snRNP. eLife. 2022; 11:e70534. PubMed PMC

Crispino  J.D., Blencowe  B.J., Sharp  P.A.  Complementation by SR proteins of pre-mRNA splicing reactions depleted of U1 snRNP. Science. 1994; 265:1866–1869. PubMed

Tarn  W.Y., Steitz  J.A.  SR proteins can compensate for the loss of U1 snRNP functions in vitro. Genes Dev.  1994; 8:2704–2717. PubMed

Crispino  J.D., Sharp  P.A.  A U6 snRNA:pre-mRNA interaction can be rate-limiting for U1-independent splicing. Genes Dev.  1995; 9:2314–2323. PubMed

Crispino  J.D., Mermoud  J.E., Lamond  A.I., Sharp  P.A.  Cis-acting elements distinct from the 5′ splice site promote U1- independent pre-mRNA splicing. RNA. 1996; 2:664–673. PubMed PMC

Fukumura  K., Taniguchi  I., Sakamoto  H., Ohno  M., Inoue  K.  U1-independent pre-mRNA splicing contributes to the regulation of alternative splicing. Nucleic Acids Res.  2009; 37:1907–1914. PubMed PMC

Black  C.S., Whelan  T.A., Garside  E.L., Macmillan  A.M., Fast  N.M., Rader  S.D.  Spliceosome assembly and regulation: insights from analysis of highly reduced spliceosomes. RNA. 2023; 29:531–550. PubMed PMC

Charenton  C., Wilkinson  M.E., Nagai  K.  Mechanism of 5′ splice site transfer for human spliceosome activation. Science. 2019; 364:362–367. PubMed PMC

Lesser  C.F., Guthrie  C.  Mutations in U6 snRNA that alter splice site specificity: implications for the active site. Science. 1993; 262:1982–1988. PubMed

Kandels-Lewis  S., Séraphin  B.  Role of U6 snRNA in 5′ splice site selection. Science. 1993; 262:2035–2039. PubMed

Zhan  X., Yan  C., Zhang  X., Lei  J., Shi  Y.  Structures of the human pre-catalytic spliceosome and its precursor spliceosome. Cell Res.  2018; 28:1129–1140. PubMed PMC

Bertram  K., Agafonov  D.E., Dybkov  O., Haselbach  D., Leelaram  M.N., Will  C.L., Urlaub  H., Kastner  B., Lührmann  R., Stark  H.  Cryo-EM structure of a pre-catalytic Human spliceosome primed for activation. Cell. 2017; 170:701–713. PubMed

Bertram  K., Agafonov  D.E., Liu  W.T., Dybkov  O., Will  C.L., Hartmuth  K., Urlaub  H., Kastner  B., Stark  H., Lührmann  R.  Cryo-EM structure of a human spliceosome activated for step 2 of splicing. Nature. 2017; 542:318–323. PubMed

Zhang  X., Yan  C., Hang  J., Finci  L.I., Lei  J., Shi  Y.  An atomic structure of the Human spliceosome. Cell. 2017; 169:918–929. PubMed

Hiller  M., Huse  K., Szafranski  K., Jahn  N., Hampe  J., Schreiber  S., Backofen  R., Platzer  M.  Widespread occurrence of alternative splicing at NAGNAG acceptors contributes to proteome plasticity. Nat. Genet.  2004; 36:1255–1257. PubMed

Hiller  M., Szafranski  K., Backofen  R., Platzer  M.  Alternative splicing at NAGNAG acceptors: simply noise or noise and more? [1]. PLoS Genet.  2006; 2:e207. PubMed PMC

Zavolan  M., Kondo  S., Schönbach  C., Adachi  J., Hume  D.A., Arakawa  T., Carninci  P., Kawai  J., Hayashizaki  Y., Gaasterland  T.  Impact of alternative initiation, splicing, and termination on the diversity of the mRNA transcripts encoded by the mouse transcriptome. Genome Res.  2003; 13:1290–1300. PubMed PMC

Hiller  M., Platzer  M.  Widespread and subtle: alternative splicing at short-distance tandem sites. Trends Genet.  2008; 24:246–255. PubMed

Hujová  P., Souček  P., Radová  L., Kramárek  M., Kováčová  T., Freiberger  T.  Nucleotides in both donor and acceptor splice sites are responsible for choice in NAGNAG tandem splice sites. Cell. Mol. Life Sci.  2021; 78:6979–6993. PubMed PMC

Hiller  M., Huse  K., Szafranski  K., Rosenstiel  P., Schreiber  S., Backofen  R., Platzer  M.  Phylogenetically widespread alternative splicing at unusual GYNGYN donors. Genome Biol.  2006; 7:R65. PubMed PMC

Turton  K.B., Annis  D.S., Rui  L., Esnault  S., Mosher  D.F.  Ratios of four STAT3 splice variants in human eosinophils and diffuse large B cell lymphoma cells. PLoS One. 2015; 10:e0127243. PubMed PMC

Sinha  R., Lenser  T., Jahn  N., Gausmann  U., Friedel  S., Szafranski  K., Huse  K., Rosenstiel  P., Hampe  J., Schuster  S.  et al. .  TassDB2 - A comprehensive database of subtle alternative splicing events. BMC Bioinf.  2010; 11:216. PubMed PMC

O’Reilly  D., Dienstbier  M., Cowley  S.A., Vazquez  P., Drozdz  M., Taylor  S., James  W.S., Murphy  S.  Differentially expressed, variant U1 snRNAs regulate gene expression in human cells. Genome Res.  2013; 23:281–291. PubMed PMC

Mabin  J.W., Lewis  P.W., Brow  D.A., Dvinge  H.  Human spliceosomal snRNA sequence variants generate variant spliceosomes. RNA. 2021; 27:1186–1203. PubMed PMC

Vazquez-Arango  P., Vowles  J., Browne  C., Hartfield  E., Fernandes  H.J.R., Mandefro  B., Sareen  D., James  W., Wade-Martins  R., Cowley  S.A.  et al. .  Variant U1 snRNAs are implicated in human pluripotent stem cell maintenance and neuromuscular disease. Nucleic Acids Res.  2016; 44:10960–10973. PubMed PMC

Hwang  D.Y., Cohen  J.B.  U1 snRNA promotes the selection of nearby 5′ splice sites by U6 snRNA in mammalian cells. Genes Dev.  1996; 10:338–350. PubMed

Brackenridge  S., Wilkie  A.O.M., Screaton  G.R.  Efficient use of a ‘dead-end’ GA 5′ splice site in the human fibroblast growth factor receptor genes. EMBO J.  2003; 22:1620–1631. PubMed PMC

Artemyeva-Isman  O.V., Porter  A.C.G.  U5 snRNA interactions with exons ensure splicing precision. Front. Genet.  2021; 12:676971. PubMed PMC

Alanis  E.F., Pinotti  M., Mas  A.D., Balestra  D., Cavallari  N., Rogalska  M.E., Bernardi  F., Pagani  F.  An exon-specific U1 small nuclear RNA (snRNA) strategy to correct splicing defects. Hum. Mol. Genet.  2012; 21:2389–2398. PubMed PMC

Sawa  H., Abelson  J.  Evidence for a base-pairing interaction between U6 small nuclear RNA and the 5′ splice site during the splicing reaction in yeast. Proc. Natl. Acad. Sci. U.S.A.  1992; 89:11269–11273. PubMed PMC

Zhang  X., Yan  C., Zhan  X., Li  L., Lei  J., Shi  Y.  Structure of the human activated spliceosome in three conformational states. Cell Res.  2018; 28:307–322. PubMed PMC

Wilkinson  M.E., Charenton  C., Nagai  K.  RNA splicing by the spliceosome. Annu. Rev. Biochem.  2020; 89:359–388. PubMed

Wan  R., Bai  R., Zhan  X., Shi  Y.  How is precursor messenger RNA spliced by the spliceosome?. Annu. Rev. Biochem.  2020; 89:333–358. PubMed

Henning  L.M., Santos  K.F., Sticht  J., Jehle  S., Lee  C.T., Wittwer  M., Urlaub  H., Stelzl  U., Wahl  M.C., Freund  C.  A new role for FBP21 as regulator of Brr2 helicase activity. Nucleic Acids Res.  2017; 45:7922–7937. PubMed PMC