Mutations in the first nucleotide of exons (E(+1)) mostly affect pre-mRNA splicing when found in AG-dependent 3' splice sites, whereas AG-independent splice sites are more resistant. The AG-dependency, however, may be difficult to assess just from primary sequence data as it depends on the quality of the polypyrimidine tract. For this reason, in silico prediction tools are commonly used to score 3' splice sites. In this study, we have assessed the ability of sequence features and in silico prediction tools to discriminate between the splicing-affecting and non-affecting E(+1) variants. For this purpose, we newly tested 16 substitutions in vitro and derived other variants from literature. Surprisingly, we found that in the presence of the substituting nucleotide, the quality of the polypyrimidine tract alone was not conclusive about its splicing fate. Rather, it was the identity of the substituting nucleotide that markedly influenced it. Among the computational tools tested, the best performance was achieved using the Maximum Entropy Model and Position-Specific Scoring Matrix. As a result of this study, we have now established preliminary discriminative cut-off values showing sensitivity up to 95% and specificity up to 90%. This is expected to improve our ability to detect splicing-affecting variants in a clinical genetic setting.

Institute of Biostatistics and Analyses Masaryk University Brno Czech Republic

International Centre for Genetic Engineering and Biotechnology Trieste Italy

Molecular Genetics Laboratory Centre for Cardiovascular Surgery and Transplantation Brno Czech Republic

Molecular Genetics Laboratory Centre for Cardiovascular Surgery and Transplantation Brno Czech Republic ; Central European Institute of Technology Masaryk University Brno Czech Republic

Molecular Genetics Laboratory Centre for Cardiovascular Surgery and Transplantation Brno Czech Republic ; Central European Institute of Technology Masaryk University Brno Czech Republic ; Institute of Clinical Immunology and Allergology St Anne's University Hospital and Masaryk University Brno Czech Republic

Zobrazit více v PubMed

Hastings ML, Krainer AR (2001) Pre-mRNA splicing in the new millennium. Curr Opin Cell Biol 13: 302–309. PubMed

Baralle D, Baralle M (2005) Splicing in action: assessing disease causing sequence changes. J Med Genet 42: 737–748. PubMed PMC

Fu Y, Masuda A, Ito M, Shinmi J, Ohno K (2011) AG-dependent 3′-splice sites are predisposed to aberrant splicing due to a mutation at the first nucleotide of an exon. Nucleic Acids Res 39: 4396–4404. PubMed PMC

Reed R (1989) The organization of 3′ splice-site sequences in mammalian introns. Genes Dev 3: 2113–2123. PubMed

Guth S, Martínez C, Gaur RK, Valcárcel J (1999) Evidence for substrate-specific requirement of the splicing factor U2AF(35) and for its function after polypyrimidine tract recognition by U2AF(65). Mol Cell Biol 19: 8263–8271. PubMed PMC

Wu S, Romfo CM, Nilsen TW, Green MR (1999) Functional recognition of the 3′ splice site AG by the splicing factor U2AF35. Nature 402: 832–835. PubMed

Shapiro MB, Senapathy P (1987) RNA splice junctions of different classes of eukaryotes: sequence statistics and functional implications in gene expression. Nucleic Acids Res 15: 7155–7174. PubMed PMC

Reese MG, Eeckman FH, Kulp D, Haussler D (1997) Improved splice site detection in Genie. J Comput Biol 4: 311–323. PubMed

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

Kol G, Lev-Maor G, Ast G (2005) Human-mouse comparative analysis reveals that branch-site plasticity contributes to splicing regulation. Hum Mol Genet 14: 1559–1568. PubMed

Schwartz SH, Silva J, Burstein D, Pupko T, Eyras E, et al. (2008) Large-scale comparative analysis of splicing signals and their corresponding splicing factors in eukaryotes. Genome Res 18: 88–103. PubMed PMC

Fairbrother WG, Yeh RF, Sharp PA, Burge CB (2002) Predictive identification of exonic splicing enhancers in human genes. Science 297: 1007–1013. PubMed

Zhang XH, Chasin LA (2004) Computational definition of sequence motifs governing constitutive exon splicing. Genes Dev 18: 1241–1250. PubMed PMC

Cartegni L, Wang J, Zhu Z, Zhang MQ, Krainer AR (2003) ESEfinder: A web resource to identify exonic splicing enhancers. Nucleic Acids Res 31: 3568–3571. PubMed PMC

Wang Z, Rolish ME, Yeo G, Tung V, Mawson M, et al. (2004) Systematic identification and analysis of exonic splicing silencers. Cell 119: 831–845. PubMed

Schwartz S, Hall E, Ast G (2009) SROOGLE: webserver for integrative, user-friendly visualization of splicing signals. Nucleic Acids Res 37: W189–192. PubMed PMC

Sickmier EA, Frato KE, Shen H, Paranawithana SR, Green MR, et al. (2006) Structural basis for polypyrimidine tract recognition by the essential pre-mRNA splicing factor U2AF65. Mol Cell 23: 49–59. PubMed PMC

Mackereth CD, Madl T, Bonnal S, Simon B, Zanier K, et al. (2011) Multi-domain conformational selection underlies pre-mRNA splicing regulation by U2AF. Nature 475: 408–411. PubMed

Jenkins JL, Agrawal AA, Gupta A, Green MR, Kielkopf CL (2013) U2AF65 adapts to diverse pre-mRNA splice sites through conformational selection of specific and promiscuous RNA recognition motifs. Nucleic Acids Res 41: 3859–3873. PubMed PMC

Graveley BR, Hertel KJ, Maniatis T (1998) A systematic analysis of the factors that determine the strength of pre-mRNA splicing enhancers. EMBO J 17: 6747–6756. PubMed PMC

Doktor TK, Schroeder LD, Vested A, Palmfeldt J, Andersen HS, et al. (2011) SMN2 exon 7 splicing is inhibited by binding of hnRNP A1 to a common ESS motif that spans the 3′ splice site. Hum Mutat 32: 220–230. PubMed

Roscigno RF, Weiner M, Garcia-Blanco MA (1993) A mutational analysis of the polypyrimidine tract of introns. Effects of sequence differences in pyrimidine tracts on splicing. J Biol Chem 268: 11222–11229. PubMed

Coolidge CJ, Seely RJ, Patton JG (1997) Functional analysis of the polypyrimidine tract in pre-mRNA splicing. Nucleic Acids Res 25: 888–896. PubMed PMC

Chen M, Manley JL (2009) Mechanisms of alternative splicing regulation: insights from molecular and genomics approaches. Nat Rev Mol Cell Biol 10: 741–754. PubMed PMC

Spellman R, Smith CW (2006) Novel modes of splicing repression by PTB. Trends Biochem Sci 31: 73–76. PubMed

Graveley BR, Hertel KJ, Maniatis T (2001) The role of U2AF35 and U2AF65 in enhancer-dependent splicing. RNA 7: 806–818. PubMed PMC

Zhang WJ, Wu JY (1996) Functional properties of p54, a novel SR protein active in constitutive and alternative splicing. Mol Cell Biol 16: 5400–5408. PubMed PMC

Shen H, Green MR (2004) A pathway of sequential arginine-serine-rich domain-splicing signal interactions during mammalian spliceosome assembly. Mol Cell 16: 363–373. PubMed

Pacheco TR, Coelho MB, Desterro JM, Mollet I, Carmo-Fonseca M (2006) In vivo requirement of the small subunit of U2AF for recognition of a weak 3′ splice site. Mol Cell Biol 26: 8183–8190. PubMed PMC

Shen H, Kan JL, Green MR (2004) Arginine-serine-rich domains bound at splicing enhancers contact the branchpoint to promote prespliceosome assembly. Mol Cell 13: 367–376. PubMed

Bennett M, Michaud S, Kingston J, Reed R (1992) Protein components specifically associated with prespliceosome and spliceosome complexes. Genes Dev 6: 1986–2000. PubMed

Cartegni L, Chew SL, Krainer AR (2002) Listening to silence and understanding nonsense: exonic mutations that affect splicing. Nat Rev Genet 3: 285–298. PubMed

Shariat N, Holladay CD, Cleary RK, Phillips JA, Patton JG (2008) Isolated growth hormone deficiency type II caused by a point mutation that alters both splice site strength and splicing enhancer function. Clin Genet 74: 539–545. PubMed PMC

Gaildrat P, Krieger S, Di Giacomo D, Abdat J, Révillion F, et al. (2012) Multiple sequence variants of BRCA2 exon 7 alter splicing regulation. J Med Genet 49: 609–617. PubMed

Auclair J, Busine MP, Navarro C, Ruano E, Montmain G, et al. (2006) Systematic mRNA analysis for the effect of MLH1 and MSH2 missense and silent mutations on aberrant splicing. Hum Mutat 27: 145–154. PubMed

Lastella P, Surdo NC, Resta N, Guanti G, Stella A (2006) In silico and in vivo splicing analysis of MLH1 and MSH2 missense mutations shows exon- and tissue-specific effects. BMC Genomics 7: 243. PubMed PMC

Vorechovský I (2006) Aberrant 3′ splice sites in human disease genes: mutation pattern, nucleotide structure and comparison of computational tools that predict their utilization. Nucleic Acids Res 34: 4630–4641. PubMed PMC

Buratti E, Chivers M, Královicová J, Romano M, Baralle M, et al. (2007) Aberrant 5′ splice sites in human disease genes: mutation pattern, nucleotide structure and comparison of computational tools that predict their utilization. Nucleic Acids Res 35: 4250–4263. PubMed PMC

Abovich N, Rosbash M (1997) Cross-intron bridging interactions in the yeast commitment complex are conserved in mammals. Cell 89: 403–412. PubMed

Berglund JA, Abovich N, Rosbash M (1998) A cooperative interaction between U2AF65 and mBBP/SF1 facilitates branchpoint region recognition. Genes Dev 12: 858–867. PubMed PMC

Houdayer C, Dehainault C, Mattler C, Michaux D, Caux-Moncoutier V, et al. (2008) Evaluation of in silico splice tools for decision-making in molecular diagnosis. Hum Mutat 29: 975–982. PubMed

Zampieri S, Buratti E, Dominissini S, Montalvo AL, Pittis MG, et al. (2011) Splicing mutations in glycogen-storage disease type II: evaluation of the full spectrum of mutations and their relation to patients’ phenotypes. Eur J Hum Genet 19: 422–431. PubMed PMC

Splicing Enhancers at Intron-Exon Borders Participate in Acceptor Splice Sites Recognition

Impact of acceptor splice site NAGTAG motif on exon recognition

DNA mutation motifs in the genes associated with inherited diseases