Splicing-affecting mutations can disrupt gene function by altering the transcript assembly. To ascertain splicing dysregulation principles, we modified a minigene assay for the parallel high-throughput evaluation of different mutations by next-generation sequencing. In our model system, all exonic and six intronic positions of the SMN1 gene's exon 7 were mutated to all possible nucleotide variants, which amounted to 180 unique single-nucleotide mutants and 470 double mutants. The mutations resulted in a wide range of splicing aberrations. Exonic splicing-affecting mutations resulted either in substantial exon skipping, supposedly driven by predicted exonic splicing silencer or cryptic donor splice site (5'ss) and de novo 5'ss strengthening and use. On the other hand, a single disruption of exonic splicing enhancer was not sufficient to cause major exon skipping, suggesting these elements can be substituted during exon recognition. While disrupting the acceptor splice site led only to exon skipping, some 5'ss mutations potentiated the use of three different cryptic 5'ss. Generally, single mutations supporting cryptic 5'ss use displayed better pre-mRNA/U1 snRNA duplex stability and increased splicing regulatory element strength across the original 5'ss. Analyzing double mutants supported the predominating splicing regulatory elements' effect, but U1 snRNA binding could contribute to the global balance of splicing isoforms. Based on these findings, we suggest that creating a new splicing enhancer across the mutated 5'ss can be one of the main factors driving cryptic 5'ss use.

Faculty of Informatics Masaryk University Brno Czech Republic

Faculty of Medicine Masaryk University Brno Czech Republic

Medical Genomics RG Central European Institute of Technology Masaryk University Brno Czech Republic

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

Zobrazit více v PubMed

Wang Z, Burge CB.. Splicing regulation: from a parts list of regulatory elements to an integrated splicing code. RNA. 2008;14:802–813. PubMed PMC

Bruun GH, Doktor TK, Borch-Jensen J, et al. Global identification of hnRNP A1 binding sites for SSO-based splicing modulation. BMC Biol. 2016;14:54. PubMed PMC

Grodecká L, Buratti E, Freiberger T. Mutations of pre-mRNA splicing regulatory elements: are predictions moving forward to clinical diagnostics? Int J Mol Sci. 2017;18:1668. PubMed PMC

König J, Zarnack K, Rot G, et al. iCLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution. Nat Struct Mol Biol. 2010;17:909–915. PubMed PMC

Tacke R, Manley JL. The human splicing factors ASF/SF2 and SC35 possess distinct, functionally significant RNA binding specificities. Embo J. 1995;14:3540–3551. PubMed PMC

Ke S, Shang S, Kalachikov SM, et al. Quantitative evaluation of all hexamers as exonic splicing elements. Genome Res. 2011;21:1360–1374. PubMed PMC

Rosenberg AB, Patwardhan RP, Shendure J, et al. Learning the sequence determinants of alternative splicing from millions of random sequences. Cell. 2015;163:698–711. PubMed

Xiong HY, Alipanahi B, Lee LJ, et al. RNA splicing. The human splicing code reveals new insights into the genetic determinants of disease. Science. 2015;347:1254806. PubMed PMC

Lerner MR, Boyle JA, Mount SM, et al. Are snRNPs involved in splicing? Nature. 1980;283:220–224. PubMed

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

Kondo Y, Oubridge C, van Roon A-M-M, et al. Crystal structure of human U1 snRNP, a small nuclear ribonucleoprotein particle, reveals the mechanism of 5ʹ splice site recognition. Elife. 2015;4:e04986. PubMed PMC

Roca X, Akerman M, Gaus H, et al. Widespread recognition of 5ʹ splice sites by noncanonical base-pairing to U1 snRNA involving bulged nucleotides. Genes Dev. 2012;26:1098–1109. PubMed PMC

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

Tan J, Ho JXJ, Zhong Z, et al. Noncanonical registers and base pairs in human 5ʹ splice-site selection. Nucleic Acids Res. 2016;44:3908–3921. PubMed PMC

Brackenridge S, Wilkie AOM, Screaton GR. Efficient use of a “dead-end” GA 5ʹ splice site in the human fibroblast growth factor receptor genes. Embo J. 2003;22:1620–1631. PubMed PMC

Cohen JB, Snow JE, Spencer SD, et al. Suppression of mammalian 5ʹ splice-site defects by U1 small nuclear RNAs from a distance. Proc Natl Acad Sci USA. 1994;91:10470–10474. PubMed PMC

Lorson CL, Strasswimmer J, Yao JM, et al. SMN oligomerization defect correlates with spinal muscular atrophy severity. Nat Genet. 1998;19:63–66. PubMed

Vezain M, Gérard B, Drunat S, et al. A leaky splicing mutation affecting SMN1 exon 7 inclusion explains an unexpected mild case of spinal muscular atrophy. Hum Mutat. 2011;32:989–994. PubMed

Singh NN, Del Rio-Malewski JB, Luo D, et al. Activation of a cryptic 5ʹ splice site reverses the impact of pathogenic splice site mutations in the spinal muscular atrophy gene. Nucleic Acids Res. 2017;45:12214–12240. PubMed PMC

Lorson CL, Hahnen E, Androphy EJ, et al. A single nucleotide in the SMN gene regulates splicing and is responsible for spinal muscular atrophy. Proc Natl Acad Sci USA. 1999;96:6307–6311. PubMed PMC

Kashima T, Rao N, David CJ, et al. hnRNP A1 functions with specificity in repression of SMN2 exon 7 splicing. Hum Mol Genet. 2007;16:3149–3159. PubMed

Cartegni L, Krainer AR. Disruption of an SF2/ASF-dependent exonic splicing enhancer in SMN2 causes spinal muscular atrophy in the absence of SMN1. Nat Genet. 2002;30:377–384. PubMed

Chen Y-C, Chang J-G, Jong Y-J, et al. High expression level of Tra2-β1 is responsible for increased SMN2 exon 7 inclusion in the testis of SMA mice. PLoS ONE. 2015;10:e0120721. PubMed PMC

Hofmann Y, Lorson CL, Stamm S, et al. Htra2-beta 1 stimulates an exonic splicing enhancer and can restore full-length SMN expression to survival motor neuron 2 (SMN2). Proc Natl Acad Sci USA. 2000;97:9618–9623. PubMed PMC

Young PJ, DiDonato CJ, Hu D, et al. SRp30c-dependent stimulation of survival motor neuron (SMN) exon 7 inclusion is facilitated by a direct interaction with hTra2 beta 1. Hum Mol Genet. 2002;11:577–587. PubMed

Cho S, Moon H, Loh TJ, et al. PSF contacts exon 7 of SMN2 pre-mRNA to promote exon 7 inclusion. Biochim Biophys Acta. 2014;1839:517–525. PubMed PMC

Hofmann Y, Wirth B. hnRNP-G promotes exon 7 inclusion of survival motor neuron (SMN) via direct interaction with Htra2-beta1. Hum Mol Genet. 2002;11:2037–2049. PubMed

Bose JK, Wang I-F, Hung L, et al. TDP-43 overexpression enhances exon 7 inclusion during the survival of motor neuron pre-mRNA splicing. J Biol Chem. 2008;283:28852–28859. PubMed PMC

Singh NN, Singh RN, Androphy EJ. Modulating role of RNA structure in alternative splicing of a critical exon in the spinal muscular atrophy genes. Nucleic Acids Res. 2007;35:371–389. PubMed PMC

Miyajima H, Miyaso H, Okumura M, et al. Identification of a cis-acting element for the regulation of SMN exon 7 splicing. J Biol Chem. 2002;277:23271–23277. PubMed

Singh NN, Seo J, Ottesen EW, et al. TIA1 prevents skipping of a critical exon associated with spinal muscular atrophy. Mol Cell Biol. 2011;31:935–954. PubMed PMC

Beusch I, Barraud P, Moursy A, et al. Tandem hnRNP A1 RNA recognition motifs act in concert to repress the splicing of survival motor neuron exon 7. Elife. 2017;6:e25736. PubMed PMC

Piva F, Giulietti M, Burini AB, et al. SpliceAid 2: a database of human splicing factors expression data and RNA target motifs. Hum Mutat. 2012;33:81–85. PubMed

Raponi M, Kralovicova J, Copson E, et al. Prediction of single-nucleotide substitutions that result in exon skipping: identification of a splicing silencer in BRCA1 exon 6. Hum Mutat. 2011;32:436–444. PubMed

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

Erkelenz S, Theiss S, Otte M, et al. Genomic HEXploring allows landscaping of novel potential splicing regulatory elements. Nucleic Acids Res. 2014;42:10681–10697. PubMed PMC

Ronchi D, Previtali SC, Sora MGN, et al. Novel splice-site mutation in SMN1 associated with a very severe SMA-I phenotype. J Mol Neurosci. 2015;56:212–215. PubMed

Lefebvre S, Bürglen L, Reboullet S, et al. Identification and characterization of a spinal muscular atrophy-determining gene. Cell. 1995;80:155–165. PubMed

Wu S, Li Y-L, Cheng N-Y, et al. c.835-5T>G variant in SMN1 gene causes transcript exclusion of exon 7 and spinal muscular atrophy. J Mol Neurosci. 2018;65:196–202. PubMed

Sheng-Yuan Z, Xiong F, Chen Y-J, et al. Molecular characterization of SMN copy number derived from carrier screening and from core families with SMA in a Chinese population. Eur J Hum Genet. 2010;18:978–984. PubMed PMC

Qu Y-J, Bai J-L, Cao -Y-Y, et al. A rare variant (c.863G>T) in exon 7 of SMN1 disrupts mRNA splicing and is responsible for spinal muscular atrophy. Eur J Hum Genet. 2016;24:864–870. PubMed PMC

Julien P, Miñana B, Baeza-Centurion P, et al. The complete local genotype-phenotype landscape for the alternative splicing of a human exon. Nat Commun. 2016;7:11558. PubMed PMC

Ke S, Anquetil V, Zamalloa JR, et al. Saturation mutagenesis reveals manifold determinants of exon definition. Genome Res. 2018;28:11–24. PubMed PMC

Braun S, Enculescu M, Setty ST, et al. Decoding a cancer-relevant splicing decision in the RON proto-oncogene using high-throughput mutagenesis. Nat Commun. 2018;9:3315. PubMed PMC

Kalle E, Kubista M, Rensing C. Multi-template polymerase chain reaction. Biomol Detect Quantif. 2014;2:11–29. PubMed PMC

Cohen RN, Van der Aa MAEM, Macaraeg N, et al. Quantification of plasmid DNA copies in the nucleus after lipoplex and polyplex transfection. J Control Release. 2009;135:166–174. PubMed PMC

Kucherlapati RS, Spencer J, Moore PD. Homologous recombination catalyzed by human cell extracts. Mol Cell Biol. 1985;5:714–720. PubMed PMC

Sharp PA. Trans splicing: variation on a familiar theme? Cell. 1987;50:147–148. PubMed

Chen -H-H, Chang J-G, Lu R-M, et al. The RNA binding protein hnRNP Q modulates the utilization of exon 7 in the survival motor neuron 2 (SMN2) gene. Mol Cell Biol. 2008;28:6929–6938. PubMed PMC

Schaal TD, Maniatis T. Multiple distinct splicing enhancers in the protein-coding sequences of a constitutively spliced pre-mRNA. Mol Cell Biol. 1999;19:261–273. PubMed PMC

Hertel KJ, Maniatis T. The function of multisite splicing enhancers. Mol Cell. 1998;1:449–455. PubMed

Tacke R, Tohyama M, Ogawa S, et al. Human Tra2 proteins are sequence-specific activators of pre-mRNA splicing. Cell. 1998;93:139–148. PubMed

Tsuda K, Someya T, Kuwasako K, et al. Structural basis for the dual RNA-recognition modes of human Tra2-β RRM. Nucleic Acids Res. 2011;39:1538–1553. PubMed PMC

Jobbins AM, Reichenbach LF, Lucas CM, et al. The mechanisms of a mammalian splicing enhancer. Nucleic Acids Res. 2018;46:2145–2158. PubMed PMC

Hicks MJ, Mueller WF, Shepard PJ, et al. Competing upstream 5ʹ splice sites enhance the rate of proximal splicing. Mol Cell Biol. 2010;30:1878–1886. PubMed PMC

Dal Mas A, Rogalska ME, Bussani E, et al. Improvement of SMN2 pre-mRNA processing mediated by exon-specific U1 small nuclear RNA. Am J Hum Genet. 2015;96:93–103. PubMed PMC

Jamison SF, Pasman Z, Wang J, et al. U1 snRNP-ASF/SF2 interaction and 5ʹ splice site recognition: characterization of required elements. Nucleic Acids Res. 1995;23:3260–3267. PubMed PMC

Förch P, Puig O, Martínez C, et al. The splicing regulator TIA-1 interacts with U1-C to promote U1 snRNP recruitment to 5ʹ splice sites. Embo J. 2002;21:6882–6892. PubMed PMC

Spena S, Tenchini ML, Buratti E. Cryptic splice site usage in exon 7 of the human fibrinogen Bbeta-chain gene is regulated by a naturally silent SF2/ASF binding site within this exon. RNA. 2006;12:948–958. PubMed PMC

Wassarman KM, Steitz JA. Association with terminal exons in pre-mRNAs: a new role for the U1 snRNP? Genes Dev. 1993;7:647–659. PubMed

Filipi T, Mazura P, Janda L, et al. Engineering the cytokinin-glucoside specificity of the maize β-D-glucosidase Zm-p60.1 using site-directed random mutagenesis. Phytochemistry. 2012;74:40–48. PubMed

Liao Y, Smyth GK, Shi W. The Subread aligner: fast, accurate and scalable read mapping by seed-and-vote. Nucleic Acids Res. 2013;41:e108. PubMed PMC

Case D, Babin V, Berryman J, et al. AMBER 14. San Francisco: University of California; 2014.

Zgarbová M, Otyepka M, Sponer J, et al. Refinement of the cornell nucleic acids force field based on reference quantum chemical calculations of glycosidic torsion profiles. J Chem Theory Comput. 2011;7:2886–2902. PubMed PMC

Leontis NB, Stombaugh J, Westhof E. The non-Watson-Crick base pairs and their associated isostericity matrices. Nucleic Acids Res. 2002;30:3497–3531. PubMed PMC

Aduri R, Psciuk BT, Saro P, et al. 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, 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. 2010;38:299–313. PubMed PMC

Humphrey W, Dalke A, Schulten K. VMD: visual molecular dynamics. J Mol Graph. 1996;14:33–8, 27–8. PubMed

Lavery R, Moakher M, Maddocks JH, et al. Conformational analysis of nucleic acids revisited: curves+. Nucleic Acids Res. 2009;37:5917–5929. PubMed PMC

Kashima T, Manley JL. A negative element in SMN2 exon 7 inhibits splicing in spinal muscular atrophy. Nat Genet. 2003;34:460–463. PubMed

Splicing analysis of STAT3 tandem donor suggests non-canonical binding registers for U1 and U6 snRNAs