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High-throughput analysis revealed mutations' diverging effects on SMN1 exon 7 splicing.
RNA Biol 2019; 16(10):1364-1376RB

Abstract

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.

Authors+Show Affiliations

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.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.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.Molecular Genetics Laboratory, Centre for Cardiovascular Surgery and Transplantation , Brno , Czech Republic.Medical Genomics RG, Central European Institute of Technology, Masaryk University , Brno , Czech Republic.Faculty of Informatics, Masaryk University , Brno , Czech Republic.Molecular Genetics Laboratory, Centre for Cardiovascular Surgery and Transplantation , 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. Faculty of Medicine, Masaryk University , Brno , Czech Republic.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

31213135

Citation

Souček, Přemysl, et al. "High-throughput Analysis Revealed Mutations' Diverging Effects On SMN1 Exon 7 Splicing." RNA Biology, vol. 16, no. 10, 2019, pp. 1364-1376.
Souček P, Réblová K, Kramárek M, et al. High-throughput analysis revealed mutations' diverging effects on SMN1 exon 7 splicing. RNA Biol. 2019;16(10):1364-1376.
Souček, P., Réblová, K., Kramárek, M., Radová, L., Grymová, T., Hujová, P., ... Freiberger, T. (2019). High-throughput analysis revealed mutations' diverging effects on SMN1 exon 7 splicing. RNA Biology, 16(10), pp. 1364-1376. doi:10.1080/15476286.2019.1630796.
Souček P, et al. High-throughput Analysis Revealed Mutations' Diverging Effects On SMN1 Exon 7 Splicing. RNA Biol. 2019;16(10):1364-1376. PubMed PMID: 31213135.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - High-throughput analysis revealed mutations' diverging effects on SMN1 exon 7 splicing. AU - Souček,Přemysl, AU - Réblová,Kamila, AU - Kramárek,Michal, AU - Radová,Lenka, AU - Grymová,Tereza, AU - Hujová,Pavla, AU - Kováčová,Tatiana, AU - Lexa,Matej, AU - Grodecká,Lucie, AU - Freiberger,Tomáš, Y1 - 2019/06/19/ PY - 2019/6/20/pubmed PY - 2019/6/20/medline PY - 2019/6/20/entrez KW - 5′ss KW - SMN1 KW - U1 snRNA KW - cryptic splice sites KW - splicing-affecting mutation SP - 1364 EP - 1376 JF - RNA biology JO - RNA Biol VL - 16 IS - 10 N2 - 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. SN - 1555-8584 UR - https://www.unboundmedicine.com/medline/citation/31213135/High_throughput_analysis_revealed_mutations'_diverging_effects_on_SMN1_exon_7_splicing_ L2 - http://www.tandfonline.com/doi/full/10.1080/15476286.2019.1630796 DB - PRIME DP - Unbound Medicine ER -