High-throughput analysis revealed mutations' diverging effects on SMN1 exon 7 splicing

RNA Biol. 2019 Oct;16(10):1364-1376. doi: 10.1080/15476286.2019.1630796. Epub 2019 Jun 19.


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.

Keywords: 5′ss; SMN1; U1 snRNA; cryptic splice sites; splicing-affecting mutation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alternative Splicing*
  • Cell Line
  • Computational Biology / methods
  • Exons*
  • High-Throughput Nucleotide Sequencing
  • Humans
  • Molecular Dynamics Simulation
  • Mutagenesis
  • Mutation*
  • Nucleic Acid Conformation
  • Protein Binding
  • RNA Splice Sites
  • RNA, Small Nuclear / chemistry
  • RNA, Small Nuclear / genetics
  • RNA, Small Nuclear / metabolism
  • Survival of Motor Neuron 1 Protein / chemistry
  • Survival of Motor Neuron 1 Protein / genetics*
  • Survival of Motor Neuron 1 Protein / metabolism


  • RNA Splice Sites
  • RNA, Small Nuclear
  • SMN1 protein, human
  • Survival of Motor Neuron 1 Protein
  • U1 small nuclear RNA

Grants and funding

This work was supported by the Grantová Agentura České Republiky [GA16-11619S/2016]; Ministerstvo Zdravotnictví České Republiky [16-34414A]; Ministerstvo Školství, Mládeže a Tělovýchovy [LM2015085]; Ministerstvo Školství, Mládeže a Tělovýchovy [LM2015091]; Ministerstvo Školství, Mládeže a Tělovýchovy [LM2015042]; Ministerstvo Školství, Mládeže a Tělovýchovy [MUNI/A/1298/2018].