SRSF1 and hnRNP H antagonistically regulate splicing of COLQ exon 16 in a congenital myasthenic syndrome

Sci Rep. 2015 Aug 18:5:13208. doi: 10.1038/srep13208.

Abstract

The catalytic subunits of acetylcholinesterase (AChE) are anchored in the basal lamina of the neuromuscular junction using a collagen-like tail subunit (ColQ) encoded by COLQ. Mutations in COLQ cause endplate AChE deficiency. An A-to-G mutation predicting p.E415G in COLQ exon 16 identified in a patient with endplate AChE deficiency causes exclusive skipping of exon 16. RNA affinity purification, mass spectrometry, and siRNA-mediated gene knocking down disclosed that the mutation disrupts binding of a splicing-enhancing RNA-binding protein, SRSF1, and de novo gains binding of a splicing-suppressing RNA-binding protein, hnRNP H. MS2-mediated artificial tethering of each factor demonstrated that SRSF1 and hnRNP H antagonistically modulate splicing by binding exclusively to the target in exon 16. Further analyses with artificial mutants revealed that SRSF1 is able to bind to degenerative binding motifs, whereas hnRNP H strictly requires an uninterrupted stretch of poly(G). The mutation compromised splicing of the downstream intron. Isolation of early spliceosome complex revealed that the mutation impairs binding of U1-70K (snRNP70) to the downstream 5' splice site. Global splicing analysis with RNA-seq revealed that exons carrying the hnRNP H-binding GGGGG motif are predisposed to be skipped compared to those carrying the SRSF1-binding GGAGG motif in both human and mouse brains.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetylcholinesterase / genetics*
  • Collagen / genetics*
  • Exons / genetics*
  • Gene Expression Regulation / genetics
  • HeLa Cells
  • Heterogeneous-Nuclear Ribonucleoprotein Group F-H / genetics*
  • Humans
  • Muscle Proteins / genetics*
  • Mutation / genetics
  • Myasthenic Syndromes, Congenital / genetics*
  • Protein Binding / genetics
  • RNA Splice Sites / genetics*
  • Serine-Arginine Splicing Factors / genetics*

Substances

  • HNRNPH2 protein, human
  • Heterogeneous-Nuclear Ribonucleoprotein Group F-H
  • Muscle Proteins
  • RNA Splice Sites
  • SRSF1 protein, human
  • Serine-Arginine Splicing Factors
  • Collagen
  • Acetylcholinesterase
  • COLQ protein, human