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

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.
AuthorsMohammad Alinoor Rahman, Yoshiteru Azuma, Farhana Nasrin, Jun-ichi Takeda, Mohammad Nazim, Khalid Bin Ahsan, Akio Masuda, Andrew G Engel, Kinji Ohno
JournalScientific reports (Sci Rep) Vol. 5 Pg. 13208 (Aug 18 2015) ISSN: 2045-2322 [Electronic] England
PMID26282582 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't)
Chemical References
  • 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
Topics
  • 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)

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