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.

• Authors: Mohammad Alinoor Rahman, Yoshiteru Azuma, Farhana Nasrin, Jun-ichi Takeda, Mohammad Nazim, Khalid Bin Ahsan, Akio Masuda, Andrew G Engel, Kinji Ohno
• Journal: Scientific reports (Sci Rep) Vol. 5 Pg. 13208 (Aug 18 2015) ISSN: 2045-2322 [Electronic] England
• PMID: 26282582 (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)