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

Mohammad Alinoor Rahman,Mohammad Nazim,Kinji Ohno,Farhana Nasrin,Jun-Ichi Takeda,Andrew G Engel,Akio Masuda,Yoshiteru Azuma,Khalid Bin Ahsan

doi:10.1038/srep13208

Mohammad Alinoor Rahman, Mohammad Nazim + Show 7 more

Open Access

https://doi.org/10.1038/srep13208

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Journal: Scientific Reports	Publication Date: Aug 18, 2015
Citations: 33	License type: CC BY 4.0

Affiliation: Nagoya University, Mayo Clinic

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.

Highlights

RNA splicing is a highly specialized process especially evolved in humans and other higher metazoans to achieve intricate regulation of gene expressions and to expand the proteome diversity
Based on the position of the mutation and the effect on AChE expression, COLQ mutations can fall into four categories[6]: (1) N-terminal mutations compromising the association of AChET with ColQ; (2) truncation mutations in the collagen domain disrupting the collagenic tail of AChE; (3) C-terminal domain (CTD) missense mutations disrupting triple helical conformation of ColQ; and (4) CTD mutations affecting anchoring of ColQ at neuromuscular junction (NMJ)
We previously reported a missense mutation (p.E415G) in the CTD of COLQ in a patient with endplate AChE deficiency, which causes aberrant skipping of a constitutively spliced exon 16 encoding a part of the ColQ CTD21

Summary

Introduction

RNA splicing is a highly specialized process especially evolved in humans and other higher metazoans to achieve intricate regulation of gene expressions and to expand the proteome diversity. We exploited specific binding of the HSPBD to perlecan[7] and of the CTD to MuSK8 to develop a protein-anchoring therapy for Colq-knockout mice[9], but there is no rational therapy for human endplate AChE deficiency except for partial mitigation of the symptoms with ephedrine[10] or albuterol[11]. We previously reported a missense mutation (p.E415G) in the CTD of COLQ in a patient with endplate AChE deficiency, which causes aberrant skipping of a constitutively spliced exon 16 encoding a part of the ColQ CTD21. In this manuscript, we investigate the mechanism underlying aberrant exon skipping. We find that the mutation impairs recruitment of U1 snRNP (U1-70K) to the downstream 5′ splice site

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