SRSF1 and hnRNP H antagonistically regulate splicing of COLQ exon 16 in a congenital myasthenic syndrome.
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.
Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan.
Mohammad Alinoor
MA
Rahman
Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan.
Yoshiteru
Y
Azuma
Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan.
Farhana
F
Nasrin
Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan.
Jun-ichi
J
Takeda
Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan.
Mohammad
M
Nazim
Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan.
Khalid
K
Bin Ahsan
Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan.
Akio
A
Masuda
Department of Neurology, Mayo Clinic, Rochester, MN, USA.
Andrew G
AG
Engel
Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Nagoya, Aichi, Japan.
Kinji
K
Ohno
D000110:Acetylcholinesterase / Q000235:genetics*; D003094:Collagen / Q000235:genetics*; D005091:Exons / Q000235:genetics*; D005786:Gene Expression Regulation / Q000235:genetics; D006367:HeLa Cells; D034541:Heterogeneous-Nuclear Ribonucleoprotein Group F-H / Q000235:genetics*; D006801:Humans; D009124:Muscle Proteins / Q000235:genetics*; D009154:Mutation / Q000235:genetics; D020294:Myasthenic Syndromes, Congenital / Q000235:genetics*; D011485:Protein Binding / Q000235:genetics; D022821:RNA Splice Sites / Q000235:genetics*; D000068103:Serine-Arginine Splicing Factors / Q000235:genetics*
C116747:HNRNPH2 protein, human; D034541:Heterogeneous-Nuclear Ribonucleoprotein Group F-H; D009124:Muscle Proteins; D022821:RNA Splice Sites; C000595779:SRSF1 protein, human; D000068103:Serine-Arginine Splicing Factors; D003094:Collagen; D000110:Acetylcholinesterase; C114027:COLQ protein, human