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.