Regulatory mechanisms of aberrant splicing in the deseases

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Myotonic dystrophy type 1 (DM1) is an autosomal dominant neuromuscular disorder associated with an expansion of CTG trinucleotide repeats in the 3'-untranslated region of the myotonic dystrophy protein kinase (DMPK) gene. The RNA gain-of-function hypothesis proposes that mutant DMPK mRNA alters the function and localization of alternative splicing regulators, which are critical for normal RNA processing. Previously, we found alternative splicing variants of sarcoplasmic/endoplasmic reticulum Ca2+-ATPase 1 (SERCA1), which excluded exon 22, in skeletal muscle of DM1 patients. In the present study, we analyzed the molecular mechanisms responsible for the splicing dysregulation of SERCA1. Five 'YGCU(U/G)Y' motifs that could potentially serve as Muscleblind, (MBNL)-binding motifs, are included downstream from the SERCA1 exon 22. Exon trapping experiments showed that MBNL acts on the 'YGCU(U/G)Y' motif, and positively regulates the exon 22 splicing. Of the five MBNL motifs in intron 22, the second and third sites were important for regulation of exon 22 splicing, but the other three binding sites were not required. Overexpression of the CUG repeat expansion of DMPK mRNA resulted in exclusion of the exon 22 of SERCA1. These results suggest that sequestration of MBNL into the CUG repeat expansion of DMPK mRNA could cause the exclusion of SERCA1 exon 22, and expression of this aberrant splicing form of SERCA1 could affect the regulation of Ca2+ concentration of sarcoplasmic reticulum in the DM patients.