Regulatory mechanisms of aberrant splicing in the deseases

强直性肌营养不良1型(DM1)是一种常染色体显性遗传性神经肌肉疾病，与强直性肌营养不良蛋白激酶(DMPK)基因3‘非翻译区CTG三核苷酸重复序列的扩增有关。RNA功能增益假说认为，突变的DMPK mRNA改变了选择性剪接调节子的功能和定位，这对正常的RNA加工至关重要。以前，我们在DM1患者的骨骼肌中发现了肌浆/内质网钙-ATPase 1(SERCA1)的选择性剪接变体，但不包括外显子22。在本研究中，我们分析了SERCA1剪接失调的分子机制。5个‘YGCU(U/G)Y’基序可能作为(MBNL)结合基序，包括在SERCA1外显子22的下游。外显子捕获实验表明，MBNL作用于‘YGCU(U/G)Y’基序，并正向调节外显子22的剪接。在内含子22的5个MBNL基序中，第二个和第三个位点对调控外显子22的剪接起重要作用，但其他三个结合位点不是必需的。DMPK基因CUG重复序列扩增过表达导致SERCA1基因外显子22缺失。提示MBNL与DMPK基因的CUG重复扩增结合可导致SERCA1外显子22的排斥，这种异常剪接形式的SERCA1表达可能影响DM患者肌浆网钙离子浓度的调节。

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.