MECHANISM OF MIS-SPLICING IN MYOTONIC DYSTROPHY 1

描述（由申请人提供）：强直性肌营养不良（DM）是一种常染色体显性遗传疾病，全球每8500人中就有1人患病。糖尿病患者表现出进行性肌营养不良、心律失常和中枢神经系统损伤。突变分别是DMPK和ZNF 9基因非翻译区CTG或CCTG重复扩增。扩展的重复序列表达为CUG（DM 1）或CCUG（DM 2）重复序列RNA，它们在细胞核中积累为离散的焦点。DM 1的发病机制涉及可变剪接的错误调节，导致表达不适当的蛋白质亚型，引起特定症状。然而，扩展的重复序列改变前mRNA选择性剪接的机制尚不清楚。两种RNA结合蛋白与DM 1发病机制有关：MBNL 1和CUG-BP 1。CUG-BP 1调节DM 1横纹肌中错误剪接的心肌肌钙蛋白T（cTNT）、胰岛素受体（IR）和氯离子通道（CIC-1）前体mRNA的选择性剪接。CIC-1和IR的错误剪接的功能后果分别与DM 1中观察到的肌强直和胰岛素抵抗直接相关。观察到的这些前体mRNA的剪接模式与DM 1横纹肌中观察到的CUG-BP 1活性增加一致。MBNL蛋白与DM 1细胞中扩增的CUG重复RNA的病灶结合并共定位。MBNL蛋白直接调节cTNT和IR选择性外显子的剪接。因此，MBNL 1和CUG-BP 1蛋白是DM 1中错误调节的前体mRNA的选择性剪接的主要调节剂。该提案的目标是了解扩展的CUG重复RNA如何破坏CUG-BP 1和MBNL 1的调节。在这项研究中，我将：i）建立和表征诱导表达CUG重复RNA的稳定细胞系，ii）表征CUG重复RNA对CUG-BP 1和MBNL 1的稳态水平和核质分布的影响，iii）确定CUG重复RNA是否影响CUG-BP 1和MBNL 1的翻译后修饰，并表征这些修饰对可变剪接的意义。

DESCRIPTION (provided by applicant): Myotonic dystrophy (DM) is an autosomal dominant disorder affecting 1 in 8500 people worldwide. Individuals with DM exhibit progressive muscular dystrophy, arrhythmias and CNS damage. Mutations are expanded CTG or CCTG repeats in untranslated regions of DMPK and ZNF9 genes respectively. The expanded repeats are expressed as CUG (DM1) or CCUG (DM2) repeat RNA, which accumulate as discrete foci in nuclei. The pathogenesis of DM1 involves misregulation of alternative splicing resulting in expression of inappropriate protein isoforms causing specific symptoms. However, the mechanism by which expanded repeats alter pre-mRNA alternative splicing is unclear. Two RNA binding proteins have been implicated in DM1 pathogenesis: MBNL1 and CUG-BP1. CUG-BP1 regulates alternative splicing of cardiac troponin T (cTNT), insulin receptor (IR) and chloride channel (CIC-1) pre-mRNAs that are mis-spliced in DM1 striated muscle. The functional consequences of mis-splicing of CIC-1 and IR directly correlate with myotonia and insulin resistance, respectively, observed in DM1. The splicing patterns observed for these pre-mRNAs are consistent with increased CUG-BP1 activity observed in DM1 striated muscle. MBNL proteins bind and colocalize with the foci of expanded CUG repeat RNA in DM1 cells. MBNL proteins directly regulate splicing of cTNT and IR alternative exons. Therefore, MBNL1 and CUG-BP1 proteins are the major regulators of alternative splicing of pre-mRNAs that are misregulated in DM1. The goal of this proposal is to understand how expanded CUG repeat RNA disrupts the regulation by CUG-BP1 and MBNL1. In this study I will: i) establish and characterize stable cell lines which inducibly express CUG repeat RNA, ii) characterize the effects of CUG repeat RNA on steady state levels and nuclearcytoplasmic distribution of CUG-BP1 and MBNL1, iii) determine whether CUG repeat RNA affects post-translational modifications of CUG-BP1 and MBNL1 and characterize the significance of these modifications on alternative splicing.