MECHANISM OF MIS-SPLICING IN MYOTONIC DYSTROPHY 1

描述(申请人提供)：强直性肌营养不良症(DM)是一种常染色体显性遗传性疾病，全世界每8500人中就有一人患有此病。糖尿病患者表现为进行性肌营养不良、心律失常和中枢神经系统损害。突变分别是DMPK和ZNF9基因非翻译区的CTG或CCTG重复序列。扩展的重复序列被表达为CUG(DM1)或CCUG(DM2)重复RNA，它们以离散的焦点聚集在细胞核中。DM1的发病机制涉及选择性剪接的错误调节，导致不适当的蛋白亚型表达，从而导致特定的症状。然而，扩展的重复序列改变Pre-mRNA选择性剪接的机制尚不清楚。有两种RNA结合蛋白参与了DM1的发病：MBNL1和CUG-BP1。CuG-BP1调控心肌肌钙蛋白T(CTnT)、胰岛素受体(IR)和氯通道(CIC-1)前mRNAs的选择性剪接，这些基因在DM1横纹肌中错误剪接。错误剪接CIC-1和IR的功能后果分别与DM1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mRNAs的剪接模式与在DM1横纹肌中观察到的CUG-BP1活性增加一致。在DM1细胞中，MBNL蛋白与扩展的CUG重复RNA的焦点结合并共定位。MBNL蛋白直接调控cTnT和IR替代外显子的剪接。因此，MBNL1和CUG-BP1蛋白是DM1中被错误调控的前mRNAs选择性剪接的主要调节因子。该提案的目标是了解扩展的CUG重复RNA如何破坏CUG-BP1和MBNL1的调控。在本研究中，我将：i)建立和鉴定稳定的可诱导表达CUG重复RNA的细胞系；ii)研究CUG重复RNA对CUG-BP1和MBNL1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-BP1和MBNL1的翻译后修饰，并表征这些修改在选择性剪接中的意义。

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.