Mechanisms of decay of toxic CUGn RNA in DM1 patients

描述(申请人提供)：强直性肌营养不良症1(DM1)是一种复杂的、遗传的、无法治愈的神经肌肉疾病。DM1是由位于染色体19q的DMPK基因3‘非编码区内未翻译的CTG重复序列的扩增引起的。我们和其他人发现，DM1突变通过积累未翻译的RNA CUG重复而导致疾病，这会扰乱患者细胞中的RNA处理。含有扩展的CUG重复序列的突变的DMPK mRNA正在形成核灶，其分解有望逆转疾病的表型。预计突变的DMPK mRNA的降解将降低CUG重复序列的毒性。然而，最新的数据表明，突变的DMPK mRNA降解的短产物比未降解的RNA毒性更大。(1)最近发现，正常大小的短CUG重复序列在小鼠体内的多个拷贝的积累产生了DM1样表型(肌强直、肌营养不良和心脏传导缺陷)，而在核病灶中组织的长CUG重复序列拷贝数低的小鼠没有症状；(2)我们最近对DM2的机制的研究数据表明，正常大小的RNA CCUG重复序列的积累会导致DM2特异的异常。根据这些数据，我们假设CUG重复扩增对DM1患者的毒性与突变DMPK mRNA降解的产物有关，而未降解的突变RNA是无毒的。为了验证这一假设，我们建议比较未降解(稳定)和腐烂的CUG转录本对DM1特定RNA处理模式(翻译和剪接)的影响。这些研究都是在特定的目标下设计的：1.突变的cugn RNA衰变的时间曲线将在含有cugn的转录本使用tet反应启动子的转录脉冲后确定。我们将研究核CUG焦点是由非降解的还是降解的突变CUG重复形成的，以及CUG焦点的形成是否增加或降低了CuGN重复的毒性。我们将确定CuGN突变转录本的生命周期，在此期间它具有最高的毒性。调节正常和突变DMPK mRNA稳定性和衰变的机制将在特定的目标2中进行研究。DMPK 3‘非编码区内调控DMPK mRNA周转的区域将被确定。调节正常和突变的DMPK mRNA稳定性和衰败的蛋白质因子将被识别。本研究的结果将确定突变的Cugn重复序列在这些转录物变毒时的状态，并将有助于开发通过控制突变的DMPK mRNA的衰退来降低Cugn RNA毒性的方法。与公共卫生相关。DM1是一种与含有重复CUG重复序列的未翻译RNA的毒性有关的破坏性疾病。该项目将确定当CUG重复序列变得有毒时CUG重复序列的状态，并将开发在Cugn RNA稳定性水平上降低这些重复序列毒性的方法。

DESCRIPTION (provided by applicant): Myotonic Dystrophy 1 (DM1) is a complex, inherited, neuro-muscular disease without cure. DM1 is caused by an expansion of untranslated CTG repeats within the 3' UTR of DMPK gene located on the chromosome 19q. We and others found that DM1 mutation causes a disease through accumulation of untranslated RNA CUG repeats which disrupt RNA processing in patients' cells. Mutant DMPK mRNA containing expanded CUG repeats is forming nuclear foci, disassembly of which is expected to reverse the disease phenotype. It is also expected that degradation of mutant DMPK mRNA would reduce toxicity of CUG repeats. However, latest data suggest that the short products of degradation of the mutant DMPK mRNA are more toxic than un-degraded RNA. (1) It has been recently found that the accumulation of multiple copies of short CUG repeats of normal size in mice produced DM1-like phenotype (myotonia, muscular dystrophy and cardiac conduction defects), while mice with low number of copies of long CUG repeats organized in nuclear foci were asymptomatic; (2) Our recent data on the mechanism of DM2, which is associated with expansion of untranslated CCTG repeats showed that accumulation of normal size of RNA CCUG repeats leads to DM2-specific abnormalities. Based on these data, we hypothesize that toxicity of CUG repeat expansion in DM1 patients is associated with the products of degradation of the mutant DMPK mRNA, while undegraded mutant RNA is not toxic. To examine this hypothesis, we propose to compare the effect of un-degraded (stable) and decayed CUG transcripts on DM1-specific pattern of RNA processing (translation and splicing). These studies are designed in Specific Aim 1. Time curve for mutant CUGn RNA decay will be determined after transcription pulse of CUGn-containing transcripts using tet-responsive promoter. We will examine whether nuclear CUG foci are formed by non-degraded or degraded mutant CUG repeats and whether CUG foci formation increases or reduces toxicity of CUGn repeats. We will determine the period of life of CUGn mutant transcript during which it has highest toxicity. The mechanisms regulating stability and decay of normal and mutant DMPK mRNA will be examined in Specific Aim 2. Regulatory regions within the 3' UTR of DMPK controlling DMPK mRNA turnover will be determined. Protein factors regulating normal and mutant DMPK mRNA stability and decay will be identified. The results of this study will identify status of mutant CUGn repeats when these transcripts become toxic and will help to develop approach to reduce CUGn RNA toxicity by controlling decay of the mutant DMPK mRNA. PUBLIC HEALTH RELEVANCE. DM1 is a devastating disease associated with toxicity of untranslated RNA containing repetitive CUG repeats. This project will identify status of CUG repeats when CUG repeats become toxic and will develop approach to reduce toxicity of these repeats at the level of CUGn RNA stability.