Mechanisms of decay of toxic CUGn RNA in DM1 patients

描述（由申请人提供）：1型肌强直性营养不良症（DM1）是一种复杂的、遗传性的、无法治愈的神经肌肉疾病。DM1是由位于19q染色体上的DMPK基因3' UTR内未翻译的CTG重复扩增引起的。我们和其他人发现DM1突变通过非翻译RNA CUG重复序列的积累导致疾病，这破坏了患者细胞中的RNA加工。含有扩增CUG重复序列的突变DMPK mRNA正在形成核病灶，其分解有望逆转疾病表型。此外，预计突变体DMPK mRNA的降解将降低CUG重复序列的毒性。然而，最新数据表明，突变DMPK mRNA降解的短产物比未降解的RNA毒性更大。(1)近期研究发现，小鼠体内正常大小的短CUG重复序列的多拷贝积累会产生dm1样表型（肌强直、肌营养不良和心脏传导缺陷），而在核病灶组织的长CUG重复序列拷贝数较少的小鼠则无症状；(2)我们最近关于DM2机制的数据表明，DM2与未翻译的CCTG重复序列的扩增有关，正常大小的RNA CCUG重复序列的积累导致DM2特异性异常。基于这些数据，我们假设DM1患者CUG重复扩增的毒性与突变DMPK mRNA降解的产物有关，而未降解的突变RNA没有毒性。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特异性RNA加工模式（翻译和剪接）的影响。这些研究是在Specific Aim 1中设计的。利用响应性启动子对含CUGn的转录物进行转录脉冲后，确定突变体CUGn RNA衰变的时间曲线。我们将研究核CUG病灶是否由未降解或降解的突变CUG重复序列形成，以及CUG病灶的形成是否增加或减少CUG重复序列的毒性。我们将确定CUGn突变转录物的生命周期，在此期间它具有最高的毒性。调节正常和突变DMPK mRNA稳定性和衰减的机制将在Specific Aim 2中进行研究。将确定DMPK 3' UTR内控制DMPK mRNA周转的调控区域。将确定调节正常和突变DMPK mRNA稳定性和衰减的蛋白质因子。本研究的结果将确定突变CUGn重复序列在这些转录物变得有毒时的状态，并将有助于开发通过控制突变DMPK mRNA的衰变来降低CUGn RNA毒性的方法。公共卫生相关性。DM1是一种毁灭性疾病，与含有重复CUG重复的未翻译RNA的毒性相关。本项目将确定CUG重复序列在CUG重复序列具有毒性时的状态，并将开发在CUG 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.