RNA DOMINANCE IN HUMAN DISEASE

描述(逐字摘自调查人员摘要)：强直性肌无力 营养不良(DM)是最常见的成人型肌营养不良症。DM是 一种常染色体显性遗传性神经肌肉疾病，由(CTG)n重复引起 DM蛋白激酶(DMPK)基因3‘端非编码区的扩增。从长远来看 这项拟议研究的目的是阐明三联体是如何重复的 基因3‘非编码区的扩展会导致一种显性遗传性疾病。 目前的证据表明，糖尿病的发病机制与 DMPK突变等位基因转录本在细胞核内的积累。我们的工作 “隔离”假说认为，糖尿病是一种RNA显性疾病，其中 (Cug)n膨胀形成一种异常稳定的双链RNA(DsRNA) 发夹结构。这种不同寻常的RNA发夹起到了高亲和力结合的作用 可能发挥作用的三联重复扩增dsRNA结合蛋白的位置 在核质RNA输出中的重要作用。大的重复扩展 与严重疾病相关导致这些蛋白在DMPK上被隔离 突变的等位基因转录本和对其他基因输出的显性负面影响 RNA。本提案的重点是测试这种RNA优势模型 几种不同的实验方法。首先，假设(CUG)n 扩增RNA对信使核糖核酸输出有显性负面影响将直接 用RNA显微注射技术检测蛙卵母细胞和哺乳动物成纤维细胞 原子核。第二，封存假说预测扩张绑定 蛋白质应与DMPK突变体一起在核内积聚 成绩单。因此，我们将完成几个方面的刻画 优先识别大的(CUG)n扩展的蛋白质，并确定 这些蛋白在正常细胞和糖尿病患者细胞中的亚细胞分布。 第三，这些扩展结合蛋白的首选RNA结合部位将是 以体外和体内分析为特征的，特别强调 识别正常情况下与这些蛋白质相关的RNA。第四，我们将 通过以下方式确定扩展结合蛋白是否参与了mRNA输出 将单抗和重组蛋白的使用与 在第一个目标中开发了微注射系统。第五，RNA的相关性 对其他神经肌肉和神经疾病的优势将是 调查过了。这些研究对于阐明以下问题具有重要意义 糖尿病发病机制的分子机制及细胞策略 促进核和核之间的遗传信息交换 细胞质。

DESCRIPTION (appended verbatim from investigator's abstract): Myotonic dystrophy (DM) is the most common form of adult onset muscular dystrophy. DM is an autosomal dominant neuromuscular disorder that is caused by a (CTG)n repeat expansion in the 3' UTR of the DM protein kinase (DMPK) gene. The long term objective of the proposed research is to elucidate how a triplet repeat expansion in the 3' UTR of a gene leads to a dominantly inherited disease. Current evidence suggests that DM pathogenesis is associated with the accumulation of DMPK mutant allele transcripts within the nucleus. Our working 'sequestration' hypothesis is that DM is an RNA dominant disease in which the (CUG)n expansion forms an exceptionally stable double stranded RNA (dsRNA) hairpin structure. This unusual RNA hairpin acts as a high affinity binding site for triplet repeat expansion dsRNA binding proteins that possibly play important roles in nucleocytoplasmic RNA export. Large repeat expansions associated with severe disease lead to sequestration of these proteins on DMPK mutant allele transcripts and a dominant negative effect on the export of other RNAs. This proposal is focused on testing this RNA dominance model using several different experimental approaches. First, the hypothesis that (CUG)n expansion RNAs have a dominant negative effect on mRNA export will be directly examined using RNA microinjection into frog oocyte and mammalian fibroblast nuclei. Second, the sequestration hypothesis predicts that expansion binding proteins should accumulate in nuclear foci together with DMPK mutant transcripts. Therefore, we will complete the characterization of several proteins that preferentially recognize large (CUG)n expansions, and determine the subcellular distribution of these proteins in normal and DM patient cells. Third, preferred RNA binding sites for these expansion binding proteins will be characterized by in vitro and in vivo analyses with particular emphasis on identifying RNAs that normally associate with these proteins. Fourth, we will determine if expansion binding proteins are involved in mRNA export by combining the use of monoclonal antibodies and recombinant proteins with the microinjection system developed in the first aim. Fifth, the relevance of RNA dominance to other neuromuscular and neurological diseases will be investigated. These studies have important implications for elucidating molecular mechanisms involved in DM pathogenesis and cellular strategies which facilitate the exchange of genetic information between the nucleus and cytoplasm.