Molecular mechanism of dipeptide repeat protein production from hexanucleotide repeats in C9ORF72-related ALS and FTD

项目摘要 C9 orf 72非编码区的六核苷酸重复扩增是两者最常见的遗传原因。 肌萎缩侧索硬化症（ALS）和额颞叶痴呆（FTD）。一种潜在的致病机制 是重复相关非AUG产生的二肽重复（DPR）蛋白的异常积累 (RAN)在两种意义的所有六个阅读框架（poly-GA、poly-GR、poly-PA、poly-PR和poly-PG）中的翻译 和反义RNA。这些DPR蛋白的异常胞质内含物已在C9 ORF 72中被发现。 患者组织和细胞。几条证据表明，某些形式的DPR蛋白可以被 致病性，但很少有人知道DPR是如何从扩增的重复序列中产生的。这个目标 建议是了解分子机制，并确定DPR生产的遗传修饰剂， 正义和反义重复序列，结合基因组规模的CRISPR/Cas9敲除筛选技术， 生物化学和分子学方法。我们将主要关注重复RNA的两个主要步骤 可能影响最终蛋白质表达水平的加工：RNA核输出和RAN翻译。我们 将剖析有义和反义重复的核输出的分子途径，什么因素 介导这一过程，以及C9介导的核质转运毒性如何影响这一过程。我们将 破译C9 RAN翻译的分子机制，并检查是否有共同的修饰符 有义和反义重复序列之间的区别总而言之，这些研究可以提供关于DPR如何 C9重复序列产生的蛋白质，以及可以采取什么方法来防止它。这将指导 研究工具，以了解DRPs如何有助于疾病的发病机制。它还可能提供 潜在的治疗靶点，以通过抑制其产生来降低DPR介导的毒性。

PROJECT SUMMARY Hexanucleotide repeat expansion in a non-coding region of C9orf72 is the most common genetic cause of both amyotrophic lateral sclerosis (ALS) and frontal temporal dementia (FTD). One potential pathogenic mechanism is the aberrant accumulation of dipeptide repeat (DPR) proteins produced by repeat-associated non-AUG (RAN) translation in all six reading frames (poly-GA, poly-GR, poly-PA, poly-PR and poly-PG) of both sense and antisense RNAs. Abnormal cytoplasmic inclusions of these DPR proteins have been found in C9ORF72 patient tissues and cells. Several lines of evidence indicate that some forms of the DPR proteins can be pathogenic, yet little is known how the DPRs are generated from the expanded repeats. The goal of this proposal is to understand the molecular mechanisms and identify genetic modifiers of DPR production from both sense and antisense repeats, combining the genome-scale CRISPR/Cas9 knockout screening technology with biochemical and molecular approaches. We will primarily focus on two major steps of repeat RNA processing that could influence the final protein expression level: RNA nuclear export and RAN translation. We will dissect the molecular pathways of nuclear export for both sense and antisense repeats, what factors mediate this process, and how this is affected by C9-mediated toxicity on nucleocytoplasmic transport. We will decipher the molecular mechanisms of C9 RAN translation, and examine whether there are common modifiers between sense and antisense repeats. Altogether, these studies can provide novel insights on how DPRs are produced from C9 repeats and what approaches can be taken to prevent it. This will guide the development of research tools to understand how DRPs contribute to the disease pathogenesis. It will also possibly provide potential therapeutic targets to reduce DPR-mediated toxicity by inhibiting their production.