Understanding and Manipulating the Degradation of the C9orf72 Repeat Expansion RNA

项目总结/摘要 理解和操纵C9 orf 72重复扩增RNA的降解 肌萎缩侧索硬化症（ALS）和额颞叶痴呆症（FTD）是进行性的，致命的 神经退行性疾病遗传性ALS和FTD最常见的原因是G4 C2的扩增。 C9 orf 72基因（c9 ALS/FTD）内含子中的序列。c9 ALS/FTD的标志性病理特征是 在神经元核中存在重复扩增RNA灶，这是不寻常的，因为内含子通常 降解得太快而不容易被检测到。积累的G4 C2重复扩增RNA被认为是 通过两种方式促进c9 ALS/FTD疾病的发展：它可以隔离RNA结合蛋白， 它们的功能，它可以输出到细胞质翻译成有毒的二肽重复蛋白。 重复扩增RNA的持续存在表明它形成了一种稳定的RNA结构， 正常的内含子降解机制。然而，真核细胞含有特定的机制， 降解具有强二级结构的RNA。这种RNA是用3'端RNA进行转录后修饰的。 寡聚尿苷或寡聚腺苷尾，然后募集进行性3'至5'核酸外切酶以降解RNA。我 假设C9 orf 72重复RNA是正常内含子降解途径的不良底物， 3'末端拖尾决定其衰变速率。为了验证这一点，本提案的目标是双重的：我将确定 重复扩增RNA的组成，积累到核灶，以了解内含子 降级正在停滞（目标1），我将检查对 G4 C2重复扩增RNA响应于3'末端加尾、去尾和RNA降解的扰动 (Aim 2）。总之，这些研究提供了一个令人兴奋的机会来发现 促进毒性重复扩增RNA的降解，并潜在地加速其衰变， 降低毒性。如果成功，从这项工作中获得的知识可以指导未来的治疗设计， c9ALS/FTD。

PROJECT SUMMARY/ABSTRACT Understanding and Manipulating the Degradation of the C9orf72 Repeat Expansion RNA Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are progressive, fatal neurodegenerative diseases. The most common cause of hereditary ALS and FTD is an expansion of the G4C2 sequence in an intron of the C9orf72 gene (c9ALS/FTD). A hallmark pathologic feature of c9ALS/FTD is the presence of repeat expansion RNA foci in neuronal nuclei, which is unusual because introns are usually degraded too rapidly to be easily detected. The accumulating G4C2 repeat expansion RNA is thought to contribute to c9ALS/FTD disease development in two ways: it can sequester RNA-binding proteins and disrupt their function, and it can be exported into the cytoplasm for translation into toxic dipeptide repeat proteins. The persistence of the repeat expansion RNA suggests that it forms a stable RNA structure that resists the normal intron degradation machinery. However, eukaryotic cells contain specific mechanisms for the degradation of RNAs with strong secondary structures. Such RNAs are post-transcriptionally modified with 3' oligo-uridine or oligo-adenosine tails that then recruit processive 3' to 5' exonucleases to degrade the RNA. I hypothesize that the C9orf72 repeat RNA is a poor substrate for normal intronic degradation pathways and that 3’ end tailing determines its rate of decay. To test this, the goal of this proposal is two-fold: I will determine the composition of the repeat expansion RNA that accumulates into nuclear foci to learn where intronic degradation is stalling (Aim 1), and I will examine the changes to the sequence and levels of the G4C2 repeat expansion RNA in response to perturbations to 3’ end tailing, tail removal, and RNA degradation (Aim 2). Taken together, these studies present an exciting opportunity to discover the mechanisms that promote degradation of the toxic repeat expansion RNA and, potentially, the ability to accelerate its decay to reduce toxicity. If successful, the knowledge gained from this work could guide future therapeutic design for c9ALS/FTD.