Development of a chemogenetically controlled RNA aggregation system and its application in elucidating the role of RNA foci in repeat expansion disease

摘要 重复扩张性疾病是一组超过40种严重的神经系统疾病， 三核苷酸或六核苷酸重复序列的异常扩增。虽然有几个建议， 重复扩增疾病的发病机制，它被广泛认为是RNA获得的功能毒性 是几种重复扩张疾病发病机制的主要贡献者。RNA功能获得毒性是 其特征在于RNA结合蛋白（RBP）的重复扩增RNA螯合，并引起全球性的 剪接事件和细胞毒性的失调。重复扩增RNA也已知形成核， 扩增RNA和RBP的相分离聚集体，也称为RNA焦点。RNA的作用 对RNA功能获得毒性的病灶了解甚少。这是因为现有的工具 基于扩增重复序列表达和RNA焦点相关性研究RNA功能获得毒性 形成与RBP螯合和细胞毒性。使用上述方法的几项研究 显示较长的形成病灶的重复序列的表达与 短的，非病灶形成的，重复的。然而，目前还不清楚细胞毒性是否依赖于形成 的RNA病灶达到毒性水平的RBP螯合，或是否增加RBP结合位点的时间较长 重复足以达到RBP螯合的毒性水平，而RNA灶的形成仅仅是一个过程。 副产品通过比较形成病灶的细胞与形成病灶的细胞的细胞毒性和蛋白质螯合水平， 不形成病灶，但具有相同长度的重复RNA，我们可以直接评估RNA病灶的形成是否是 在重复扩增疾病中细胞毒性所需的。要做到这一点，诱导RNA焦点的形成是必需的。 最近，我开发了一种方法，首次允许化学遗传控制RNA 聚合来这项技术提供了机会，以确定是否RNA焦点的形成是负责 重复扩张性疾病的发病机制。在这个建议中，我将优化我以前开发的 可诱导RNA聚集系统，以改进其作为研究重复序列中RNA焦点工具的可用性 膨胀病然后，我将使用这个系统来确定RNA焦点的形成是否是 负责增加水平的RBP螯合和细胞毒性的重复扩增 疾病C9 orf 72型肌萎缩侧索硬化症（ALS）和额颞叶痴呆（FTD）。我 研究结果将确定RNA病灶的破坏是否会直接为C9 orf 72型 ALS/FTD和潜在的其他重复扩增疾病。此外，第一个工具的出版， RNA聚集的化学遗传学控制将开创研究RNA聚集的全新途径 分子生物学和疾病中的事件。

ABSTRACT Repeat expansion diseases are a group of over 40 severe neurological disorders that are caused by an abnormal expansion of trinucleotide or hexanucleotide repeat sequences. While there are several proposed mechanisms of repeat expansion disease pathogenesis, it is widely accepted that RNA-gain-of-function toxicity is the major contributor to pathogenesis in several repeat expansion diseases. RNA-gain-of-function toxicity is characterized by repeat expansion RNA sequestering of RNA-binding proteins (RBPs) and causing global dysregulation in splicing events and cellular toxicity. Repeat expansion RNA is also known to form nuclear, phase-separated aggregates of expansion RNA and RBPs, also known as RNA foci. However, the role of RNA foci in RNA-gain-of-function toxicity is poorly understood. This is due to the fact that the only existing tools for studying RNA-gain-of-function toxicity rely on expression of expansion repeats and correlation of RNA foci formation with RBP sequestration and cellular toxicity. Several studies using the aforementioned methods have shown that expression of long, foci-forming repeats elicits higher cellular toxicity compared to expression of short, non-foci forming, repeats. However, it is unclear whether cellular toxicity is dependent on the formation of RNA foci to reach toxic levels of RBP sequestration, or whether the increase RBP binding sites of longer repeats is sufficient to reach toxic levels of RBP sequestration and the formation of RNA foci is merely a byproduct. By comparing cellular toxicity and levels of protein sequestration in cells that form foci, to cells that do not form foci but harbor the same length repeat RNA, we can directly assess whether RNA foci formation is required for cellular toxicity in repeat expansion diseases. To do this, inducible RNA foci formation is required. Recently, I have developed an approach that allows, for the first time, for chemogenetic controlled RNA aggregation. This technology presents the opportunity to determine whether RNA foci formation is responsible for repeat expansion disease pathogenesis. In this proposal, I will optimize my previously developed inducible RNA aggregation system to improve its usability as a tool to study RNA foci in repeat expansion disease. Then, I will use this system to determine whether RNA foci formation is responsible for increased levels of RBP sequestration and cellular toxicity in the repeat expansion disease C9orf72-type amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). My findings will determine whether disruption of RNA foci will directly provide therapeutic benefit for C9orf72-type ALS/FTD and potentially other repeat expansion diseases. In addition, publication of the first ever tool for chemogenetic control of RNA aggregation will pioneer an entirely new approach for studying RNA aggregation events in molecular biology and disease.