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

化学遗传学控制的RNA聚集系统的开发及其在阐明RNA焦点在重复扩增疾病中的作用中的应用

• 批准号: 10387069
• 负责人: Mildred Johanna Unti
• 金额: $ 3.8万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-01 至 2023-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10387069
• 关键词: Affinity; Binding Proteins; Binding Sites; Biophysics; C9ORF72; Cells; Development; Devices; Dimerization; Disease; Event; Genetic Transcription; Length; Methods; Modeling; Molecular Biology; Molecular Disease; Nuclear; Pathogenesis; Phase; Proteins; Publications; RNA; RNA Binding; RNA Splicing; RNA-Binding Proteins; Role; System; Technology; Tetracyclines; Therapeutic; Time; Toxic effect; Work; aptamer; base; cytotoxicity; design; frontotemporal lobar dementia-amyotrophic lateral sclerosis; gain of function; genome-wide; improved; nervous system disorder; novel strategies; prevent; response; stem; tool; tool development; usability

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.

抽象的 重复扩张疾病是一组由 40 多种严重神经系统疾病引起的疾病 三核苷酸或六核苷酸重复序列的异常扩增。虽然有几个提议 重复扩增疾病发病机制中，人们普遍认为 RNA 功能获得性毒性 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 其特点是重复扩增 RNA 隔离 RNA 结合蛋白 (RBP) 并导致全局 剪接事件和细胞毒性的失调。重复扩增 RNA 也可形成核， 扩增 RNA 和 RBP 的相分离聚集体，也称为 RNA 焦点。然而，RNA 的作用 RNA 功能获得毒性的焦点尚不清楚。这是因为现有的唯一工具 研究 RNA 功能获得毒性依赖于扩展重复的表达和 RNA 焦点的相关性 形成 RBP 隔离和细胞毒性。一些使用上述方法的研究已经 结果表明，与表达长的、形成灶的重复序列相比，表达会引起更高的细胞毒性。 短、​​不形成焦点、重复。然而，尚不清楚细胞毒性是否依赖于形成 RNA 焦点达到 RBP 隔离的毒性水平，或者是否增加 RBP 结合位点的时间更长 重复序列足以达到 RBP 隔离的毒性水平，并且 RNA 焦点的形成仅仅是一个 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隔离水平， 不形成焦点但具有相同长度的重复RNA，我们可以直接评估RNA焦点的形成是否是 重复扩增疾病中细胞毒性所需的。为此，需要形成可诱导的 RNA 灶。 最近，我开发了一种方法，首次允许化学遗传学控制 RNA 聚合。这项技术提供了确定 RNA 焦点形成是否是其原因的机会 为重复扩张病的发病机制。在这个提案中，我将优化我之前开发的 诱导性 RNA 聚集系统，提高其作为重复研究 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 导致重复扩增中 RBP 隔离和细胞毒性水平升高 疾病 C9orf72 型肌萎缩侧索硬化症 (ALS) 和额颞叶痴呆 (FTD)。我的 研究结果将确定 RNA 焦点的破坏是否会直接为 C9orf72 型提供治疗益处 ALS/FTD 和其他潜在的重复扩张疾病。此外，还发布了第一个工具 RNA聚集的化学遗传学控制将开创一种研究RNA聚集的全新方法 分子生物学和疾病中的事件。