RNA helicases to combat RNA phase transitions in repeat expansion disorders

RNA解旋酶对抗重复扩增障碍中的RNA相变

基本信息

批准号：
10640592
负责人：
Linamarie Miller
金额：
$ 6.87万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-12-01 至 2026-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10640592
关键词：
Address Affect Affinity Age of Onset Antisense Oligonucleotides Binding Biochemical Biological Assay CAG repeat CRISPR/Cas technology Catalogs Cell Nucleus Cells Central Nervous System Central Nervous System Diseases Chemicals Clinical Trials Code Collaborations Collection Cytoplasm Cytoplasmic Granules Disease Event Failure Fellowship Future General Population Genetic Genome Human Huntington Disease In Vitro Knock-out Language Length Liquid substance Mass Spectrum Analysis Mediating Methods MicroRNAs Molecular Monitor Neurodegenerative Disorders Nuclear Nuclear RNA Nucleotides Pathologic Pathology Pediatric Hospitals Pennsylvania Peptides Phase Phase Transition Philadelphia Physical condensation Proteins Protocols documentation RNA RNA Helicase RNA Splicing RNA metabolism RNA purification RNA-Binding Proteins Recombinants Regulation Research Institute Research Personnel Ribonucleoproteins Role Severity of illness Spinocerebellar Ataxias Techniques Therapeutic Toxic effect Training Transcript Translations Universities Untranslated RNA Visuospatial Work candidate identification cellular targeting combat design executive function helicase in vivo information processing insight multimodality mutant nervous system disorder neurotoxic new therapeutic target novel therapeutics physical property processing speed protein function spinal and bulbar muscular atrophy stress granule tool transcriptome sequencing

项目摘要

Abstract Repeat-expansion disorders are a collection of more than 40 rare but devastating diseases that largely effect the central nervous system. Currently, they are incurable. The genetic basis for these disorders is repeat expansions, the length of which determines disease severity and age of onset. In these disorders, repeat sequences cause pathology in a multimodal fashion. The translation of repeats located in coding regions can disrupt normal protein function and produce toxic repeat peptides. These neurotoxic peptides can be produced even when the repeats are in noncoding regions via repeat associated non-AUG translation. Another agent of pathology is the repeat RNA transcript itself, where aberrant binding of the repeat RNA to RNA-binding proteins leads to issues like deregulation of the miRNA machinery, aberrant translation, and mis-splicing events. Repeat RNAs also enable transcripts to undergo liquid-liquid phase separation (LLPS) and form nuclear RNA foci in cells. These foci can sequester RNA-binding proteins, which may contribute to the observed repeat RNA toxicity. It has been well established that RNA helicases regulate the formation of phase separated ribonucleoprotein (RNP) granules in cells, but no work exists to understand how helicases affect the RNA foci in repeat-expansion disorders. Considering first that repeat RNAs cause potentially pathological RNA foci and that second, helicases are important for the formation of other cellular RNP granules, we hypothesize that RNA helicases can also modulate the formation of RNA foci in repeat-expansion disorders. The proposed work is focused on the deleterious RNA foci that form in the repeat-expansion disorders caused by CAG trinucleotide expansions like Huntington’s Disease and several spinocerebellar ataxias. This proposed study aims to identify RNA helicases that modulate these deleterious RNA foci and understand the mechanism by which they do so. Knockout screens in human HAP1 cells expressing CAG repeat constructs which form RNA foci have identified candidate RNA helicases that affect RNA foci size and number. This work will utilize in vitro approaches to understand the mechanism underlying how these candidate helicases effect RNA foci. After the recent failure of two candidate therapies for Huntington’s Disease in clinical trials, it is especially necessary to consider new therapeutic targets for these disorders. This work will expand our understanding of RNA helicase function in repeat-expansion disorders and offer a new therapeutic tool to explore for combatting these diseases. The proposed fellowship will be conducted at the University of Pennsylvania in the lab of Dr. James Shorter, a leading researcher in the molecular basis of neurodegenerative diseases and protein disaggregases. Completion of the proposal in this lab will provide rigorous training in biochemical assay design and in vitro protein and RNA handling techniques.

抽象的重复扩张障碍是 40 多种罕见但毁灭性疾病的集合，这些疾病在很大程度上影响目前，这些疾病的遗传基础是无法治愈的。扩展，其长度决定疾病的严重程度和发病年龄在这些疾病中，重复。位于编码区的重复序列的翻译可以以多模式方式引起序列病理。破坏正常的蛋白质功能并产生有毒的重复肽这些神经毒性肽。即使当重复位于非编码区域时，也通过重复相关的非 AUG 翻译。病理学是重复RNA转录本本身，其中重复RNA与RNA结合蛋白的异常结合导致 miRNA 机制失调、翻译异常和重复剪接事件等问题。 RNA 还使转录物能够进行液-液相分离 (LLPS) 并在细胞核中形成 RNA 焦点这些焦点可以隔离 RNA 结合蛋白，这可能导致观察到的重复 RNA 毒性。众所周知，RNA 解旋酶调节相分离核糖核蛋白的形成 (RNP) 颗粒在细胞中，但目前还没有工作来了解解旋酶如何影响重复扩增中的 RNA 焦点首先考虑重复RNA会导致潜在的病理性RNA病灶，其次考虑解旋酶。对于其他细胞 RNP 颗粒的形成很重要，我们发现 RNA 解旋酶也可以调节重复扩增紊乱中 RNA 灶的形成。拟议的工作重点是由 CAG 三核苷酸扩增引起的重复扩增紊乱中形成的有害 RNA 灶，例如亨廷顿病和几种脊髓小脑性共济失调本研究旨在鉴定 RNA 解旋酶。调节这些有害的 RNA 焦点并了解它们的敲除筛选机制。在表达形成 RNA 焦点的 CAG 重复结构的人类 HAP1 细胞中，已鉴定出候选 RNA 这项工作将利用体外方法来了解影响 RNA 焦点大小和数量的解旋酶。在最近两个候选解旋酶失败后，这些候选解旋酶如何影响 RNA 焦点的机制。亨廷顿病的治疗方法进入临床试验，尤其需要考虑新的治疗靶点这项工作将扩大我们对重复扩增中 RNA 解旋酶功能的理解。疾病并提供一种新的治疗工具来探索对抗这些疾病。拟议的奖学金将在宾夕法尼亚大学 James Shorter 博士的实验室进行。神经退行性疾病和蛋白质解聚的分子基础领域的领先研究员。该实验室的提案将提供生化分析设计以及体外蛋白质和 RNA 方面的严格培训处理技术。