Mechanisms of RNA binding proteins rescuing FUS induced toxicity in yeast

RNA 结合蛋白拯救 FUS 诱导的酵母毒性的机制

基本信息

批准号：
10202952
负责人：
Shulin Ju
金额：
$ 45万
依托单位：
WRIGHT STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-04-01 至 2025-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10202952
关键词：
ALS pathology Affect Amyotrophic Lateral Sclerosis Automobile Driving Biological Process C-terminal C9ORF72 Cells Collaborations Color Cues Cytosol DNA Data Defect Development Disease Dose Environment Eukaryota Fluorescence Fluorescence Microscopy Functional disorder Gene Library Genes Genetic Genetic Models Growth Home Homologous Gene Human Image Impairment Individual Institution Laboratories Length Libraries Light Mammalian Cell Mammals Mediating Methods Modeling Monitor Mus N-terminal Neurodegenerative Disorders Neurons Partner in relationship Pathologic Pathway interactions Point Mutation Proteins RNA RNA Binding RNA metabolism RNA-Binding Protein FUS RNA-Binding Proteins Rattus Regulation Research Resources Ribonucleoproteins Role Stress Structure Suppressor Genes TAF15 gene Testing Toxic effect Two-Hybrid System Techniques Western Blotting Work Yeast Model System Yeasts base cytotoxicity experimental study expression vector fitness gene cloning genome-wide insight interest live cell imaging mutant new therapeutic target novel novel therapeutics overexpression protein TDP-43 protein aggregation protein biomarkers response screening stress granule success therapeutically effective tool undergraduate student

项目摘要

PROJECT SUMMARY Identification of two RNA-binding proteins (RBPs), FUS and TDP-43, as causative factors of Amyotrophic Lateral Sclerosis (ALS) resulted in a paradigm shift centered on RNA dysfunction as a disease-driving mechanism. We previously established a yeast model of FUS cytotoxicity. Using this model, we carried out genome-wide overexpression screen and identified five yeast genes that rescue FUS toxicity. Three out of five suppressor genes have human homologs and all three encode RBPs. We found that hUPF1, the human homolog of one of the three RBPs, rescues the toxicity of FUS in mouse primary neurons and a rat model of ALS. These findings support the value of our study of FUS toxicity in yeast and uncovered a novel pathway, currently under development as new therapeutics for ALS. Motivated by this success, we constructed a new genome-scale library, containing 13,570 full-length sequence verified human gene clones individually cloned in an inducible yeast-expression vector. Using this library and a newly developed efficient screening method, we identified 37 human genes, when overexpressed, robustly rescuing FUS induced toxicity in yeast. Genes encoding RBPs are highly enriched among suppressors (12 out of 37). Strikingly, all 12 RBP suppressors have known connections to stress granules (SG). The objective of this application is to define mechanisms of FUS toxicity by studying how the 12 RBPs work to rescue cellular defects induced by FUS. We hypothesize that toxicity of FUS involves direct impairment of SG function, and human RBP suppressors rescue FUS toxicity by alleviating detrimental effect of FUS on SG. Two specific Aims are proposed: i) examine the effect of suppressor RBPs on FUS aggregation and localization;; and ii) examine the effects of suppressor RBPs on SG. Our hypothesis is consistent with findings that FUS protein aggregates mis-localize to SG in yeast and in mammalian cells. Based on our preliminary data on TAF15, one of the human RBP suppressors, we reason that the suppressor mechanisms likely involve interactions between FUS and the suppressors as well as alterations in SG structure and function. Regulation and mis-regulation of SG is a pathway that is of immense interest to ALS research field. Completion of this project will provide deeper understanding of FUS mediated cytotoxicity, its connection to RNA metabolism, particularly with respect to aberrant SG function. Projects proposed here will not only expose more undergraduate students to meritorious research in the PI’s laboratory but also help promote discussion on scientific discoveries in an upper lever undergraduate course, regularly offered by the PI. Both serve to strengthen the research environment at the PI’s home institution.

项目概要鉴定出两种 RNA 结合蛋白 (RBP)：FUS 和 TDP-43，作为肌萎缩侧索硬化症的致病因素侧索硬化症 (ALS) 导致了以 RNA 功能障碍为中心的范式转变，这是一种疾病驱动因素我们之前建立了 FUS 细胞毒性的酵母模型，并利用该模型进行了研究。全基因组过度表达筛选并鉴定出 5 种酵母基因，可解救 FUS 毒性中的 3 种。五个抑制基因具有人类同源物，并且所有三个都编码 RBP，即 hUPF1。三种 RBP 之一的人类同源物，可消除 FUS 对小鼠原代神经元和大鼠的毒性这些发现支持了我们对酵母中 FUS 毒性的研究的价值，并发现了一种新的方法。受这一成功的激励，我们目前正在开发 ALS 的新疗法。构建了一个新的基因组规模的文库，包含13,570个经过验证的全长序列的人类基因使用该文库和新开发的克隆在诱导型酵母表达载体中单独克隆。有效的筛选方法，我们鉴定了37个人类基因，当过度表达时，有力地拯救了FUS 酵母中诱导的毒性。编码 RBP 的基因在抑制因子中高度富集（37 个中的 12 个）。引人注目的是，所有 12 种 RBP 抑制剂都与应激颗粒 (SG) 存在联系。该应用程序是通过研究 12 个 RBP 如何拯救细胞来定义 FUS 毒性机制我们假设 FUS 的毒性涉及 SG 功能的直接损害，和人类 RBP 抑制剂通过减轻 FUS 对 SG 的有害影响来缓解 FUS 的毒性。提出了具体目标： i) 检查抑制 RBP 对 FUS 聚集和定位的影响； ii) 检查抑制剂 RBP 对 SG 的影响。我们的假设与 FUS 的发现一致。根据我们的初步数据，蛋白质聚集体在酵母和哺乳动物细胞中错误定位到 SG。 TAF15 是人类 RBP 抑制剂之一，我们认为该抑制机制可能涉及其中。 FUS 和抑制因子之间的相互作用以及 SG 结构和功能的改变。 SG 的调控和误调控是 ALS 研究领域非常感兴趣的途径。该项目的完成将更深入地了解 FUS 介导的细胞毒性及其与 RNA 代谢，特别是关于异常 SG 功能的项目不仅会。让更多本科生接触 PI 实验室的优秀研究，同时也有助于促进 PI 定期提供的高级本科课程中关于科学发现的讨论。两者都有助于加强 PI 所在机构的研究环境。