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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.

项目总结