Identification of Genetic Suppressors of RNA Granules

RNA 颗粒遗传抑制因子的鉴定

基本信息

批准号：
10432294
负责人：
Natalie Gilks Farny
金额：
$ 7.37万
依托单位：
WORCESTER POLYTECHNIC INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-06-01 至 2024-02-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10432294
关键词：
Address Affect Alpha Granule Alzheimer&apos s Disease Amyotrophic Lateral Sclerosis Area Biological Biological Process Biology Cell Line Cells Cellular Stress Clustered Regularly Interspaced Short Palindromic Repeats Cytoplasmic Granules Data Data Set Defect Disease Drosophila genus Embryonic Development Environmental Risk Factor Future Genes Genetic Genetic Determinism Germ Germ Cells Goals Hand Human Image Image Analysis Investigation Knock-out Knowledge Libraries Link Malignant Neoplasms Manuals Maps Messenger RNA Modeling Molecular Neurodegenerative Disorders Neurons Normal Cell Pathogenicity Pathway interactions Physiological Play Poly A Poly(A)+ RNA Positioning Attribute Proteins Publishing RNA RNA Interference RNA Probes RNA interference screen Regulation Research Role TDP-43 aggregation Testing Therapeutic Visualization Work analysis pipeline automated image analysis biological adaptation to stress design genome-wide granule cell interest mRNA Export oligo (dT)programs protein TDP-43 protein aggregation screening stress granule tau aggregation whole genome

项目摘要

SUMMARY/ABSTRACT Polyadenylated (Poly(A)+) RNA containing cytoplasmic granules affect numerous biological functions and disease states. In normal cells, poly(A)+ granules take many forms, such as stress granules (SGs), processing bodies (PBs), neuronal granules, and germ cell granules. These granules affect mRNA localization, stability, and/or translational control under various physiological contexts. Pathogenic protein aggregates are a unifying feature of many neurodegenerative diseases (ND), including amyotrophic lateral sclerosis (ALS). The relationships between normal and pathogenic aggregates is still being deciphered, however clear links between the two are known. Thus, the co-aggregation of poly(A)+ RNA and protein in cytoplasmic granules under both normal and disease conditions is of broad biological importance. Yet, the genetic suppressors of poly(A)+ RNA granule formation have not been identified on a genome-wide scale. The long-term goal of this research program is to understand both the genetic and the environmental factors that regulate SG formation, and to understand how SGs relate to other types of aggregates and disease states. The overarching hypothesis is that genetic regulators of poly(A)+ RNA granule formation can be used to modulate RNA granule formation in specific biological and disease-related contexts. As a first step to testing this hypothesis, the primary objective of this proposal is to perform whole-genome screening to identify genes that suppress poly(A)+ RNA granule formation in both Drosophila and human cells. RNA granule suppressing genes, when depleted by RNAi, will cause the spontaneous formation of poly(A)+ RNA granules. To obtain the primary objective, the following two specific aims will be pursued. Aim 1: To identify genes that suppress poly(A)+ RNA granules in Drosophila S2R+ cells. An image analysis pipeline will be adapted to re-analyze images from a previously published whole-genome RNAi screen in Drosophila S2R+ cells (Farny et al., 2008). Aim 2: To examine the evolutionary conservation of poly(A)+ RNA granule suppressors in human cells. Using the primary screen hits from Aim 1 as a guide, a targeted CRISPR knockout library will be screened to identify human genes that suppress poly(A)+ RNA granules U2OS cells. This aim will also address the working hypothesis that genetic silencing of poly(A)+ RNA granule suppressors will result in an increase in endogenous TDP-43 aggregation in U2OS cells. The rationale for this approach is that, given the known links between poly(A)+ RNA granules and disease-causing aggregates, suppressors of poly(A)+ aggregation could be harnessed in future studies to suppress pathogenic aggregates. Given the known links between normal and pathogenic aggregate formation, understanding the genetic determinants of poly(A)+ RNA granule formation will provide new avenues of investigation for the genetic control of pathogenic aggregate formation. Thus the knowledge gained here will be of broad interest and importance.

摘要/摘要含有细胞质颗粒的聚腺苷酸 (Poly(A)+) RNA 影响多种生物学功能和疾病状态。在正常细胞中，Poly(A)+ 颗粒有多种形式，例如应激颗粒 (SG)、加工体 (PB)、神经元颗粒和生殖细胞颗粒。这些颗粒影响 mRNA 定位，各种生理环境下的稳定性和/或翻译控制。致病性蛋白质聚集体是许多神经退行性疾病（ND）的共同特征，包括肌萎缩侧索硬化症（ALS）。这正常聚集体和致病聚集体之间的关系仍在破译中，但联系却很明确两者之间已知。因此，细胞质颗粒中 Poly(A)+ RNA 和蛋白质的共聚集在正常和疾病条件下都具有广泛的生物学重要性。然而，基因抑制因子尚未在全基因组范围内鉴定出 Poly(A)+ RNA 颗粒的形成。本次活动的长远目标研究计划是了解调节 SG 形成的遗传和环境因素，并了解 SG 如何与其他类型的聚集体和疾病状态相关。首要的假设是 Poly(A)+ RNA 颗粒形成的遗传调节因子可用于调节 RNA 颗粒在特定的生物和疾病相关背景下形成。作为检验这一假设的第一步，该提案的主要目标是进行全基因组筛选，以确定抑制基因果蝇和人类细胞中聚 (A)+ RNA 颗粒的形成。 RNA颗粒抑制基因，当被 RNAi 耗尽后，将导致 Poly(A)+ RNA 颗粒自发形成。为了获得初级为实现这一目标，将实现以下两个具体目标。目标 1：鉴定抑制 Poly(A)+ RNA 的基因果蝇 S2R+ 细胞中的颗粒。图像分析管道将适用于重新分析来自先前发表的果蝇 S2R+ 细胞全基因组 RNAi 筛选（Farny 等，2008）。目标 2：检查人类细胞中 Poly(A)+ RNA 颗粒抑制因子的进化保守性。使用初级以 Aim 1 的筛选结果为指导，将筛选靶向 CRISPR 敲除文库以识别人类抑制 Poly(A)+ RNA 颗粒 U2OS 细胞的基因。这一目标还将解决工作假设 Poly(A)+ RNA 颗粒抑制因子的基因沉默将导致内源性 TDP-43 增加 U2OS细胞中的聚集。这种方法的基本原理是，考虑到 poly(A)+ 之间的已知联系 RNA 颗粒和致病聚集体、poly(A)+ 聚集抑制因子可用于未来抑制致病聚集体的研究。鉴于正常和致病之间已知的联系聚集体形成，了解 Poly(A)+ RNA 颗粒形成的遗传决定因素将提供致病聚集体形成的遗传控制研究的新途径。因而知识在这里获得的成果将具有广泛的兴趣和重要性。