Identification of Genetic Suppressors of RNA Granules

总结/摘要 含有多聚腺苷酸化（Poly（A）+）RNA的细胞质颗粒影响许多生物学功能 和疾病状态。在正常细胞中，poly（A）+颗粒具有多种形式，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， 稳定性和/或在各种生理环境下的翻译控制。致病性蛋白质聚集体是一种 许多神经退行性疾病（ND）的统一特征，包括肌萎缩侧索硬化症（ALS）。的 正常和致病聚集体之间的关系仍在破译，但明确的联系 两者之间的关系是已知的。因此，poly（A）+ RNA和蛋白质在细胞质颗粒中的共聚集 在正常和疾病条件下都具有广泛的生物学重要性。然而，遗传抑制因子 poly（A）+ RNA颗粒形成尚未在全基因组范围内鉴定。其长期目标是 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。总体 假设poly（A）+ RNA颗粒形成遗传调节因子可用于调节RNA颗粒 在特定的生物学和疾病相关的背景下形成。作为检验这一假设的第一步， 这项建议的主要目的是进行全基因组筛选，以确定基因，抑制 果蝇和人类细胞中的poly（A）+ RNA颗粒形成。RNA颗粒抑制基因，当 被RNAi耗尽的聚腺苷酸（poly（A）+ RNA）将引起聚腺苷酸+ RNA颗粒的自发形成。获得原发性 为了实现这一目标，将努力实现以下两个具体目标。目的1：鉴定抑制poly（A）+ RNA的基因 果蝇S2 R+细胞中的颗粒。图像分析流水线将适于重新分析来自 先前公开的在果蝇S2 R+细胞中的全基因组RNAi筛选（Farny等，2008年）。目标2： 研究人类细胞中poly（A）+ RNA颗粒抑制因子的进化保守性。使用主 筛选来自Aim 1的命中作为指导，将筛选靶向CRISPR敲除文库以鉴定人CRISPR基因敲除文库。 抑制poly（A）+ RNA颗粒U2 OS细胞的基因。这一目标也将解决工作假设 poly（A）+ RNA颗粒抑制子的遗传沉默将导致内源性TDP-43的增加， 在U2 OS细胞中聚集。这种方法的基本原理是，考虑到聚（A）+之间的已知联系， RNA颗粒和致病聚集体，poly（A）+聚集的抑制剂可以被利用， 未来的研究，以抑制致病性聚集。鉴于正常和致病之间的已知联系 聚合物的形成，了解聚（A）+ RNA颗粒形成的遗传决定因素将提供 研究致病性聚集体形成的遗传控制的新途径。所以，知识 在这里取得的成果将具有广泛的兴趣和重要性。

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.