Defining the Role of Germ Granules in Gene Regulation

定义胚芽颗粒在基因调控中的作用

• 批准号: RGPIN-2020-06235
• 负责人: Claycomb, Julie
• 金额: $ 4.23万
• 依托单位: University of Toronto
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=717978
• 关键词: Defining; Role; Germ; Granules; Gene

Regulation of gene expression is a tightly controlled multistep process that is central to the development of all organisms. Such regulation is particularly important within the germline, which makes sperm and eggs. Errors in germline gene regulation can be transmitted to future generations and have major implications for species survival. Notably, phase-separated cytoplasmic sites of RNA localization and regulation, called germ granules, have emerged as key but poorly understood hubs for regulating gene expression in animal germlines. Small RNA mediated gene regulatory pathways (sRNA pathways) also play key roles in germline gene expression and epigenetic inheritance. At the core of these pathways are the Argonaute (AGO) proteins, which are guided in a sequence specific manner to their target transcripts by sRNAs (18-30 nucleotides). C. elegans has made critical contributions to understanding sRNA mechanisms, and a robust network of sRNA pathways, including four types of sRNA and 15 AGOs contributes to proper germline gene regulation. In fact, eight germline AGOs localize to germ granules, and loss of some agos leads to disruption of the granules, along with dysfunction of epigenetic inheritance of sRNAs, and sterility. Conversely, loss of known germ granule components results in a deficiency to mount an RNAi silencing response and sterility. At least three different types of germ granules have been discovered in worms, including P granules, Mutator foci, and Z granules. However, the molecular mechanisms governing the interplay between germ granules and sRNA gene regulatory pathways are poorly understood. Therefore, we aim to dissect the role(s) of germ granules in gene regulation and their relationship to sRNA pathways, using an integrated approach that combines our strengths in C. elegans sRNA biology, genomics, genetics, and molecular, cellular and developmental biology. Our goals are to: 1. Define germ granule components and the roles of these components in gene regulation Approach: We will use microscopy and in vivo proximity labeling (BioID) to comprehensively identify germ granule proteins and their protein-protein interactions. 2. Characterize the localization patterns of AGO/sRNA target transcripts Approach: We will use smFISH and develop live imaging of transcripts in the worm germline to determine where sRNA target transcripts localize 3. Identify the RNA signatures required for routing transcripts into AGO pathways Approach: We will use computational methods and comparative genomics to determine the features of mRNAs that enable them to be routed into one sRNA pathway versus any other. These studies will uncover novel biological insights into mechanisms of the essential process of germline gene regulation. Moreover, they will deliver new datasets (proteome of germ granules) and tools (germline BioID and live RNA imaging) for studying the C. elegans germline that be widely utilized by the entire field.

基因表达的调控是一个严格控制的多步骤过程，是所有生物体发育的核心。这种调节在生殖细胞中尤其重要，生殖细胞制造精子和卵子。生殖系基因调控的错误可以传递给后代，并对物种生存产生重大影响。值得注意的是，相分离的RNA定位和调节的细胞质位点，称为生殖颗粒，已经成为调节动物生殖系基因表达的关键但知之甚少的枢纽。 小RNA介导的基因调控途径（sRNA途径）也在生殖系基因表达和表观遗传中发挥关键作用。这些途径的核心是Argonaute（AGO）蛋白，其通过sRNA（18-30个核苷酸）以序列特异性方式引导至其靶转录物。C.线虫为理解sRNA机制做出了重要贡献，而sRNA途径的强大网络，包括四种类型的sRNA和15种AGO，有助于正确的生殖系基因调控。事实上，八个生殖系AGO定位于胚芽颗粒，并且一些AGO的损失导致颗粒的破坏，沿着sRNA的表观遗传遗传功能障碍和不育。相反，已知的胚芽颗粒组分的损失导致RNAi沉默反应的缺乏和不育。 在蠕虫中至少发现了三种不同类型的生殖颗粒，包括P颗粒、突变灶和Z颗粒。然而，控制胚芽颗粒和sRNA基因调控途径之间相互作用的分子机制知之甚少。因此，我们的目标是剖析的作用（S）的胚芽颗粒在基因调控和它们的关系，sRNA途径，使用一个综合的方法，结合我们的优势，在C。线虫sRNA生物学、基因组学、遗传学以及分子、细胞和发育生物学。 我们的目标是： 1.定义胚粒成分和这些成分在基因调控中的作用 方法：我们将使用显微镜和体内邻近标记（BioID）来全面鉴定胚芽颗粒蛋白及其蛋白质-蛋白质相互作用。 2.表征AGO/sRNA靶转录物的定位模式 方法：我们将使用smFISH和开发蠕虫生殖系中转录本的实时成像，以确定sRNA靶转录本的定位 3.确定将转录本路由到AGO途径所需的RNA签名 方法：我们将使用计算方法和比较基因组学来确定mRNAs的特征，这些特征使它们能够被路由到一个sRNA通路而不是任何其他通路。 这些研究将揭示新的生物学见解生殖细胞基因调控的基本过程的机制。此外，他们还将提供新的数据集（胚芽颗粒蛋白质组）和工具（种系BioID和活RNA成像），用于研究C。elegans种系，被整个领域广泛利用。