Defining the Role of Germ Granules in Gene Regulation

基因表达调控是一个严格控制的多步骤过程，对所有生物体的发育都是至关重要的。这种调节在产生精子和卵子的生殖系中尤为重要。生殖系基因调控中的错误可能会遗传给后代，并对物种的生存产生重大影响。值得注意的是，被称为生殖粒的RNA定位和调控的相分离细胞质位置已经成为调节动物种系中基因表达的关键但鲜为人知的枢纽。小RNA介导的基因调控通路(sRNA通路)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ArgAerte(AGO)蛋白，它们由sRNAs(18-30个核苷酸)以序列特异性的方式引导到其目标转录本。线虫在理解sRNA机制方面做出了重要贡献，一个强大的sRNA通路网络，包括4种类型的sRNA和15个agos，有助于适当的生殖系基因调控。事实上，8个生殖系agos定位于生殖粒，一些agos的丢失会导致颗粒的破坏，以及sRNA的表观遗传功能障碍，从而导致不育。相反，已知的细菌颗粒成分的丢失会导致缺乏RNAi沉默反应和不育。在蠕虫中至少发现了三种不同类型的细菌颗粒，包括P颗粒、突变焦点和Z颗粒。然而，控制生殖细胞颗粒和sRNA基因调控途径相互作用的分子机制却知之甚少。因此，我们的目标是结合我们在线虫sRNA生物学、基因组学、遗传学以及分子、细胞和发育生物学方面的优势，使用一种综合的方法来剖析胚粒在基因调控中的作用(S)及其与sRNA途径的关系。我们的目标是：1.确定胚芽颗粒的组成成分及其在基因调控方法中的作用：我们将使用显微镜和活体邻近标记(BioID)来全面鉴定胚芽颗粒蛋白及其蛋白-蛋白质相互作用。2.鉴定AGO/sRNA靶向转录本的定位模式：我们将使用smFISH并对蠕虫胚系中的转录本进行实时成像，以确定sRNA靶向转录本定位在哪里。3.确定将转录本送入AGO途径所需的RNA签名：我们将使用计算方法和比较基因组学来确定mRNAs的功能，使它们能够被路由到一条sRNA途径而不是另一条sRNA途径。这些研究将揭示对生殖系基因调控的基本过程的新的生物学见解。此外，他们将提供新的数据集(胚芽颗粒蛋白质组)和工具(胚系BioID和实时RNA成像)，用于研究被整个领域广泛利用的线虫种系。

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.