Defining the Role of Germ Granules in Gene Regulation

基因表达的调控是一个严格控制的多步骤过程，对所有生物体的发育至关重要。这种调节在生殖细胞中尤为重要，生殖细胞产生精子和卵子。生殖系基因调控中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，相分离的细胞质RNA定位和调控位点，称为胚粒，已经成为调节动物种系基因表达的关键枢纽，但人们对其知之甚少。小RNA介导的基因调控通路（sRNA通路）在种系基因表达和表观遗传中也起着关键作用。这些途径的核心是Argonaute （AGO）蛋白，它通过sRNAs（18-30个核苷酸）以序列特定的方式引导到它们的目标转录物。秀丽隐杆线虫在理解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，有助于适当的种系基因调控。事实上，有8个种系的AGOs定位于胚芽颗粒，其中一些AGOs的丢失会导致胚芽颗粒的破坏，同时导致sRNAs表观遗传功能障碍和不育。相反，失去已知的细菌颗粒成分会导致缺乏RNAi沉默反应和不育。在蠕虫中至少发现了三种不同类型的胚芽颗粒，包括P颗粒、突变灶和Z颗粒。然而，调控胚芽颗粒与sRNA基因调控途径之间相互作用的分子机制尚不清楚。因此，我们的目标是利用综合我们在秀丽隐杆线虫sRNA生物学、基因组学、遗传学、分子、细胞和发育生物学方面的优势的综合方法，剖析细菌颗粒在基因调控中的作用及其与sRNA通路的关系。我们的目标是：1。定义细菌颗粒成分及其在基因调控中的作用方法：我们将使用显微镜和体内接近标记（BioID）来全面鉴定细菌颗粒蛋白及其蛋白-蛋白相互作用。2. 鉴定AGO/sRNA靶转录物的定位模式方法：我们将使用smFISH并在蠕虫种系中开发转录物的实时成像，以确定sRNA靶转录物的定位3。方法：我们将使用计算方法和比较基因组学来确定mrna的特征，这些特征使它们能够被路由到一个sRNA途径而不是任何其他途径。这些研究将为种系基因调控基本过程的机制揭示新的生物学见解。此外，他们将为秀丽隐杆线虫种系研究提供新的数据集（胚粒蛋白质组）和工具（种系BioID和活RNA成像），这些数据集和工具将被整个领域广泛使用。

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.