Defining the Role of Germ Granules in Gene Regulation

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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.