TurboID-based proximity labeling identifies novel germline proteins that maintain E granule integrity and small RNA homeostasis in C. elegans.

Germ granules are biomolecular condensates composed of RNA and proteins that play crucial roles in RNA metabolism and post-transcriptional gene regulation. C. elegans germ granules consist of multiple distinct subcompartments, including P granules, Mutator foci, Z granules, SIMR foci, P-bodies, D granules, and E granules. Among these condensates, the E granule, which is nonrandomly positioned within the germ granule, is required for the production of a specialized class of small interfering RNAs (siRNAs). However, the mechanisms underlying E granule formation and its functional significance remain largely unexplored. In this study, via the use of TurboID-based proximity labeling technology combined with an RNAi-based reverse genetic screen, we identified two novel components of the E granule, EGC-2/C27B7.5 and EGC-3/F59G1.8, which initiate E granule assembly. The depletion of EGC-2 or EGC-3 disrupts the perinuclear localization of the EGO and PICS complexes, both of which are enriched in E granules and are required for E-class siRNA and piRNA biogenesis, respectively. Small RNAomic analyses revealed that both EGC-2 and EGC-3 promote the production of 5' E-class siRNA, whereas piRNA accumulation is inhibited by EGC-3. Taken together, our results elucidate the roles of EGC-2 and EGC-3 in maintaining E granule integrity and small RNA homeostasis. Additionally, the combination of proximity labeling technology and reverse genetic screening provides a robust strategy for studying the assembly of biomolecular condensates.