eIF4e Assembly into Germ Granules is Essential for its Repressive Function

In metazoans, germ cells play a fundamental role in ensuring the continuity of animal life. Germ cells contain germ granules, cytoplasmic non-membrane bound organelles also referred to as RNA-protein condensates. These germ granules house a variety of RNAs and proteins essential for germline identity, maintenance, and fertility. In C. elegans, P granules are a specific type of germ granule that localize to the nuclear periphery of developing germ cells and regulate mRNA expression. Proper P granule assembly is dependent on the PGL family of granule-forming proteins. PGL-1, the flagship P granule assembly protein and PGL member, contains two dimerization domains and a C-terminus region. Numerous proteins localize to P granules, most of which are RNA-binding or regulatory proteins. For example, IFE-1 is a eIF4E family member that binds the 5’ cap of mRNAs, directly interacts with PGL-1 in vivo and in vitro, and localizes to P granules. The precise molecular function of PGL and their associated proteins remains unclear; they may act solely as granule assembly proteins, serve as scaffolds to recruit other proteins, or directly regulate RNA. Here, genetics and imaging methods were used to molecularly dissect PGL-1 in vivo to probe how different protein regions are required for granule assembly, P granule binding partner recruitment, and germ cell development. We used CRISPR to (a) tag IFE-1 protein with an mScarlet fluorescent protein, (b) delete protein coding regions of pgl-1 to investigate IFE-1 binding and to understand how IFE-1 assembly into P granules is crucial for its role in germ cell development. Deletion of PGL-1 dimerization domains significantly disrupted P granule formation and IFE-1 recruitment. Removal of the C-terminus region led to abnormal P granule formation, which profoundly affected IFE-1 localization. These findings reveal that the previously uncharacterized C-terminal region plays a critical role in the integrity and functionality of P granules. We are performing immunoprecipitation assays to verify the direct binding of IFE-1 and PGL-1. Our study provides novel insights into the molecular mechanisms governing P granule protein interactions, highlighting their essential biological relevance and potential impact on understanding germ cell development. eCLIP was performed to identify the RNA targets of the C. elegans eIF4e homolog, IFE-1.