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. Overall design: eCLIP was performed to identify the RNA targets of the C. elegans eIF4e homolog, IFE-1.