Project Summary The pluripotent potential of a cell is generally assumed to be a product of its nucleus, regulated by gene expression and chromatin state. This view informs most approaches to drug discovery, where an ideal therapeutic would target nuclear processes to induce pluripotency and proliferation to heal a wound or inhibit pluripotency and proliferation in cancer. Often overlooked are somatic cell nuclear transfer experiments that definitively show cellular potential is also a product of the cytoplasm, specifically, the cytoplasm of germ cells. The long-term objectives of this research are to understand how cellular pluripotency and regenerative capacity are retained in germ cells through their specialized cytoplasm. We expect these findings to inform and expand approaches to drug discovery and lead to therapeutics that function by targeting the cytoplasmic milieu. Cytoplasmic processes unique to germ cells often occur in protein/RNA assemblies that reside on or near the nuclear periphery. These assemblies are collectively called germ granules. Because core germ-granule composition is conserved from nematodes to humans, we can use C. elegans nematodes to visualize germ granules in vivo and to expedite studies into their function. Mutations that displace or severely disrupt germ granules in C. elegans cause sterility and germline-to-somatic reprogramming. In this proposal, we look at the earliest events of somatic reprogramming to determine how processes within germ granules antagonize this fate. We investigate the contribution of glycine-rich repeat motifs to the specialized germ-granule microenvironment. We then describe two novel interactions with a core germ-granule protein called GLH-1 (DDX4 in humans). Lastly, we follow up on another core germ-granule protein that we named LOTR-1 (TDRD5/7 in humans) and its LOTUS-domain-dependent interaction with a 3’UTR binding complex. Together, these findings will provide an understanding of cytoplasmic processes within germ granules that ensure the pluripotent potential of germ cells. The parent grant proposed the creation of numerous new C. elegans and bacterial lines, along with the maintenance of existing C. elegans strains and bacterial RNAi libraries. Currently, these reagents are stored in a -80°C ultra-low temperature (ULT) freezer, which we inherited when we established our lab in 2012. We estimate this freezer to be over 15 years old and approaching the end of its useful life. The administrative supplement will replace the aging freezer with a modern, energy-efficient model, ensuring the continued preservation of our strains, nucleic acids, protein samples, and other reagents necessary for required resource sharing and the continuation of the experiments outlined in the parent grant.