In many animal species, oocytes arrest in meiosis until they are fertilized. As oocytes age changes occur in their cytoplasm, and fertility diminishes. Our goal is to better understand the regulation and function of ribonucleoprotein (RNP) granules in the oocytes of Caenorhabditis nematodes that are arrested in meiosis. The assembly of large RNP granules is hypothesized to maintain oocyte quality when fertilization is delayed by regulating mRNA metabolism (Jud et al., 2008). We have taken two approaches to identify regulators of RNP granules, a targeted RNAi screen and a candidate approach. In our functional RNAi screen we identified 143 candidate regulators of RNP granule assembly. We are currently performing a secondary screen to identify regulators that control RNP granules without altering meiotic arrest. Interestingly, when we knock-down several regulators to inhibit RNP granule assembly in arrested oocytes, oocyte quality appears to decline.The first part of our candidate approach is based on experiments in Drosophila and other systems that indicate a role for microtubule motor proteins in regulating RNP granule dynamics in the germ line. In C. elegans, meiotically-arrested oocytes undergo a dramatic reorganization of their microtubules at the same time RNP granules assemble (Harris et al., 2006). We targeted sixteen kinesin and dynein motor protein components to characterize the role of microtubule motor proteins in the assembly and dissociation of RNP granules. Our preliminary results indicate several motor proteins promote the assembly of RNP granules. We are also exploring the role of the actin cytoskeleton in regulating RNP granules by targeting six genes related to
wsp-1, a homolog of the human WASP complex that was identified in the RNAi screen. The second avenue of our candidate approach targets the MEG proteins that were recently identified as regulators of P granule assembly in embryos (Wang et al., 2014). Our preliminary data suggest the MEG proteins also regulate the assembly of the large RNP granules in oocytes. We are currently investigating the role of phosphorylation in regulating RNP granule dynamics.