ERK-mediated regulation of RNA binding protein condensation during female germ cell development

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Project Summary A fundamental gap exists in understanding the mechanisms that maintain the maternal mRNAs necessary for oocyte growth and early embryogenesis. Birth defects and infertility can arise if mRNAs are not properly regulated. Many mRNAs and RNA binding proteins undergo regulated phase separation that can result in granules. In the human ovary, infertility is associated with ectopic granules of RNA binding proteins. Despite their critical nature, the molecular mechanisms that regulate phase separations of oogenic RNA binding proteins are not yet well understood. The long-term goal is to identify mechanisms that allow oocytes to maintain their quality throughout oogenesis. The primary objective of this application is to identify the ERK (extracellular signal regulated kinase)-mediated mechanisms that modulate RNA binding protein phase transitions and function in oogenesis. The two central hypotheses are: 1) the canonical Ras-ERK signaling pathway acts in oocytes to inhibit the condensation of RNA binding proteins into RNP granules which is necessary for the translational regulation of mRNAs, and 2) ERK modulates condensation via regulation of the CCT chaperonin and/or remodeling of the ER or cytoskeleton. These hypotheses have been formulated based on results in the PI’s lab showing depletion of ERK activity results in ectopic granules of two RNA binding proteins, and cellular remodeling is associated with RNP granules. The overall approach is to investigate the condensation of RNA binding proteins using an outstanding in vivo model of oogenesis in the nematode C. elegans. The approach is educationally innovative because it involves a large number of undergraduates, some of whom will be involved in authentic research in an elective lab course. The strategy also integrates a powerful combination of genetics, cell biology, and microscopy. The first aim is to elucidate the role of the canonical Ras-ERK signaling pathway in regulating RNA binding protein condensation. The role of the G protein signaling pathway in the sheath cells will also be determined, as well as the effect of depleted ERK activity on the translational regulation of maternal mRNAs. The second aim is to characterize the composition and dynamics of ectopic RNP condensates and identify candidate ERK substrates that mediate the inhibition of condensation. The effects of depleting ERK activity on diverse classes of RNA binding proteins and mRNA will be determined. A combination of genetics and transmission electron microscopy will be used to determine if ERK regulates remodeling of the ER and cytoskeleton. These results will be used to screen candidate ERK substrates to identify downstream ERK targets. Overall, this contribution will provide an understanding of the regulation of RNA binding proteins and maternal mRNAs that may have critical importance in maintaining gamete quality. This project is significant as it will uniquely contribute to the field by uncovering upstream ERK pathways that regulate the phase transitions of RNA binding proteins in oogenesis. Candidate ERK substrates will provide targets for future biochemical studies and to modulate the pathway for interventions.