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

项目摘要 在理解维持母体mRNA的机制方面存在着根本性的差距， 是卵母细胞生长和早期胚胎发生所必需的。如果基因表达不正常， 适当监管。许多mRNA和RNA结合蛋白经历受调节的相分离， 导致颗粒。在人类卵巢中，不育与RNA结合蛋白的异位颗粒有关。 尽管它们具有重要的性质，但调节卵子RNA相分离的分子机制 结合蛋白尚未被很好地理解。长期目标是确定允许卵母细胞发育的机制 在整个卵子发生过程中保持其质量。本申请的主要目的是鉴定ERK （细胞外信号调节激酶）介导的调节RNA结合蛋白相的机制 卵子发生的过渡和功能。两个中心假设是：1）典型的Ras-ERK信号传导 在卵母细胞中，RNA通路抑制RNA结合蛋白凝结成RNP颗粒， 对于mRNA的翻译调节是必需的，和2）ERK通过调节mRNA的翻译调节蛋白质的表达来调节缩合。 CCT伴侣蛋白和/或ER或细胞骨架的重塑。这些假设是基于 PI实验室的结果显示ERK活性的缺失导致两种RNA结合的异位颗粒， 蛋白质，和细胞重塑与RNP颗粒。总体方法是调查 RNA结合蛋白的缩合使用一个出色的体内模型卵子发生在线虫C。 优雅这种方法在教育上是创新的，因为它涉及大量的本科生， 他们中的一些人将参与选修实验室课程的真实研究。该战略还纳入了 遗传学、细胞生物学和显微镜的强大组合。第一个目的是阐明 经典的Ras-ERK信号通路调节RNA结合蛋白的缩合。国集团的作用 还将确定鞘细胞中的蛋白质信号传导途径，以及缺失ERK的影响 对母体mRNAs的翻译调节活性。第二个目的是表征组成 和动力学的异位RNP缩合物，并确定候选ERK底物介导的抑制， 凝减少ERK活性对不同种类的RNA结合蛋白和mRNA的影响将 下定决心。将结合遗传学和透射电子显微镜来确定是否 ERK调节ER和细胞骨架的重塑。这些结果将用于筛选候选ERK 底物以鉴定下游ERK靶点。总的来说，这一贡献将提供一个了解 RNA结合蛋白和母体mRNA的调节，可能对维持 配子质量该项目意义重大，因为它将通过揭示上游ERK而对该领域做出独特贡献 调控卵子发生中RNA结合蛋白相变的途径。候选ERK底物 将为未来的生物化学研究提供目标，并调节干预途径。

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.