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

项目摘要 在理解维持母体mRNAs的机制方面存在着根本的差距。 是卵母细胞生长和早期胚胎发育所必需的。如果mRNA不是，就可能出现出生缺陷和不孕不育 监管得当。许多mRNAs和RNA结合蛋白经历了受调控的相分离，这可以 结果形成颗粒。在人类卵巢中，不孕症与异位的RNA结合蛋白颗粒有关。 尽管它们具有关键的性质，但调节卵源RNA相分离的分子机制 结合蛋白还没有被很好地理解。长期目标是确定允许卵母细胞 在整个卵子发生过程中保持它们的质量。此应用程序的主要目标是识别ERK (细胞外信号调节激酶)介导的调节RNA结合蛋白相的机制 卵子发生中的过渡和功能。两个中心假设是：1)规范的RAS-ERK信号 在卵母细胞中，途径作用于抑制RNA结合蛋白凝聚成RNP颗粒，这是 翻译调节mRNAs所必需的，以及2)ERK通过调节 CCT伴侣和/或内质网或细胞骨架重塑。这些假设是基于 PI的实验室结果显示ERK活性耗尽导致两个RNA结合的异位颗粒 蛋白质，细胞重塑与RNP颗粒有关。总体方法是调查 利用线虫C。 优雅女装。这种方法在教育上是创新的，因为它涉及到大量的本科生， 其中一些人将在一门选修的实验室课程中参与真实的研究。该战略还整合了一个 遗传学、细胞生物学和显微镜的强大结合。第一个目的是阐明 调节RNA结合蛋白缩合的典型Ras-ERK信号通路。G的作用 还将确定鞘细胞中的蛋白质信号通路，以及耗尽的ERK的作用 母体mRNAs的翻译调控活性。第二个目标是确定组成的特征。 和动力学的异位RNP凝聚体和确定候选ERK底物，介导抑制 冷凝。ERK活性耗竭对不同类别的RNA结合蛋白和mRNA的影响 要下定决心。遗传学和电子显微镜的结合将被用来确定 ERK调节内质网和细胞骨架的重构。这些结果将用于筛选候选人ERK 底物以识别下游ERK靶点。总体而言，这一贡献将提供对 对RNA结合蛋白和母体mRNAs的调节可能对维持 配子质量。该项目意义重大，因为它将通过发现上游ERK来为该领域做出独特贡献 卵子发生过程中调节RNA结合蛋白相变的途径。候选ERK底物 将为未来的生化研究提供目标，并调整干预途径。

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.