Mechanisms of RNP granule function in the germ line

描述(由申请人提供)：在理解RNP颗粒动力学在发生在老化或减数分裂停滞的卵母细胞中的卵母细胞质量下降过程中的作用方面存在着根本的差距。如果不能更好地了解老年卵母细胞中卵母细胞质量的调节，帮助更多患有年龄相关性不孕问题的女性的能力将受到阻碍。长期目标是确定在受精延迟时维持卵母细胞质量的关键机制，例如老年卵母细胞或减数分裂停滞的卵母细胞。本应用的主要目的是确定RNP颗粒组装和解离的直接调节因子，并确定RNP颗粒的分子功能。这一建议的中心假设是RNP颗粒介导的mRNA代谢调节促进了卵母细胞的质量。这一假设是基于PI实验室产生的令人兴奋的初步数据而提出的。总体方法是利用线虫研究胚系RNP颗粒的动力学和功能。这种方法是创新的，因为它利用了多细胞系统，在卵母细胞中自然发生RNP颗粒的组装和解离。RNAi筛选的初步结果确定了143个RNP颗粒组装的候选调控因子，为这些研究提供了良好的基础。第一个目标是从143个候选者中确定RNP颗粒组装的直接调控因子。成熟率分析和免疫荧光实验将用于确定不影响卵母细胞减数分裂成熟率的RNP颗粒组装的调节因子，以及控制多个RNP颗粒成分定位的调节因子。第二个目的是确定微管和肌动蛋白细胞骨架网络在调节RNP颗粒组装和解离中的相对作用。实时成像实验将检查RNP颗粒组装和解离时的细胞骨架。对微管马达蛋白突变体和WASP复合体突变体的有针对性的分析将确定RNP颗粒组装的其他调节因子。第三个目的是确定RNP颗粒是否通过调节mRNA代谢来提高卵母细胞质量。当RNP颗粒组装被破坏时，将检测mRNA水平、转录激活、mRNA多腺化和翻译抑制。综上所述，这些研究将为深入了解RNP颗粒的组装、解离和功能机制提供依据。这个项目意义重大，因为它检验了新的中心假设，即RNP颗粒成分通过调节mRNA代谢在维持减数分裂停止的卵母细胞质量方面起到保护作用。由于RNP颗粒成分的保守性很强，这一结果可能对无脊椎动物和脊椎动物产生深远的影响。预计这一结果将与理解女性随着年龄增长而发生不孕不育的基础有关。

DESCRIPTION (provided by applicant): A fundamental gap exists in understanding the role of RNP granule dynamics during the decline of oocyte quality that occurs in old-aged or meiotically-arrested oocytes. Without a better understanding of the regulation of oocyte quality in old-aged oocytes, the ability to assist a larger number of women with age-related infertility problems will be hampered. The long-term goal is to identify the critical mechanisms that maintain oocyte quality when fertilization is delayed, as for old-aged or meiotically- arrested oocytes. The primary objectives of this application are to identify direct regulators of RNP granule assembly and dissociation and to determine the molecular function of RNP granules. The central hypothesis of this proposal is that RNP granule-mediated regulation of mRNA metabolism promotes oocyte quality. This hypothesis has been formulated based on exciting preliminary data produced in the PI's lab. The overall approach is to investigate germ line RNP granule dynamics and function using the nematode C. elegans. The approach is innovative because it takes advantage of a multicellular system with naturally-occurring RNP granule assembly and dissociation in oocytes. Preliminary results from an RNAi screen identify 143 candidate regulators of RNP granule assembly that provide an excellent foundation for these studies. The first aim is to identify direct regulators of RNP granule assembly from among the 143 candidates. Maturation rate assays and immunofluorescence experiments will be used to identify regulators of RNP granule assembly that do not also affect oocyte meiotic maturation rates, and those that control the localization of multiple RNP granule components. The second aim is to determine the relative roles of the microtubule and actin cytoskeleton networks in regulating RNP granule assembly and dissociation. Live-imaging experiments will examine the cytoskeleton as RNP granules assemble and dissociate. A targeted analysis of microtubule motor protein mutants and WASP complex mutants will identify additional regulators of RNP granule assembly. The third aim is to determine if RNP granules promote oocyte quality by modulating mRNA metabolism. mRNA levels, transcriptional activation, mRNA polyadenylation, and de- repression of translation will be assayed when RNP granule assembly is disrupted. Taken together, these studies will provide insight into the mechanisms of RNP granule assembly, dissociation, and function. This project is significant because it tests the novel, central hypothesis that RNP granule components play a protective role in maintaining the quality of meiotically arrested oocytes by modulating mRNA metabolism. Due to the strong conservation of RNP granule components, the results are likely to have far-reaching impact in invertebrates and vertebrates. It is expected the results will have relevance to understanding the basis for infertility that occurs as women age.