Mechanisms of RNP granule function in the germ line

描述（由申请人提供）：在理解RNP颗粒动力学在老年或减数分裂停止的卵母细胞中发生的卵母细胞质量下降过程中的作用方面存在根本差距。如果对老年卵母细胞中卵母细胞质量的调节没有更好的了解，那么帮助更多有年龄相关不孕问题的妇女的能力将受到阻碍。长期目标是确定当受精延迟时维持卵母细胞质量的关键机制，如老年或减数分裂停止的卵母细胞。本应用程序的主要目的是确定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。该建议的中心假设是RNP颗粒介导的mRNA代谢调节促进卵母细胞质量。这一假设是根据PI实验室产生的令人兴奋的初步数据制定的。总体方法是利用线虫研究生殖系RNP颗粒的动力学和功能。该方法具有创新性，因为它利用了卵母细胞中自然发生的RNP颗粒组装和解离的多细胞系统。RNAi筛选的初步结果确定了143个候选RNP颗粒组装调节因子，为这些研究提供了良好的基础。第一个目标是从143个候选物中确定RNP颗粒组装的直接调节因子。成熟率测定和免疫荧光实验将用于鉴定不影响卵母细胞减数分裂成熟率的RNP颗粒组装调节因子，以及那些控制多个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颗粒组装和分离时的细胞骨架。对微管运动蛋白突变体和WASP复合物突变体的靶向分析将确定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代谢来促进卵母细胞质量。当RNP颗粒组装被破坏时，将检测mRNA水平、转录激活、mRNA聚腺苷化和翻译的去抑制。综上所述，这些研究将提供对RNP颗粒组装、解离和功能机制的深入了解。该项目意义重大，因为它验证了一个新的中心假设，即RNP颗粒成分通过调节mRNA代谢在维持减数分裂阻滞的卵母细胞质量中发挥保护作用。由于RNP颗粒组分具有较强的保守性，研究结果可能对无脊椎动物和脊椎动物产生深远的影响。预计研究结果将有助于了解随着女性年龄增长而发生的不孕症的基础。

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.