Regulation and Function of Germline RNP Granules

描述(申请人提供)：核糖核蛋白(RNP)复合体组装成显微镜下可见的颗粒是RNA新陈代谢(加工体)正常细胞调节的一部分，也是对应激(应激颗粒)的反应。应激或延迟受精也会在生殖系中诱导大的RNP颗粒。这项应用的长期目标是了解RNPs在生殖细胞中的功能，并解决它们是作为RNA新陈代谢改变的原因还是结果发挥作用的问题。总的方法是利用线虫作为体内多细胞模型系统来研究RNP的功能，假设在卵母细胞中形成大的RNP颗粒，以维持RNA的稳定，抑制翻译，并维持整体卵母细胞的存活率/生育力。该提案的第一个具体目标是识别和表征RNP颗粒组装和解离所需的基因。将进行微阵列分析，以确定在卵母细胞中存在和不存在RNP颗粒的蠕虫之间的差异表达基因。这些基因和其他以前被认为是“生殖系固有基因”的基因将成为功能性RNAi筛查的靶点，以确定当排卵停止时MEX-3RNA结合蛋白组装成大颗粒所需的基因。免疫荧光实验对阳性HITS的表征将决定每个基因是调节MEX-3对颗粒的特异性定位，还是控制更多的全局RNP颗粒组装。微管破坏的影响，对eIF2a的依赖，以及多聚体稳定性在RNP颗粒组装中的作用，将通过抑制剂微量注射和靶向RNAi实验相结合来确定。第二个具体目的是直接检验RNP颗粒功能的假说。受精率将在与卵母细胞中RNP颗粒形成中断的蠕虫交配后进行评估。RNA稳定性和翻译抑制将类似地分别使用qRT-PCR和免疫荧光实验来确定。第三个特定目标包括对生殖系RNPs的超微结构进行表征，以确定当排卵停止或压力存在时，核孔、线粒体和核糖体是否在生殖系中存在差异定位。综上所述，这些研究将为了解RNPs在生殖细胞中的调控和功能提供重要的见解。这一结果可能与理解女性随着年龄增长而发生不孕不育的基础有关，以及当压力发生时细胞采用的机制和一般策略。 与公共卫生相关：这个项目的广泛目标是深入了解细胞在应激状态下调节蛋白质翻译的机制。RNA和RNA结合蛋白(RNP)的大复合体积聚成应激颗粒来调节对应激的反应，类似的RNP颗粒在卵子(卵母细胞)中组装，当它们必须等待较长时间才能受精或经历环境胁迫时。这项研究测试了一种假设，即大的RNP颗粒在维持卵母细胞存活率和生育力方面起到保护作用，并可能与理解随着女性年龄增长而发生不孕不育的基础有关。

DESCRIPTION (provided by applicant): The assembly of ribonucleoprotein (RNP) complexes into microscopically visible granules occurs as part of the normal cellular regulation of RNA metabolism (processing bodies) and in response to stress (stress granules). Large RNP granules are also induced in the germline by stress or delayed fertilization. The long-term objectives of this application are to understand the function of RNPs in germ cells and resolve the question if they function as a cause or consequence of altered RNA metabolism. The overall approach is to investigate RNP function utilizing Caenorhabditis as an in vivo, multicellular model system, with the hypothesis that large RNP granules form in oocytes in order to maintain RNA stability, repress translation, and maintain overall oocyte viability/ fertility. The first specific aim of the proposal is to identify and characterize the genes required for assembly and dissociation of RNP granules. Microarray analyses will be performed to identify genes differentially expressed between worms with and without RNP granules present in oocytes. These genes and others previously identified as "germline intrinsic" will be targeted in a functional RNAi screen to identify genes required for the MEX-3 RNA-binding protein to assemble into large granules when ovulation is arrested. Characterization of the positive hits by immunofluorescence experiments will determine whether each gene regulates MEX-3 localization to granules specifically or controls more global RNP granule assembly. The effect of microtubule disruption, the dependence on eIF2a, and the role of polysome stability in RNP granule assembly will be determined using a combination of inhibitor microinjections and targeted RNAi experiments. The second specific aim is to directly test the hypothesis for RNP granule function. Fertility will be assessed after mating into worms with disrupted RNP granule formation in oocytes. RNA stability and translational repression will similarly be determined using qRT-PCR and immunofluorescence experiments, respectively. The third specific aim includes an ultrastructural characterization of germline RNPs to determine if nuclear pores, mitochondria, and ribosomes are differentially localized in germlines when ovulation is arrested or stress is present. Taken together, these studies will provide significant insight into the regulation and function of RNPs in germ cells. The results may have relevance to understanding the basis for infertility that occurs as women age and the mechanisms and general strategies cells employ when stresses occur. PUBLIC HEALTH RELEVANCE: The broad goal of this project is to gain insight into the mechanisms used by cells to regulate protein translation during stress. Large complexes of RNA and RNA-binding proteins (RNPs) accumulate into stress granules to regulate translation in response to stress, and similar RNP granules assemble in eggs (oocytes) when they must wait a prolonged period to be fertilized or undergo an environmental stress. This research tests the hypothesis that large RNP granules play a protective role in maintaining oocyte viability and fertility and may be relevant to understanding the basis for infertility that occurs as women age.