Regulation and Function of Germline RNP Granules

描述（由申请人提供）：核糖核蛋白（RNP）复合物组装成显微镜可见的颗粒，是RNA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。大的RNP颗粒也诱导在生殖系的压力或延迟受精。本申请的长期目标是了解RNP在生殖细胞中的功能，并解决它们是否作为RNA代谢改变的原因或结果发挥作用的问题。总体方法是利用小杆线虫作为体内多细胞模型系统来研究RNP功能，假设卵母细胞中形成大的RNP颗粒，以维持RNA稳定性，抑制翻译，并维持总体卵母细胞活力/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，以确定卵母细胞中存在和不存在RNP颗粒的蠕虫之间差异表达的基因。这些基因和其他先前被鉴定为“生殖系固有”的基因将在功能性RNAi筛选中被靶向，以鉴定当排卵被阻止时MEX-3 RNA结合蛋白组装成大颗粒所需的基因。通过免疫荧光实验对阳性命中的表征将确定每个基因是否特异性地调节MEX-3定位于颗粒或控制更全面的RNP颗粒组装。微管破坏的影响，对eIF 2a的依赖性，以及多核糖体稳定性在RNP颗粒组装中的作用将使用抑制剂显微注射和靶向RNAi实验的组合来确定。第二个具体目标是直接检验RNP颗粒功能的假设。在与卵母细胞中RNP颗粒形成破坏的蠕虫交配后评估生育力。将分别使用qRT-PCR和免疫荧光实验类似地测定RNA稳定性和翻译抑制。第三个具体目标包括生殖系RNP的超微结构表征，以确定当排卵被阻止或存在压力时，核孔、线粒体和核糖体是否在生殖系中差异定位。总之，这些研究将为生殖细胞中RNP的调控和功能提供重要的见解。这些结果可能有助于理解随着女性年龄的增长而发生的不孕症的基础，以及当压力发生时细胞所采用的机制和一般策略。 公共卫生相关性：该项目的主要目标是深入了解细胞在压力下调节蛋白质翻译的机制。RNA和RNA结合蛋白（RNP）的大型复合物积累到应激颗粒中，以调节应激反应的翻译，当卵母细胞必须等待很长时间才能受精或经历环境应激时，类似的RNP颗粒会在卵（卵母细胞）中组装。这项研究测试了大RNP颗粒在维持卵母细胞活力和生育力方面发挥保护作用的假设，并且可能与理解随着女性年龄增长而发生的不孕症的基础有关。

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.