Components of C. elegans P-granules and regulation of their function

秀丽隐杆线虫 P-颗粒的成分及其功能调节

• 批准号: 7276289
• 负责人: Ekaterina Voronina
• 金额: $ 5.29万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7276289
• 关键词: Components; C.; elegans; granules; regulation

DESCRIPTION (provided by applicant): Germ granules are evolutionary conserved ribonucleoprotein complexes necessary for fertility present in animal germ cells. The full complement of their components is not known in any organism, but many of discovered components are conserved from C. elegans, to Drosophila, to mouse and human. This proposal focuses on identifying new components and regulators of germ granule function in a model system C. elegans. This will be carried out by a combination of genetic screening and proteomic approaches. First, I will use RNAi to functionally screen the germline-expressed genes and assess localization of fluorescently tagged P granules of C. elegans. For this visual screen, I will use two reagents already available in the Seydoux lab: a strain of C. elegans expressing a GFP fusion to the germ granule component PGL-1, and an RNAi library targeting -3,000 genes expressed preferentially in the female germline. In a second approach, I will use biochemical methods to isolate P granule complexes. I will tag P granules in select germ cell sub- types (adult gonadal stem cells and embryonic primordial germ cells) by generating transgenic worms expressing tagged P granule components PGL-1 and GLH-1 in the germline under the control of defined regulatory elements. This will permit biochemical purification of populations of P granules specific to mitotic germline of embryonic primordial germ cells. The protein components of the isolated complexes will be identified by mass-spectrometry. Validity of candidate interactors will be assessed by alternative methods, such as yeast two-hybrid assays or in-vitro GST-pulldown assays. Positives resulting from both RNAi and proteomic screens will be studied in further detail: protein localization survey will assess whether any of these contribute to P granules themselves, and their function will be analyzed by disrupting gene function in vivo, by RNAi, or by expression of dominant-interfering constructs. Conservation of known germ granule components from invertebrates to vertebrates suggests that the results of these studies will advance our understanding of germline development in a broad array of species. In a number of reported cases, disruption of germ cell function results not only in lack of fertility, but also leads to malignant transformations (cancers) in the affected individuals. Advanced knowledge of P granule members and regulators will bring forth improved understanding of both fertility as well as malignancies in humans.

描述（由申请人提供）：生殖颗粒是动物生殖细胞中存在的生育能力所必需的进化保守的核糖核蛋白复合物。它们的全部成分在任何生物体中尚不清楚，但许多已发现的成分从线虫到果蝇、小鼠和人类都是保守的。该提案的重点是确定秀丽隐杆线虫模型系统中胚芽颗粒功能的新成分和调节剂。这将通过遗传筛查和蛋白质组学方法相结合来进行。首先，我将使用 RNAi 对种系表达基因进行功能性筛选，并评估线虫荧光标记 P 颗粒的定位。对于此视觉筛选，我将使用 Seydoux 实验室中已有的两种试剂：表达与胚芽颗粒成分 PGL-1 融合的 GFP 的线虫菌株，以及针对优先在雌性种系中表达的 -3,000 个基因的 RNAi 文库。在第二种方法中，我将使用生化方法来分离 P 颗粒复合物。我将通过生成转基因蠕虫来标记选定的生殖细胞亚型（成人性腺干细胞和胚胎原始生殖细胞）中的 P 颗粒，这些转基因蠕虫在特定调控元件的控制下在种系中表达标记的 P 颗粒成分 PGL-1 和 GLH-1。这将允许对胚胎原始生殖细胞的有丝分裂种系特异的P颗粒群进行生化纯化。分离的复合物的蛋白质成分将通过质谱法进行鉴定。候选相互作用因子的有效性将通过替代方法进行评估，例如酵母双杂交测定或体外 GST 下拉测定。 RNAi 和蛋白质组学筛选产生的阳性结果将得到更详细的研究：蛋白质定位调查将评估其中任何一个是否对 P 颗粒本身有贡献，并且将通过 RNAi 或显性干扰构建体的表达破坏体内基因功能来分析它们的功能。从无脊椎动物到脊椎动物，已知的胚芽颗粒成分的保守性表明，这些研究的结果将增进我们对广泛物种种系发育的理解。在许多报道的病例中，生殖细胞功能的破坏不仅会导致缺乏生育能力，还会导致受影响个体发生恶性转化（癌症）。对 P 颗粒成员和调节因子的深入了解将增进对人类生育能力和恶性肿瘤的了解。