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

描述（由申请人提供）：生殖颗粒是进化保守的核糖核蛋白复合物，存在于动物生殖细胞中，是生育所必需的。它们的全部成分在任何生物体中都不为人所知，但从秀丽隐杆线虫到果蝇，再到小鼠和人类，许多已发现的成分都是保守的。本研究的重点是在秀丽隐杆线虫模型系统中识别新的细菌颗粒功能成分和调节因子。这将通过基因筛选和蛋白质组学方法的结合来进行。首先，我将使用RNAi对秀丽隐杆线虫的种系表达基因进行功能性筛选，并评估荧光标记P颗粒的定位。为了这个视觉筛选，我将使用赛都实验室已经提供的两种试剂：一种表达GFP融合到胚粒成分PGL-1的线虫菌株，以及一个针对在雌性种系中优先表达的- 3000个基因的RNAi文库。在第二种方法中，我将使用生化方法分离P颗粒复合物。我将在选定的生殖细胞亚型（成体性腺干细胞和胚胎原始生殖细胞）中标记P颗粒，在定义的调节元件的控制下，通过在种系中产生表达标记的P颗粒成分PGL-1和GLH-1的转基因蠕虫。这将允许对胚胎原始生殖细胞有丝分裂种系特有的P颗粒群体进行生化纯化。分离的复合物的蛋白质成分将通过质谱法进行鉴定。候选相互作用物的有效性将通过替代方法进行评估，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进一步详细研究：蛋白质定位调查将评估这些因素是否对P颗粒本身有贡献，它们的功能将通过体内破坏基因功能、RNAi或显性干扰结构的表达来分析。从无脊椎动物到脊椎动物的已知胚粒成分的保存表明，这些研究结果将促进我们对广泛物种的种系发育的理解。在一些报告的病例中，生殖细胞功能的破坏不仅导致缺乏生育能力，而且还导致受影响个体的恶性转化（癌症）。对P颗粒成员和调节因子的深入了解将提高对人类生育和恶性肿瘤的认识。

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.