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

描述(申请人提供)：生殖粒是进化保守的核糖核蛋白复合体，在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是未知的，但从线虫到果蝇，再到老鼠和人类，许多已发现的成分都是保守的。这项建议侧重于在模式系统秀丽线虫中识别新的胚芽颗粒功能的成分和调节因子。这将通过基因筛选和蛋白质组学方法相结合的方式进行。首先，我将使用RNAi从功能上筛选生殖系表达的基因，并评估线虫荧光标记的P颗粒的定位。对于这个视觉屏幕，我将使用Seydoux实验室已经提供的两种试剂：一种是表达GFP与生殖粒成分PGL-1融合的线虫菌株，另一种是针对女性生殖系优先表达的3000个基因的RNAi文库。在第二种方法中，我将使用生化方法来分离P颗粒复合体。我将在特定的生殖细胞亚型(成人性腺干细胞和胚胎原始生殖细胞)中标记P颗粒，在已定义的调控元件的控制下，在生殖系中产生表达标记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生殖细胞系特有的P颗粒群体进行生化纯化。分离的复合体的蛋白质组分将通过质谱学进行鉴定。候选交互作用的有效性将通过其他方法进行评估，例如酵母双杂交试验或体外GST-Pull-Down试验。来自RNAi和蛋白质组筛选的阳性结果将被进一步详细研究：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，它们的功能将通过扰乱体内基因功能、通过RNAi或通过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生殖粒成分的保存表明，这些研究的结果将促进我们对广泛物种中生殖系发育的理解。在一些报告的病例中，生殖细胞功能中断不仅导致生育能力不足，而且还会导致受影响个人的恶变(癌症)。对P颗粒成员和调节器的高级知识将带来对人类生育和恶性肿瘤的更好的理解。

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.