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: 7449595
• 负责人: Ekaterina Voronina
• 金额: $ 5.48万
• 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/7449595
• 关键词: Adult; Affect; Affinity Chromatography; Animals; Biochemical; Biological Assay; Biological Models; Caenorhabditis elegans; Case Study; Cell physiology; Complement; Complex; Cytoplasmic Granules; Development; Disruption; Drosophila genus; Embryo; Female; Fertility; Gene Expression; Genes; Genetic; Genetic Screening; Germ; Germ Cells; Green Fluorescent Proteins; Human; Immunoprecipitation; In Vitro; Individual; Invertebrates; Knowledge; Libraries; Life; Life Cycle Stages; Light; Malignant - descriptor; Malignant Neoplasms; Mammals; Mass Spectrum Analysis; Mediating; Methods; Mitotic; Mus; Numbers; Organism; Orthologous Gene; Population; Proteins; Proteomics; Protocols documentation; RNA; RNA Interference; Reagent; Regulation; Regulatory Element; Ribonucleoproteins; Sampling; Screening procedure; Stem cells; Structure of primordial sex cell; Surveys; Transgenic Organisms; Translational Regulation; Two-Hybrid System Techniques; Vertebrates; Visual; Yeasts; cell type; embryonic stem cell; gene function; improved; in vivo; intracellular protein transport; member; mutant; novel; positional cloning; protein localization location; tool

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生育所必需的进化保守的核糖核蛋白复合物。在任何有机体中都不知道它们的成分的全部补充，但是许多发现的成分都是从秀丽隐杆线虫到果蝇，到小鼠和人类的保守的。该建议着重于在模型系统中识别胚芽颗粒功能的新组件和调节剂。这将通过遗传筛查和蛋白质组学方法的结合来进行。首先，我将使用RNAi在功能上筛选了种系表达的基因，并评估荧座秀丽隐杆线虫的荧光标记的P颗粒的定位。对于此视觉屏幕，我将使用Seydoux实验室中已经可用的两种试剂：秀丽隐杆线虫的菌株，表达对胚芽颗粒成分PGL -1的GFP融合，以及一个靶向-3,000个基因的RNAi库在雌性种系中优先表达的基因。在第二种方法中，我将使用生化方法来分离颗粒复合物。我将通过在定义的调节元件的控制下产生表达标记的P颗粒成分PGL-1和GLH-1的转基因蠕虫来标记精选生殖细胞亚类型（成年性腺干细胞和胚胎原始生殖细胞）中的P颗粒。这将允许对特定于胚胎原始生殖细胞有丝分裂种系的P颗粒种群的生化纯化。分离的复合物的蛋白质成分将通过质谱法鉴定。候选人相互作用者的有效性将通过替代方法进行评估，例如酵母两种杂交测定法或体外GST-GST-Pulldown分析。将进一步研究由RNAI和蛋白质组学筛查产生的阳性：蛋白质定位调查将评估这些蛋白质定位调查是否有助于P颗粒本身，并通过RNAI中的基因功能，或通过表达显性交互构建体的表达来分析其功能。从无脊椎动物到脊椎动物的已知生殖颗粒成分的保护表明，这些研究的结果将提高我们对各种物种种类发育的理解。在许多报道的病例中，生殖细胞功能的破坏不仅在缺乏生育方面，而且导致受影响个体的恶性转化（癌症）。 P颗粒成员和监管机构的先进知识将提出对人类生育能力和恶性肿瘤的了解。