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: 7638027
• 负责人: Ekaterina Voronina
• 金额: $ 5.72万
• 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/7638027
• 关键词: Adult; Affect; Affinity Chromatography; Animals; Biochemical; Biological Assay; Biological Models; Caenorhabditis elegans; Case Study; Cell physiology; Complement; Complex; Cytoplasmic Granules; Development; Drosophila genus; Embryo; Female; Fertility; Gene Expression; Genes; Genetic; Genetic Screening; Germ; Germ Cells; 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; 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; member; mutant; novel; positional cloning; 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（由申请方提供）：生殖颗粒是动物生殖细胞中存在的生育力所必需的进化保守的核糖核蛋白复合物。在任何生物体中都不知道它们的全部组分，但许多已发现的组分是从C.线虫，果蝇，老鼠和人类。该建议的重点是在一个模型系统C中鉴定新的组分和胚芽颗粒功能的调节剂。优美的这将通过遗传筛选和蛋白质组学方法相结合来进行。首先，我将利用RNAi技术对C.优雅的。对于这个视觉屏幕，我将使用Seydoux实验室中已有的两种试剂：一种C。表达与胚粒成分PGL-1融合的GFP的秀丽隐杆线虫，以及靶向在雌性种系中优先表达的约3，000个基因的RNAi库。在第二种方法中，我将使用生物化学方法分离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，方法是在确定的调控元件的控制下，在生殖系中产生表达标记的P颗粒组分PGL-1和GLH-1的转基因蠕虫。这将允许对胚胎原始生殖细胞的有丝分裂生殖系特异的P颗粒群体进行生化纯化。分离的复合物的蛋白质组分将通过质谱法鉴定。候选相互作用物的有效性将通过替代方法评估，例如酵母双杂交测定或体外GST-下拉测定。从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是否有助于P颗粒本身，并通过干扰体内基因功能，RNAi或显性干扰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胚粒组分的保护表明，这些研究的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生育能力的丧失，而且还导致受影响个体的恶性转化（癌症）。先进的知识P颗粒成员和监管机构将带来更好的理解生育能力以及人类恶性肿瘤。