Mechanistic analysis of Drosophila germ granule assembly

果蝇胚芽颗粒组装机理分析

• 批准号: 9262069
• 负责人: Matthew Gene Niepielko
• 金额: $ 5.92万
• 依托单位: PRINCETON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-04-01 至 2018-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9262069
• 关键词: 3' Untranslated Regions; Affinity; Affinity Chromatography; Animals; Behavior; Binding Proteins; Biochemical; Biological Assay; Cell Differentiation process; Cell physiology; Cells; Cellular Structures; Complex; Cytoplasmic Granules; Data; Defect; Development; Docking; Drosophila genus; Elements; Event; Fluorescent in Situ Hybridization; Gene Dosage; Gene Expression Regulation; Genes; Genetic; Genetic Materials; Genetic Techniques; Genetic Transcription; Germ; Germ Cells; Goals; Image; Image Analysis; Individual; Infertility; Label; Location; Maintenance; Mammals; Mass Spectrum Analysis; Mediating; Messenger RNA; Metabolism; Modeling; Molecular; Molecular Genetics; Molecular Structure; Mus; Mutation; Oocytes; Play; Post-Transcriptional Regulation; Process; Proteins; RNA; Regulation; Reporter; Reproduction; Research; Resolution; Ribonucleoproteins; Role; Site; Sterility; Structure of primordial sex cell; System; Techniques; Testing; Transcript; Transcriptional Regulation; Untranslated Regions; Work; Xenopus; Zebrafish; base; egg; experimental study; in vivo; insight; nano; next generation; particle; public health relevance; quantitative imaging; scaffold; single molecule

 DESCRIPTION (provided by applicant): Animal reproduction requires the development of a functional germline, the set of highly specialized cells capable of passing on genetic material to the next generation. Throughout the animal kingdom, germline function and maintenance requires formation of large, complex ribonucleoprotein particles (RNPs) called germ granules. Germ granules control the post-transcriptional regulation of many mRNAs that are essential for germline differentiation, proliferation and maintenance. Evidence for the conserved role of germ granules comes from the effect of mutations that eliminate conserved germ granule components in Drosophila, Xenopus, zebrafish, and mouse, resulting in loss of primordial germ cells. Despite their importance, the underlying mechanisms that regulate germ granule formation are unclear; elucidating these mechanisms may provide insight into germline defects such as infertility and sterility. The proposed research investigates mechanisms regulating germ granule assembly using Drosophila germ granules, called polar granules, as a model. Results from a recent study show that polar granules comprise many different mRNAs. These mRNAs are proposed to co-assemble in a process in which mRNAs of a given gene interact to form multi-copy homotypic RNPs, then different homotypic RNPs of different transcripts associate to form higher-order polar granules that are heterogeneous in both the amount and types of mRNAs. Current data support two models of polar granule assembly: 1) polar granules form by the fusion of different homotypic RNPs or 2) homotypic RNPs of different mRNAs form concurrently on predefined scaffolding. The goal of Aim 1 is to test these models by using a combination of single molecule fluorescence in situ hybridization (smFISH), quantitative image analysis, and live imaging experiments. The molecular mechanisms regulating polar granule assembly are unknown and the analysis proposed in Aim 2 seeks to identify cis-acting RNA elements that mediate RNA granule assembly. Aim 3 focuses on the isolation and characterized of proteins that regulate the polar granule assembly process using RNA and protein affinity purification strategies combined with molecular and genetic techniques. Completion of the proposed aims will provide new insight into germ granule formation and its importance in germline development. Since different classes of RNA granules have shared features and conserved components, key findings from this work may have broader impact by application to other types RNA granules.

 描述(申请人提供)：动物繁殖需要发展一个功能性生殖系，这是一组高度专业化的细胞，能够将遗传物质传递给下一代。在整个动物界，生殖系的功能和维持需要形成大的、复杂的核糖核蛋白颗粒(RNPs)，称为胚粒。生殖细胞颗粒控制着许多对生殖系分化、增殖和维持至关重要的mRNAs的转录后调控。生殖粒保守作用的证据来自于突变的效果，该突变消除了果蝇、非洲爪哇、斑马鱼和小鼠中保守的生殖粒成分，导致原始生殖细胞的丧失。尽管它们很重要，但调节生殖细胞颗粒形成的潜在机制尚不清楚；阐明这些机制可能有助于深入了解生殖系缺陷，如不育和不育。这项拟议的研究以被称为极地颗粒的果蝇胚粒为模型，研究调节胚粒组装的机制。最近的一项研究结果表明，极性颗粒由许多不同的mRNAs组成。这些mRNAs被认为是在一个过程中共同组装的，在这个过程中，给定基因的mRNAs相互作用形成多拷贝同型RNPs，然后不同转录本的不同同型RNPs结合形成在mRNAs数量和类型上都不同的高阶极性颗粒。目前的数据支持两种极性颗粒组装模型：1)不同同型RNPs融合形成极性颗粒；2)不同mRNAs在预定义支架上同时形成同型RNPs。目标1的目标是通过使用单分子荧光原位杂交(SmFISH)、定量图像分析和现场成像实验的组合来测试这些模型。调节极性颗粒组装的分子机制尚不清楚，目标2中提出的分析试图确定介导RNA颗粒组装的顺式作用RNA元件。目的3利用RNA和蛋白质亲和纯化策略，结合分子和基因技术，分离和鉴定调节极性颗粒组装过程的蛋白质。拟议目标的完成将为胚种颗粒的形成及其在生殖系发育中的重要性提供新的见解。由于不同类别的RNA颗粒具有共同的特征和保守的成分，这项工作的关键发现可能会通过应用于其他类型的RNA颗粒而产生更广泛的影响。