Mechanistic analysis of Drosophila germ granule assembly

 描述（由申请人提供）：动物繁殖需要发育功能性生殖系，即一组能够将遗传物质传递给下一代的高度特化细胞。在整个动物界，生殖细胞的功能和维持需要形成大的，复杂的核糖核蛋白颗粒（RNP）称为胚芽颗粒。生殖颗粒控制许多mRNA的转录后调节，这些mRNA对于生殖细胞分化、增殖和维持是必不可少的。生殖颗粒保守作用的证据来自于果蝇、非洲爪蟾、斑马鱼和小鼠中消除保守生殖颗粒组分的突变效应，导致原始生殖细胞的丧失。尽管它们的重要性，调节生殖颗粒形成的潜在机制尚不清楚;阐明这些机制可能会提供对生殖系缺陷（如不育和不育）的见解。拟议的研究调查机制调节胚芽颗粒组装使用果蝇胚芽颗粒，称为极性颗粒，作为一个模型。最近的一项研究结果表明，极性颗粒包含许多不同的mRNA。这些mRNA被提出在一个过程中共同组装，其中给定基因的mRNA相互作用以形成多拷贝同型RNP，然后不同转录物的不同同型RNP缔合以形成在mRNA的量和类型上均不均匀的高阶极性颗粒。目前的数据支持极性颗粒组装的两种模型：1）极性颗粒通过不同同型RNP的融合形成，或2）不同mRNA的同型RNP在预定义的支架上同时形成。目的1的目标是通过使用单分子荧光原位杂交（smFISH），定量图像分析和实时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是未知的，目的2中提出的分析旨在确定介导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，结合分子和遗传技术，分离和鉴定调节极性颗粒组装过程的蛋白质。所提出的目标的完成将提供新的见解胚芽颗粒的形成及其在种系发育的重要性。由于不同种类的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对其他类型的RNA颗粒产生更广泛的影响。

 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.