Regulation of Centrosomes by Localized RNA

项目总结/摘要 异常的中心体功能是对人类健康具有深远意义的病理学的基础，包括 生长缺陷综合征和癌症。中心体作为微管的组织中心， 有丝分裂纺锤体，形成纤毛细胞中纤毛模板的基体，并作为信号平台 包括细胞周期信号传导的级联反应。中心体由一对中心粒组成，中心粒周围有一种蛋白质 称为中心粒周围物质（PCM）的基质。PCM的组成和数量决定了微管的大小 中心体的成核活性，并在循环细胞中迅速变化。负责的机制 中心体组成和结构的快速变化还不完全清楚。有趣的是，一个屏幕 在果蝇早期胚胎中定位的mRNA鉴定了在纺锤体两极富集的多个mRNA转录物。 这些基因中的几个突变破坏了中心体功能和/或有丝分裂纺锤体，这表明， mRNA在中心体的富集和局部翻译可能是一种未被探索的机制， 中心体组成这项提案旨在通过利用基因上易处理的 果蝇早期胚胎，一个理想的系统，以显示数百个活跃的MTOC中心体。 我的中心假设是特定的RNA通过RNA结合主动定位于中心体 蛋白质来调节其结构和功能。我将用两个互补的目标来检验这个假设。在 具体目标1，我将定义中心体mRNA的作用，在中心体的组成和功能，使用 单分子荧光原位杂交，旋转圆盘共聚焦显微镜，超分辨显微镜，定量成像 分析，中心体的生化分离，以及充分表征的3 'UTR交换实验。 果蝇中心体突变体和对照。在具体目标2中，我将确定中心体的机制 通过验证果蝇胞质元件结合蛋白Orb 直系同源物和RNA结合蛋白，将mRNA靶向中心体。我将使用单个 分子FISH、RNA免疫沉淀和具有突变的Orb共有结合的嵌合报告基因 序列的在目标2中，我还将采用无偏的方法来鉴定其他RNA结合蛋白， 通过RNA干扰筛选有助于中心体mRNA定位，这将成为我的基础。 作为一名独立的研究员。这项提议利用了果蝇早期胚胎系统 为了深入了解细胞周期中中心体组成的快速变化， 与癌症发病机理和生长缺陷综合征有关。

PROJECT SUMMARY/ABSTRACT Aberrant centrosome function underlies pathologies with profound significance for human health, including growth deficiency syndromes and cancer. Centrosomes function as microtubule organizing centers, build the mitotic spindle, form the basal body to template cilia in ciliated cells, and serve as platforms for signaling cascades including cell cycle signaling. The centrosome consists of a pair of centrioles surrounded by a protein matrix termed pericentriolar material (PCM). The composition and quantity of PCM determines the microtubule nucleating activity of the centrosome and changes rapidly in cycling cells. The mechanisms responsible for rapid changes to centrosome composition and structure are incompletely understood. Intriguingly, a screen for localized mRNA in Drosophila early embryos identified multiple mRNA transcripts enriched at spindle poles. Mutations to several of these genes disrupt centrosome function and/or the mitotic spindle, suggesting that mRNA enrichment and local translation at the centrosome may be an unexplored mechanism to modulate centrosome composition. This proposal aims to fill this gap in knowledge by leveraging the genetically tractable Drosophila melanogaster early embryo, an ideal system to visualize hundreds of active MTOC centrosomes. My central hypothesis is that specific RNAs are actively localized to the centrosome by RNA binding proteins to regulate its structure and function. I will test this hypothesis with two complementary aims. In Specific Aim 1, I will define the role of centrosome mRNAs in centrosome composition and function using single molecule FISH, spinning disk confocal microscopy, super-resolution microscopy, quantitative image analysis, biochemical isolation of centrosomes, and 3’UTR swapping experiments in well-characterized Drosophila centrosome mutants and controls. In Specific Aim 2, I will identify mechanisms of centrosome mRNA localization by testing the hypothesis that Orb, a Drosophila cytoplasmic element binding protein ortholog and RNA binding protein, targets mRNAs to centrosomes. I will test this hypothesis using single molecule FISH, RNA immunoprecipitation, and chimeric reporters with mutated Orb consensus binding sequences. In Aim 2, I will also take an unbiased approach to identify other RNA binding proteins that contribute to centrosome mRNA localization via an RNA interference screen, which will form the basis for my research program as an independent investigator. This proposal exploits the Drosophila early embryo system to provide insight into the rapid changes in centrosome composition during the cell cycle, a biological process with implications for cancer pathogenesis and growth deficiency syndromes.