Regulation of Centrosomes by Localized RNA

项目摘要/摘要 中心体功能异常是对人类健康具有深远意义的病理基础，包括 生长缺陷综合症和癌症。中心体作为微管组织中心，构建 有丝分裂纺锤体，在纤毛细胞中形成基底体以模板纤毛，并作为信号传递的平台 包括细胞周期信号在内的级联反应。中心体由一对中心粒组成，中心粒周围有一种蛋白质 基质称为中心周围材料(PCM)。PCM的组成和数量决定了微管 中心体的成核活性，并在循环细胞中迅速变化。负责执行任务的机制 中心体组成和结构的快速变化尚不完全清楚。耐人寻味的是， 果蝇早期胚胎中的mRNA定位表明，多个转录本集中在纺锤体极。 其中几个基因的突变会扰乱中心体功能和/或有丝分裂纺锤体，这表明 中心体的mRNA丰富和局部翻译可能是一种未知的调节机制 中心体成分。这项提议旨在通过利用遗传上易驯服的 果蝇早期胚胎，一个理想的系统，以可视化数百个活跃的MTOC中心体。 我的中心假设是，特定的RNA通过RNA结合活跃地定位在中心体上 调节其结构和功能的蛋白质。我将用两个互补的目标来检验这一假设。在……里面 具体目标1，我将定义中心体mRNAs在中心体组成和功能中的作用 单分子FISH，旋转圆盘共聚焦显微镜，超分辨率显微镜，定量图像 中心体的分析、生化分离和3‘非编码区互换实验 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 果蝇细胞质元件结合蛋白ORB的信使核糖核酸定位 同源和RNA结合蛋白，靶向中心体的mRNAs。我将使用Single来验证这个假设 分子FISH、RNA免疫沉淀和具有突变的ORB共识结合的嵌合报告 序列。在目标2中，我还将采取一种无偏见的方法来鉴定其他RNA结合蛋白，这些蛋白 通过RNA干扰筛选促进中心体mRNA的定位，这将成为My 以独立调查员的身份参与研究计划。这一提议利用了果蝇早期胚胎系统 为了洞察细胞周期中中心体组成的快速变化，这是一个生物过程 与癌症发病机制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.