Compartmentalized Exosome Structure and Function

描述（由申请人提供）：信使RNA分子的调控加工、周转、输出和监测对于维持真核生物准确的基因表达至关重要。在这些不同的RNA代谢途径中，外泌体是一个不可或缺的参与者，它是一种由许多3‘到5’外核糖核酸酶组成的重要蛋白质复合物。然而，外泌体如何在这些途径中发挥作用的确切机制尚不清楚。在这方面，我们的初步数据表明，果蝇外泌体亚基在体内的分布是不同的，这表明外泌体复合物具有与其亚细胞区隔化相对应的特殊功能。特别是，dDisS亚基主要存在于细胞核和核外周，而dCsl4亚基则富集于细胞质病灶。在本提案中，我们通过三个特定目的探讨了外泌体底物特异性是通过区隔和组成不同的外泌体复合物实现的假设：(1)使用高分辨率固定和活细胞荧光技术确定dDis3， dCsl4和外泌体复合物在体内的相互作用；(2)通过检测内源和报告mRNA的稳定性，证明dDisS和dCsl4的亚细胞定位对细胞核和细胞质中受调节的mRNA衰变至关重要；(3)利用色谱和质谱方法鉴定和表征区隔化的dDis3-和dcsl4沉淀的外泌体复合物和相关因子。这些研究必然会对正确的dDisS和dCsl4亚细胞分布与外泌体复杂结构和功能之间的关系产生重要的见解。该研究的长期目标是在mRNA代谢途径的背景下确定专门的外泌体亚基功能和相互作用。深入了解外泌体如何处理和降解mrna对人类健康至关重要，因为异常基因表达是许多疾病状态的病因因素。

DESCRIPTION (provided by applicant): Regulated processing, turnover, export, and surveillance of messenger RNA molecules are critical to maintain accurate gene expression in eukaryotes. An integral player in these distinct RNA metabolic pathways is the exosome, an essential protein complex comprising numerous 3' to 5' exoribonucleases. Yet, the exact mechanisms underlying how the exosome functions in these pathways are unclear. In this regard, our preliminary data show that Drosophila exosome subunits are differentially distributed in vivo, suggesting that exosome complexes have specialized functions corresponding to their subcellular compartmentalization. In particular, the dDisS subunit is predominantly nuclear and nucleoperipheral whereas the dCsl4 subunit in enriched in cytoplasmic foci. In this proposal, we explore the hypothesis that exosome substrate specificity is achieved by compartmentally and compositionally distinct exosome complexes through three specific aims: (1) define the interplay among dDis3, dCsl4, and the exosome complex in vivo, using high-resolution fixed and live cell fluorescence techniques; (2) demonstrate that proper dDisS and dCsl4 subcellular localization is critical for regulated mRNA decay in the nucleus and the cytoplasm, respectively, by examining endogenous and reporter mRNA stability; and (3) identify and characterize the compartmentalized dDis3- and dCsl4-precipitated exosome complexes and associated factors using chromatographic and mass spectrometric approaches. These studies are bound to yield important insight into how proper dDisS and dCsl4 subcellular distribution correlates with exosome complex structure and function. The long-term objective of the study is to identify specialized exosome subunit functions and interactions in the context of mRNA metabolic pathways. An in-depth understanding of how the exosome processes and degrades mRNAs is fundamentally important to human health, as aberrant gene expression is an etiological factor in numerous disease states.