Compartmentalized Exosome Structure and Function

描述（由申请人提供）：信使 RNA 分子的规范加工、周转、输出和监视对于维持真核生物中准确的基因表达至关重要。这些独特的 RNA 代谢途径中不可或缺的参与者是外泌体，它是一种必需的蛋白质复合物，包含许多 3' 至 5' 外切核糖核酸酶。然而，外泌体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分布存在差异，表明外泌体复合物具有与其亚细胞区室相对应的特殊功能。特别是，dDisS 亚基主要位于核和核外周，而 dCsl4 亚基富集于细胞质灶中。在本提案中，我们通过三个具体目标探讨了外泌体底物特异性是通过区室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定义体内 dDis3、dCsl4 和外泌体复合物之间的相互作用； (2) 通过检查内源性和报告基因 mRNA 稳定性，证明适当的 dDisS 和 dCsl4 亚细胞定位分别对于细胞核和细胞质中受调节的 mRNA 衰减至关重要； (3)使用色谱和质谱方法鉴定和表征区室化的dDis3-和dCsl4-沉淀的外泌体复合物和相关因子。这些研究必将对正确的 dDisS 和 dCsl4 亚细胞分布如何与外泌体复杂结构和功能相关产生重要的见解。该研究的长期目标是确定 mRNA 代谢途径中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。

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.