Compartmentalized Exosome Structure and Function

描述(由申请人提供)：信使RNA分子的调节加工、周转、出口和监测对维持真核生物的准确基因表达至关重要。在这些不同的RNA代谢途径中，一个不可或缺的参与者是外体，它是一种重要的蛋白质复合体，由许多3‘到5’外切核酸酶组成。然而，外切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，我们的初步数据显示，果蝇外切体亚单位在体内的分布存在差异，这表明外切体复合体具有与其亚细胞区划相对应的特殊功能。特别是，dDisS亚基主要分布在核和核周，而dCsl4亚基主要分布在胞浆内。在这个建议中，我们探索了这样的假设，即外切体底物的特异性是通过三个特定的目标来实现的：(1)利用高分辨率的固定和活细胞荧光技术，确定dDis3、dCsl4和体内外切体复合体之间的相互作用；(2)通过检测内源和报告基因的稳定性，证明适当的dDisS和dCsl4的亚细胞定位对于分别调控细胞核和细胞质中的mRNA衰退至关重要；以及(3)使用色谱和质谱学方法鉴定和表征dDis3和dCsl4沉淀的外切体复合体和相关因子。这些研究必将为正确的dDiss和dCsl4亚细胞分布与外体复杂结构和功能的关系提供重要的见解。这项研究的长期目标是在mRNA代谢途径的背景下确定专门的外体亚单位的功能和相互作用。深入了解外切体如何处理和降解mRNAs对人类健康至关重要，因为异常的基因表达是许多疾病状态的致病因素。

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.