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、出口和监测对于维持真核生物中的准确基因表达至关重要。这些不同的RNA代谢途径中的一个组成部分是外泌体，一种包含许多3'至5'外切核糖核酸酶的必需蛋白质复合物。然而，外泌体在这些通路中如何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差异分布，表明外泌体复合物具有专门的功能，对应于它们的亚细胞区室化。特别地，dDisS亚基主要是核和核周的，而dCsl 4亚基富集在细胞质灶中。在该提议中，我们通过三个特定的目的探索了外泌体底物特异性是通过区室上和组成上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来定义体内dDis 3、dCs 14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mRNA的稳定性，证明适当的dDisS和dCsl 4亚细胞定位分别对于细胞核和细胞质中的调节性mRNA衰减是关键的;和（3）使用色谱和质谱方法鉴定和表征区室化的dDis 3-和dCs 14-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dCsl 4亚细胞分布如何与外泌体复合物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代谢途径背景下的专门外泌体亚基功能和相互作用。深入了解外泌体如何加工和降解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.