Compartmentalized Exosome Structure and Function

区室化的外泌体结构和功能

• 批准号: 7669381
• 负责人: ERIK D ANDRULIS
• 金额: $ 28.5万
• 依托单位: CASE WESTERN RESERVE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7669381
• 关键词: 5' -exoribonuclease; Affinity; Binding; Biochemical; Cell Nucleus; Cells; Complement; Complex; Critical Pathways; Cytoplasm; Data; Disease; Drosophila genus; Drosophila melanogaster; Epitopes; Eukaryota; Eukaryotic Cell; Fluorescence; Gene Expression; Goals; Health; Human; Individual; Life; Mass Spectrum Analysis; Messenger RNA; Metabolic Pathway; Molecular; Nonsense-Mediated Decay; Nuclear; Pathway interactions; Process; Proteins; RNA; RNA Interference; RNA Processing; Reporter; Resolution; Ribosomal RNA; Site-Directed Mutagenesis; Small Nuclear RNA; Structure; Substrate Specificity; Techniques; Testing; cofactor; in vivo; insight; mRNA Decay; mRNA Export; mRNA Stability; mutant; protein complex; research study; tissue/cell culture

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.

描述（由申请人提供）：信使RNA分子的调节处理，周转，出口和监视对于维持真核生物中准确的基因表达至关重要。在这些独特的RNA代谢途径中，一个不可或缺的参与者是外泌体，这是一种必不可少的蛋白质复合物，其中包含大量3'至5'驱虫核酸酶。然而，尚不清楚外泌体在这些途径中如何功能的确切机制。在这方面，我们的初步数据表明，果蝇外部亚基是在体内差异分布的，这表明外泌体配合物具有与其亚细胞分区化相对应的专业功能。特别是，ddiss亚基主要是核和核的，而DCSL4亚基在富含细胞质的灶中。在该提案中，我们探讨了以下假设：外泌体底物特异性是通过三个特定目的通过隔室和组成不同的外泌体复合物来实现的：（1）使用高分辨率固定固定和活细胞荧光技术在体内定义DDIS3，DCSL4和外ost体复合物之间的相互作用； （2）证明，通过检查内源性和报告基因的mRNA稳定性，分别对细胞核和细胞质中调节的mRNA衰减至关重要。 （3）使用色谱和质谱方法识别和表征隔室化的DDIS3-和DCSL4预测的外泌体配合物以及相关因素。这些研究必将对适当的DDIS和DCSL4亚细胞分布与外泌体复杂的结构和功能相关。该研究的长期目标是在mRNA代谢途径的背景下鉴定专业的外泌体亚基功能和相互作用。对外泌体过程和降解mRNA的深入了解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。