Structural and Functional Studies of Eukaryotic Exosomes

描述（由申请人提供）：RNA 的寿命和丰度是通过维持 RNA 转录和 RNA 降解之间的平衡来调节的。虽然已经发现了几种 RNA 降解途径，但它们的成分、精确的催化活性和细胞功能仍然是研究的持续焦点。真核生物中两个主要的 RNA 衰变途径之一涉及 RNA 外泌体，这是一种催化 3' 至 5' RNA 衰变的多亚基蛋白质复合物。 RNA外泌体在真核进化过程中是保守的，并且被认为以至少两种形式存在，一种是由九个或十个不同的蛋白质亚基组成的细胞质外泌体，表观质量为300-400 kDa，另一个是由十个或十一个不同的蛋白质亚基组成的核外泌体，表观质量为400-500 kDa。在芽殖酵母中，十一个基因中有十个对于生长至关重要，这表明每个基因在细胞功能中都发挥着关键作用。对单个外泌体亚基的鉴定得出了这样的假设：外泌体由多达 11 种不同的 3' 至 5' 外切核糖核酸酶组成。然而，最近利用重构和亲和纯化的细胞外泌体复合物的研究表明，只有少数亚基编码具有外切核糖核酸酶活性的多肽，这表明大多数外泌体亚基的非催化功能是保守的。与此一致的是，已知外泌体亚基与多种促进或指导 RNA 降解的蛋白质伴侣相关。虽然最近的努力揭示了真核外泌体结构和功能的许多有趣方面，但关于 RNA 衰变中外泌体亚基的个体和集体功能仍然存在许多问题。在本提案中，我们将利用体外重建的人类和酵母外泌体复合物，结合遗传和生化测定来分析其组装、催化活性和结构完整性，通过开展研究来实现以下目标：1）表征和重建人类和酵母外泌体的亚基和复合物； 2) 确定来自酵母和人类的个体、亚复合物和完整外泌体复合物的活性的生化基础，并通过芽殖酵母酿酒酵母的体内互补和分析确定观察到的活性和结构的生理重要性； 3) 确定酵母和人类外泌体亚基和复合物的结构和生物物理基础。 RNA 外泌体通过 3' 至 5' 衰变有助于细胞 RNA 稳态，从而平衡 RNA 转录与 RNA 降解。外泌体还通过多种质量控制途径参与维持 RNA 完整性，这些途径可针对异常 RNA 进行破坏。这些途径共同调节特定 RNA 的寿命，并保护细胞免受可能导致细胞病理的有害 RNA 的侵害。这些过程的缺陷与多种人类疾病有关，包括癌症、炎症和神经退行性疾病。

DESCRIPTION (provided by applicant): The lifetime and abundance of RNA is regulated by maintaining a balance between RNA transcription and RNA degradation. While several RNA degradation pathways have been discovered, their components, precise catalytic activities, and cellular function remain a continued focus of study. One of two principle RNA decay pathways in eukaryotes involves the RNA exosome, a multi-subunit protein complex that catalyzes 3' to 5' RNA decay. The RNA exosome is conserved throughout eukaryotic evolution and is thought to exist in at least two forms, a cytoplasmic exosome composed of nine or ten distinct protein subunits with an apparent mass of 300-400 kDa, and a nuclear exosome composed of ten or eleven distinct protein subunits with an apparent mass of 400-500 kDa. In budding yeast, ten of the eleven genes are essential for growth, suggesting critical roles for each in cellular function. The identification of individual exosome subunits led to the hypothesis that the exosome was composed of up to eleven distinct 3' to 5' exoribonucleases. However, recent studies utilizing reconstituted and affinity-purified cellular exosome complexes indicate that only a few of the subunits encode polypeptides with exoribonuclease activity, suggesting that most exosome subunits are conserved for their non-catalytic functions. Consistent with this, exosome subunits are known to associate with a variety of protein partners that facilitate or direct RNA degradation. While recent efforts have revealed many interesting aspects of eukaryotic exosome structure and function, many questions remain with respect to individual and collective functions for exosome subunits in RNA decay. In this proposal, we will utilize in vitro reconstituted human and yeast exosome complexes in combination with genetic and biochemical assays to analyze its assembly, catalytic activity, and structural integrity by conducting research with these aims: 1) characterize and reconstitute subunits and complexes from human and yeast exosomes; 2) determine the biochemical basis for activities ascribed to individual, sub-complexes, and intact exosome complexes from yeast and human and determine the physiological importance of the observed activities and structures through in vivo complementation and analysis in the budding yeast Saccharomyces cerevisiae; 3) determine the structural and biophysical basis for exosome subunits and complexes from yeast and human. RNA exosomes contribute to cellular RNA homeostasis through 3' to 5' decay, thus balancing RNA transcription with RNA degradation. Exosomes are also involved in maintaining RNA integrity via several quality control pathways which serve to target aberrant RNA for destruction. Together, these pathways regulate the lifetime of a particular RNA and protect the cell from deleterious RNA that could lead to cellular pathology. Defects in these processes are associated with several human diseases including cancer, inflammation, and neurodegenerative disorders.