Structural and Functional Studies of Eukaryotic Exosomes

描述(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，但它们的组成、精确的催化活性和细胞功能仍然是研究的重点。真核生物中两条主要的RNA衰变途径之一涉及RNA外体，它是一种催化3‘到5’RNA衰变的多亚单位蛋白质复合体。RNA外切体在真核生物进化过程中是保守的，被认为至少以两种形式存在：细胞质外切体由9个或10个不同的蛋白质亚基组成，表观质量为300-400 kDa；核外切体由10个或11个不同的蛋白质亚基组成，表观质量为400-500 kDa。在萌芽酵母中，11个基因中有10个是生长所必需的，这表明每个基因在细胞功能中都发挥着关键作用。对单个外切体亚单位的鉴定导致了这样的假设，即外切体由多达11个不同的3‘到5’外切核糖核酸酶组成。然而，最近利用重组和亲和纯化的细胞外切体复合体的研究表明，只有少数亚基编码具有外切核酸酶活性的多肽，这表明大多数外切体亚单位的非催化功能是保守的。与此一致的是，外切体亚单位已知与促进或指导RNA降解的各种蛋白质伙伴相关联。虽然最近的工作揭示了真核细胞外体结构和功能的许多有趣的方面，但关于外体亚单位在RNA衰变中的个体和集体功能仍然存在许多问题。在这项建议中，我们将利用体外重组的人和酵母外切体复合体，并结合遗传和生化分析来分析其组装、催化活性和结构完整性，开展的研究目的如下：1)鉴定和重组人和酵母外切体的亚单位和复合体；2)确定酵母和人类的单个、亚复合体和完整外切体复合体的活动的生化基础，并通过在萌芽酵母中的体内互补和分析确定所观察到的活性和结构的生理意义；3)确定酵母和人类的外切体亚单位和复合体的结构和生物物理基础。RNA外切体通过3‘到5’的衰变促进细胞RNA的动态平衡，从而平衡RNA转录和RNA降解。Exosome还通过几个质量控制途径参与维持RNA的完整性，这些途径可以针对异常的RNA进行破坏。这些途径共同调节特定RNA的寿命，保护细胞免受可能导致细胞病理的有害RNA的影响。这些过程中的缺陷与几种人类疾病有关，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.