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焦点。真核生物中两种主要的RNA衰变途径之一涉及RNA外泌体，这是一种多亚基蛋白质复合物，可催化3‘至5’ RNA衰变。RNA外泌体在整个真核生物进化过程中是保守的，被认为至少存在两种形式，一种是由9或10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，另一种是由10或11个不同的蛋白质亚基组成的核外泌体，表观质量为400-500 kDa。在出芽酵母中，11个基因中有10个对生长至关重要，这表明每个基因在细胞功能中都起着关键作用。单个外泌体亚基的鉴定导致了外泌体由多达11个不同的3‘至5’外核糖核酸酶组成的假设。然而，最近利用重组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核糖核酸酶活性的多肽，这表明大多数外泌体亚基因其非催化功能而被保守。与此一致的是，外泌体亚基已知与多种促进或直接RNA降解的蛋白质伴侣相关联。虽然最近的研究已经揭示了真核外泌体结构和功能的许多有趣方面，但关于外泌体亚基在RNA衰变中的个体和集体功能仍存在许多问题。在本项目中，我们将利用体外重组的人和酵母外泌体复合物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，通过以下目的进行研究：1)表征和重组来自人和酵母外泌体的亚基和复合物；2)确定酵母和人类个体、亚复合物和完整外泌体复合物活性的生化基础，并通过出芽酵母（Saccharomyces cerevisiae）的体内互补和分析确定所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′~ 5′衰变促进细胞RNA稳态，从而平衡RNA转录和RNA降解。外泌体还通过几种质量控制途径参与维持RNA的完整性，这些途径用于靶向异常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.