Structural studies of RNA processing and ubiquitin-like protein modification

 描述(申请人提供)：泛素样蛋白。信号转导通路依赖于可逆的化学修饰来在细胞内和细胞间传递信息。泛素和泛素样蛋白如SUMO(小泛素样修饰物)对蛋白质底物的共价修饰有助于调节细胞功能，包括核运输、胞质分裂、染色体分离、G2-M细胞周期进展和转录调节等。泛素(Ub)和类泛素(Ubl)蛋白的翻译后修饰需要E1激活酶、E2结合酶和E3连接酶的顺序作用，而Ub/Ub1的加工和去结合是由Ub/Ubl特异性的酶催化的。泛素和相扑结合在真核细胞的核代谢和细胞周期控制中起着不可或缺的作用，我们的研究直接关系到人类健康、癌症和美国国立卫生研究院的使命。这一建议试图通过结构、生化和遗传研究来解决泛素和相扑结合途径组件的功能意义，这些研究将为Ub/Ub1)激活奠定基础， 2)E2和E3酶的结合，3)识别Ub/Ub1结合底物的特征性受体的信号转导。组成泛素和相扑蛋白结合途径的酶、机制和因素是保守的，因此我们的研究与其他Ub/Ub1相关途径广泛相关。核糖核酸会腐烂。改变RNA转录和降解之间的平衡可以调节RNA的寿命、质量和丰度。真核生物中存在两条主要的RNA衰变途径，一条是催化5‘到3’方向的降解，另一条是沿3‘到5’方向降解RNA。3‘到5’衰变途径需要RNA外体的活性，它是一个大的多亚基蛋白质复合体，包含一个由9个亚基和2个附加亚基组成的非催化核心，催化3‘到5’RNA外切核酸酶的进行性和分布性活性。在萌芽酵母中，11个基因中有10个是生命所必需的，这表明了rna外切体及其在细胞中的活动的重要性。 功能。最近的工作阐明了真核细胞外体结构和功能的基本方面，但关于外体亚单位在RNA加工和衰退过程中的集体活动，以及它们如何与招募和修饰RNA底物的因子相结合，仍然存在许多问题。RNA衰变途径在真核细胞的核酸代谢中起着不可或缺的作用，因此我们的研究直接关系到人类的健康和NIH的使命，因为RNA加工和衰退的错误调控与癌症、炎症和神经变性等疾病有关。这项建议将通过确定单个外切体亚基的特征，通过重组多个亚基RNA外切体，并通过分析这些复合体在生化、遗传和结构研究中的活性来解决人类和酵母RNA外切体生物学的中心问题，这些研究将建立外切体的功能。 体外和体内的RNA加工和降解。

 DESCRIPTION (provided by applicant): Ubiquitin-like proteins. Signal transduction pathways rely on reversible chemical modifications to relay information within and across cells. Covalent modification of protein substrates by ubiquitin and the ubiquitin-like proteins such as SUMO (small ubiquitin-like modifier) contribute to pathways that regulate cellular functions including nuclear transport, cytokinesis, chromosome segregation, G2-M cell cycle progression and transcriptional regulation among many others. Post-translational modification by ubiquitin (Ub) and ubiquitin-like (Ubl) proteins requires the sequential action of E1 activating enzymes, E2 conjugating enzymes and E3 ligases while Ub/Ubl processing and deconjugation is catalyzed by Ub/Ubl-specific proteases. Ubiquitin and SUMO conjugation play an integral role in eukaryotic nuclear metabolism and cell cycle control and our studies are of direct relevance to human health, cancer, and the mission of the NIH. This proposal seeks to address the functional significance for components of the ubiquitin and SUMO conjugation pathways through structural, biochemical and genetic studies that will establish the basis for Ub/Ubl 1) activation, 2) conjugation by E2 and E3 enzymes, 3) and signal transduction through characterization receptors that recognize Ub/Ubl-conjugated substrates. The enzymes, mechanisms and factors that constitute ubiquitin and SUMO protein conjugation pathways are conserved so our studies are broadly relevant to other Ub/Ubl-related pathways. RNA decay. Shifting the balance between RNA transcription and degradation regulates RNA lifetime, quality and abundance. Two principle RNA decay pathways exist in eukaryotes, one catalyzes degradation 5' to 3' while the other degrades RNA in the 3' to 5' direction. The 3' to 5' decay pathway requires the activities of the RNA exosome, a large multi-subunit protein complex that contains a non-catalytic core of nine subunits and two additional subunits that catalyze processive and distributive 3' to 5' RNA exoribonuclease activities. In budding yeast, ten of the eleven genes are essential for life, suggesting the importance of the RNA exosome and its activities in cellular function. Recent efforts illuminated fundamental aspects of eukaryotic exosome structure and function, however many questions remain with respect to the collective activities for exosome subunits in RNA processing and decay and how they are coupled to factors the recruit and modify the RNA substrates. RNA decay pathways play an integral role in eukaryotic nucleic acid metabolism, so our studies are of direct relevance to human health and the mission of the NIH because misregulation of RNA processing and decay is associated with diseases such as cancer, inflammation and neurodegeneration. This proposal will address central issues of human and yeast RNA exosome biology by characterizing individual exosome subunits, by reconstituting multi-subunit RNA exosomes and by analyzing the activities of these complexes in biochemical, genetic and structural studies that will establish functions for the exosome during RNA processing and degradation in vitro and in vivo.