Structural studies of RNA processing and ubiquitin-like protein modification

RNA衰变。RNA转录和降解之间的平衡有助于RNA的调节 寿命、质量和丰富度。真核生物中存在两种主要的RNA降解途径，一种降解RNA 5'端， 另一个将RNA降解为3'至5'。3'至5'衰变途径需要RNA外泌体的活性， 一种多亚基蛋白质复合物，包含一个九亚基非催化核心和两个额外的亚基， 催化进行性和分配性3'至5' RNA核糖核酸外切酶活性。11个基因中有10个是 这是芽殖酵母中生命所必需的，表明RNA外泌体及其在细胞内活动的重要性。 功能此外，过去几年的报告表明，人类在选择性基因突变中， 3 ′到5 ′衰变途径的组分患有从运动神经元病到癌症的疾病。 真核外泌体结构和功能的基本方面已经被阐明;然而， 关于上游因素如何将底物作为降解的目标仍然存在问题。随着RNA衰变 通路在真核核酸代谢和疾病中起着重要作用，我们的研究直接 与人类健康和NIH的使命相关，因为RNA衰变的失调与癌症有关， 炎症和神经变性。这次更新将通过以下方式解决RNA外泌体生物学的核心问题： 重组或纯化RNA外泌体和上游因子以通过生物化学表征， 遗传和结构研究，以建立体外和体内3'至5'衰变的功能。 泛素样蛋白。信号转导可以依靠可逆的化学修饰来传递信息。 蛋白质底物可以被泛素和泛素样蛋白质如SUMO（小泛素）共价修饰。 泛素样修饰物）来调节诸如核转运、胞质分裂、染色体分离 G2-M细胞周期进程和转录。泛素（Ub）和泛素- 类（Ubl）蛋白需要E1激活酶、E2结合酶和E3结合酶的顺序作用。 蛋白质连接酶，而Ub/Ubl加工和解缀合由Ub/Ubl特异性蛋白酶催化。泛素 和SUMO结合在真核细胞核代谢和细胞周期控制中起着不可或缺的作用， 研究与人类健康、癌症和NIH的使命直接相关。此次更新旨在 解决泛素和SUMO结合途径组分的功能意义， 结构、生物化学和遗传学研究，将为Ub/Ubl激活、E2结合奠定基础 和E3酶，以及通过识别Ub/Ubl缀合的 印刷受体.泛素和SUMO蛋白结合的酶、机制和辅因子 这些通路是保守的，因此我们的研究与其他Ub/Ubl相关通路广泛相关。

RNA decay. The balance between RNA transcription and degradation contributes to regulation of RNA lifetime, quality and abundance. Two principle RNA decay pathways exist in eukaryotes, one degrades RNA 5’ to 3’ while the other degrades RNA 3’ to 5’. The 3’ to 5’ decay pathway requires activities of the RNA exosome, a multi-subunit protein complex that contains a nine-subunit non-catalytic core and two additional subunits that catalyze processive and distributive 3’ to 5’ RNA exoribonuclease activities. Ten of the eleven genes are essential for life in budding yeast, suggesting the importance of the RNA exosome and its activities in cellular function. In addition, reports over the last several years suggest that humans harboring mutations in select components of the 3’ to 5’ decay pathway suffer from diseases ranging from motor neuronopathies to cancer. Fundamental aspects of eukaryotic exosome structure and function have been illuminated; however, many questions remain with respect to how upstream factors target substrates for degradation. As RNA decay pathways play a fundamental role in eukaryotic nucleic acid metabolism and disease, our studies are directly relevant to human health and the NIH mission as misregulation of RNA decay is associated with cancer, inflammation and neurodegeneration. This renewal will address central issues of RNA exosome biology by reconstituting or purifying RNA exosomes and upstream factors for characterization through biochemical, genetic and structural studies to establish functions for 3’ to 5’ decay in vitro and in vivo. Ubiquitin-like proteins. Signal transduction can rely on reversible chemical modifications to relay information. Protein substrates can be covalently modified by ubiquitin and ubiquitin-like proteins such as SUMO (small ubiquitin-like modifier) to regulate processes such as nuclear transport, cytokinesis, chromosome segregation, G2-M cell cycle progression and transcription. Post-translational modification by ubiquitin (Ub) and ubiquitin- like (Ubl) proteins requires the sequential action of E1 activating enzymes, E2 conjugating enzymes and E3 protein ligases while Ub/Ubl processing and deconjugation is catalyzed by Ub/Ubl-specific proteases. Ubiquitin and SUMO conjugation play integral roles 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 renewal seeks to address the functional significance for components of the ubiquitin and SUMO conjugation pathways through structural, biochemical and genetic studies that will establish a basis for Ub/Ubl activation, conjugation by E2 and E3 enzymes, and signaling through characterization receptors that recognize Ub/Ubl-conjugated substrates. The enzymes, mechanisms and cofactors utilized for ubiquitin and SUMO protein conjugation pathways are conserved so our studies are broadly relevant to other Ub/Ubl-related pathways.