Structural basis of mRNA decapping by Dcp2: conformational changes & co-activator

Dcp2 mRNA 脱帽的结构基础：构象变化

• 批准号: 8456674
• 负责人: Jeffrey Scott Mugridge
• 金额: $ 5.22万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-02-01 至 2015-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8456674
• 关键词: Acceleration; Active Sites; Affect; Biochemical; Biological; Biological Assay; Biological Models; Catalytic Domain; Cells; Chemicals; Commit; Complex; Data; Detection; Development; Disease; Drosophila genus; Enzyme Inhibition; Enzymes; Equilibrium; Excision; Fission Yeast; Fluorescence; Fluorescence Polarization; Fluorides; Functional RNA; Gene Expression; Genetic; Goals; Guanosine; Homeostasis; Human; Hydrolysis; In Vitro; Kinetics; Lead; Link; Maintenance; Malignant Neoplasms; Mammals; Mediating; Messenger RNA; Metals; MicroRNAs; Modeling; Molecular; Molecular Conformation; NMR Spectroscopy; Pathway interactions; Phenotype; Play; Proteins; RNA; RNA Caps; RNA Decay; Reaction; Regulation; Research; Roentgen Rays; Role; Saccharomyces cerevisiae; Site; Structural Models; Structure; Surface; Testing; Transcript; X-Ray Crystallography; Yeasts; analog; base; biological adaptation to stress; cell growth regulation; decapping enzyme; design; in vivo; inorganic phosphate; protein protein interaction; public health relevance; research study; tumor progression

DESCRIPTION (provided by applicant): Cellular regulation of messenger RNA (mRNA) is crucial for proper gene expression. One of the major pathways used to regulate eukaryotic mRNA is 5'-to-3' decay. A critical step in this pathway is the removal of the protective methyl-guanosine cap found on the 5'-end of all eukaryotic mRNA, which commits the transcript to rapid degradation. Cleavage of the cap structure is catalyzed by the conserved decapping enzyme Dcp2, in combination with protein coactivators that modulate decapping activity. Dcp2 is essential for microRNA- mediated degradation of mRNA transcripts in Drosophila, and important for degradation of long non-coding RNAs in yeast. These two classes of non-coding RNAs are important for the maintenance of cellular equilibrium in mammals and abnormal levels of micro or long non-coding RNAs are found in many human cancers. Despite the biological importance of decapping and 5'-to-3' mRNA decay, the structural details of mRNA cap cleavage by Dcp2 and its protein-protein interactions with coactivators remain poorly understood. The decapping enzyme Dcp2 appears to regulate mRNA cap removal using a combination of conformational dynamics and protein-protein interactions. Recent studies suggest that the two domains of Dcp2 form a closed, composite active site that recognizes mRNA substrate and catalyzes cap cleavage, while coactivators may accelerate decapping by promoting or stabilizing the closed, catalytically-active conformation of Dcp2. In this proposal, a diverse set f biochemical, biophysical and genetics experiments will be used to test these hypotheses by constructing a comprehensive structural model for Dcp2 activity. The catalytically-active conformation of Dcp2 will be stabilized using transition state analogs (TSAs) that mimic cap phosphate hydrolysis in the active site of Dcp2. TSAs based on oxometallate or metal fluoride additives will be identified using a combination of small angle x-ray scattering, fluorescence polarization and enzyme inhibition experiments. TSAs that promote the active conformation of Dcp2 will be structurally characterized using NMR spectroscopy and X-ray crystallography. To investigate the structural role played by coactivators of decapping, NMR spectroscopy will be used to identify contacts between the catalytic domain of Dcp2 and coactivators Dcp1 and Edc1 that might be protein-protein interaction surfaces that promote the closed, active conformation of Dcp2. NMR structural assignments, in combination with other structural data obtained from TSA studies where possible, will be used to model how coactivators perturb the conformational equilibria of Dcp2 and affect decapping activity. Mutational analyses, using in vitro decapping kinetics and in vivo yeast complementation experiments, will be used to link structure to phenotype and confirm the biological relevance of the structural model. These studies will provide a molecular level understanding of how protein-protein interactions and conformational changes in the conserved decapping enzyme Dcp2 control mRNA cap cleavage and thus help regulate transcript stability.

描述（由申请人提供）：信使 RNA (mRNA) 的细胞调节对于正确的基因表达至关重要。用于调节真核 mRNA 的主要途径之一是 5' 至 3' 衰变。该途径的一个关键步骤是去除所有真核 mRNA 5' 端的保护性甲基鸟苷帽，这使得转录物快速降解。帽结构的裂解由保守的脱帽酶 Dcp2 与调节脱帽活性的蛋白质共激活剂结合催化。 DCp2 对于果蝇中 microRNA 介导的 mRNA 转录物降解至关重要，对于酵母中长非编码 RNA 的降解也很重要。这两类非编码 RNA 对于哺乳动物细胞平衡的维持非常重要，并且在许多人类癌症中发现了异常水平的微小或长非编码 RNA。尽管脱帽和 5'-to-3' mRNA 降解具有生物学重要性，但 Dcp2 裂解 mRNA 帽的结构细节及其与共激活剂的蛋白质-蛋白质相互作用仍然知之甚少。脱帽酶 Dcp2 似乎通过构象动力学和蛋白质-蛋白质相互作用的组合来调节 mRNA 帽的去除。最近的研究表明，Dcp2 的两个结构域形成一个封闭的复合活性位点，可识别 mRNA 底物并催化帽子裂解，而共激活剂可能通过促进或稳定 DCP2 的封闭催化活性构象来加速脱帽。在本提案中，将通过构建 Dcp2 活性的综合结构模型，使用多种生化、生物物理和遗传学实验来检验这些假设。 Dcp2 的催化活性构象将使用模拟 Dcp2 活性位点中的磷酸帽水解的过渡态类似物 (TSA) 来稳定。基于金属氧酸盐或金属氟化物添加剂的 TSA 将通过小角度 X 射线散射、荧光偏振和酶抑制实验的组合来鉴定。促进 Dcp2 活性构象的 TSA 将使用 NMR 光谱和 X 射线晶体学进行结构表征。为了研究脱帽共激活剂所起的结构作用，核磁共振波谱将用于识别 Dcp2 催化结构域与共激活剂 Dcp1 和 Edc1 之间的接触，这些接触可能是促进 Dcp2 闭合、活性构象的蛋白质-蛋白质相互作用表面。 NMR 结构分配，在可能的情况下，与 TSA 研究中获得的其他结构数据相结合，将用于模拟共激活剂如何扰乱 Dcp2 的构象平衡并影响脱帽活性。利用体外脱盖动力学和体内酵母互补实验的突变分析将用于将结构与表型联系起来并确认结构模型的生物学相关性。这些研究将在分子水平上了解保守的脱帽酶 Dcp2 中的蛋白质-蛋白质相互作用和构象变化如何控制 mRNA 帽切割，从而帮助调节转录稳定性。