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: 8607845
• 负责人: Jeffrey Scott Mugridge
• 金额: $ 5.51万
• 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/8607845
• 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）的细胞调节对于适当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'衰减。该途径的关键步骤是在所有真核mRNA的5'末端发现的保护性甲基 - 瓜氨酸盖，这将转录物赋予快速降解。盖结构的裂解是由保守的Decaptic DCP2催化的，结合了调节decap活性的蛋白质共激活剂。 DCP2对于果蝇中mRNA转录物的微介导的降解至关重要，对于酵母中长的非编码RNA降解至关重要。这两类的非编码RNA对于在许多人类癌症中都发现了哺乳动物的细胞平衡和微型或长的非编码RNA的水平非常重要。尽管decapping和5'至3'mRNA衰变具有生物学重要性，但DCP2的mRNA帽裂解的结构细节及其与共激活因子的蛋白质 - 蛋白质相互作用仍然知之甚少。 Decapping酶DCP2似乎使用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的两个结构域形成了一个闭合的复合活性位点，识别mRNA底物和催化帽裂解，而同时激活剂可能通过促进或稳定DCP2的封闭，催化活性的构象来加速脱皮。在此提案中，通过构建DCP2活性的综合结构模型来测试这些假设的生物化学，生物物理和遗传学实验。 DCP2的催化活性构象将使用过渡态类似物（TSA）稳定，该类似物（TSA）模仿DCP2活性位点中的磷酸磷酸磷酸盐水解。将使用小角度X射线散射，荧光偏振和酶抑制实验的组合来鉴定基于氧计或金属氟化物添加剂的TSA。促进DCP2主动构象的TSA将在结构上使用NMR光谱和X射线晶体学表征。为了研究脱皮的共激活因子所起的结构作用，NMR光谱法将用于鉴定DCP2的催化结构域与共激活剂DCP1和EDC1之间的接触，这些接触可能是蛋白质 - 蛋白质相互作用表面，这些表面可以促进DCP2的封闭，主动构型。 NMR的结构分配与从TSA研究获得的其他结构数据结合使用，将用于模拟同时激活剂如何扰动DCP2的构象平衡并影响dec骨活动。突变分析，使用体外dec酸动力学和体内酵母互补实验，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。这些研究将提供分子水平的理解，了解蛋白质 - 蛋白质相互作用和构象如何在保守的Decapping酶DCP2控制mRNA帽裂解中的变化，从而有助于调节转录本的稳定性。