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

描述（由申请人提供）：信使RNA （mRNA）的细胞调控对基因的正确表达至关重要。用于调节真核mRNA的主要途径之一是5‘到3’衰变。该途径的一个关键步骤是去除所有真核生物mRNA 5'端上的保护性甲基鸟苷帽，这使得转录物快速降解。帽状结构的切割是由保守的脱帽酶Dcp2催化的，并与调节脱帽活性的蛋白质共激活物结合。在果蝇中，Dcp2是microRNA介导的mRNA转录物降解所必需的，在酵母中，Dcp2对长链非编码rna的降解也很重要。这两类非编码rna对于维持哺乳动物的细胞平衡非常重要，在许多人类癌症中发现了异常水平的微或长链非编码rna。尽管脱帽和5‘到3’ mRNA衰变具有重要的生物学意义，但Dcp2切割mRNA帽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。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，识别mRNA底物并催化帽子裂解，而共激活因子可能通过促进或稳定Dcp2封闭的催化活性构象来加速脱帽。在本研究中，我们将通过构建Dcp2活性的综合结构模型，采用一系列不同的生化、生物物理和遗传学实验来验证这些假设。Dcp2的催化活性构象将使用过渡态类似物（TSAs）来稳定，这些类似物可以模拟Dcp2活性位点的磷酸帽水解。基于金属氧酸盐或氟化金属添加剂的tsa将通过小角度x射线散射、荧光偏振和酶抑制实验相结合来确定。促进Dcp2活性构象的tsa将使用核磁共振波谱和x射线晶体学进行结构表征。为了研究脱帽的共激活因子在结构上的作用，NMR波谱将用于鉴定Dcp2的催化结构域与共激活因子Dcp1和Edc1之间的接触，这些接触可能是促进Dcp2封闭、活性构象的蛋白质-蛋白质相互作用表面。核磁共振结构赋值，结合从TSA研究中获得的其他结构数据，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。突变分析，使用体外脱帽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。这些研究将从分子水平上了解保守脱帽酶Dcp2的蛋白-蛋白相互作用和构象变化如何控制mRNA帽切割，从而有助于调节转录物的稳定性。

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.