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。该途径中的关键步骤是去除在所有真核mRNA的5 '端上发现的保护性甲基鸟苷帽，其使转录物快速降解。帽结构的切割由保守的脱帽酶Dcp 2与调节脱帽活性的蛋白质共活化剂组合催化。Dcp 2是果蝇中microRNA介导的mRNA转录物降解所必需的，并且对酵母中长非编码RNA的降解很重要。这两类非编码RNA对于维持哺乳动物的细胞平衡非常重要，并且在许多人类癌症中发现了异常水平的微小或长链非编码RNA。尽管去帽和5 '-至-3' mRNA衰变的生物学重要性，但Dcp 2切割mRNA帽的结构细节及其与辅激活剂的蛋白质-蛋白质相互作用仍然知之甚少。脱帽酶Dcp 2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帽去除。最近的研究表明，Dcp 2的两个结构域形成一个封闭的复合活性位点，识别mRNA底物并催化帽切割，而辅活化剂可以通过促进或稳定Dcp 2的封闭的催化活性构象来加速脱帽。在这个建议中，一个多样化的生物化学，生物物理学和遗传学实验将被用来测试这些假设，通过构建一个全面的结构模型Dcp 2的活动。Dcp 2的催化活性构象将使用过渡态类似物（TSA）来稳定，所述过渡态类似物模拟Dcp 2的活性位点中的帽磷酸水解。基于金属氧酸盐或金属氟化物添加剂的TSA将使用小角X射线散射、荧光偏振和酶抑制实验的组合来鉴定。TSAs，促进Dcp 2的活性构象将使用NMR光谱和X射线晶体学进行结构表征。要调查的结构所起的作用，由coactivators的decapping，NMR光谱将被用来确定Dcp 2的催化结构域和coactivators Dcp 1和Edc 1，可能是蛋白质-蛋白质相互作用的表面，促进封闭，活性构象的Dcp 2之间的接触。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，将被用来模拟共激活剂如何扰乱Dcp 2的构象平衡，并影响decapping活性。突变分析，使用体外脱帽动力学和体内酵母互补实验，将被用来连接结构表型和确认结构模型的生物相关性。这些研究将提供一个分子水平的了解如何蛋白质-蛋白质相互作用和构象变化的保守的脱帽酶Dcp 2控制mRNA帽切割，从而帮助调节转录稳定性。

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.