Structure and Function of the Decapping Enzyme Complex

脱帽酶复合物的结构和功能

基本信息

批准号：
8889016
负责人：
John D Gross
金额：
$ 30.08万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-12-01 至 2019-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8889016
关键词：
Active Sites Adaptor Signaling Protein Address Affect Affinity Animals Binding Binding Proteins Biochemical Biological Assay Biology Catalysis Catalytic Domain Cells Chemicals Complex Crystallization Crystallography Data Development Enhancers Ensure Enzymes Excision Exoribonucleases Fission Yeast Gene Expression Genes Hereditary Disease Holoenzymes Human Hydrolysis Immune response In Vitro Inherited Kinetics Knowledge Link Lobe Mammalian Cell Maps Mediating Messenger RNA Modeling Molecular Molecular Conformation Multienzyme Complexes Mutation NMR Spectroscopy Nonsense Codon Nonsense-Mediated Decay Nuclear Obesity Pathway interactions Play Post-Translational Protein Processing Process Proline Proteins Quality Control RNA RNA Decay RNA Degradation RNA Processing RNA Sequences RNA-Binding Proteins Recruitment Activity Regulation Relative (related person)Relaxation Resolution Ribonucleoproteins Ribosomes Role Solutions Specificity Structure Trans-Activators Transcript Work Yeasts analog base cofactor decapping enzyme enzyme activity flexibility genetic analysis human disease link protein lipid biosynthesis messenger ribonucleoprotein mutant obesity in children public health relevance receptor response restraint transcriptome sequencing yeast protein

项目摘要

DESCRIPTION (provided by applicant): The decay of mRNA is a key step in eukaryotic gene expression. It is important for early animal development, differentiation, proliferation, the immune response and ensures quality control of gene expression. A critical step in eukaryotic mRNA decay is the removal of the protective 5' terminal cap structure, which precedes and permits degradation of the RNA body by exoribonucleases. The decapping holoenzyme is a ribonucleoprotein assembly containing a catalytic core comprised of Dcp2, activators, and substrate RNA. Dcp1 is an essential interaction partner of Dcp2 that directly enhances enzyme activity. It also functions as a protein interaction platform that allows Dcp1/2 to be recruited to specific transcripts, and promotes cap hydrolysis through a poorly understood mechanism. The Dcp1/2 complex can exist in an ensemble of open and closed states in solution, and prior work suggests conversion to the closed form can impart a switch-like response in activity. Flexibility has encumbered resolution of the active form of the Dcp1/2 complex by crystallography; therefore, in Aim 1 we will determine the structure of the closed, active form of Dcp1/2 bound to non-hydrolyzable cap analogue using an integrated modeling platform that incorporates restraints from structural and biochemical studies. Nonsense-mediated decay (NMD) is a quality control pathway that eliminates transcripts with premature termination codons that would otherwise encode harmful, truncated proteins. Proline-rich nuclear co-receptor 2 (PNRC2), a protein linked to adipogenesis and obesity, promotes decapping of non-sense containing transcripts during NMD and degradation of normal transcripts during Staufen mediated mRNA decay. It functions as an adaptor, linking Dcp1 to trans acting factors that recognize mRNA. PNRC2 also stimulates decapping activity and contains a deeply conserved motif that is required for this effect. In Aim 2 we will study the mechanism of PNRC2 stimulation of decapping during NMD using a combination of in vitro kinetic assays and functional studies in mammalian cells. In Aim 3 we will determine the structural basis for PNRC2 activation of the Dcp1/2 complex using x-ray crystallography in combination with SAXS. These studies will define how adaptors that link Dcp1/2 to specific mRNA decay pathways play a dual role in recruitment and activation. More broadly, the project addresses an emerging paradigm in RNA biology of how a weak, non- specific enzymatic activity can be recruited to a specific mRNA and then activated robustly by associated protein cofactors to ensure correct targeting of substrates.

描述（由申请人提供）： mRNA 的衰变是真核基因表达的关键步骤，它对于早期动物发育、分化、增殖、免疫反应以及确保基因表达的质量控制非常重要。脱帽全酶是去除保护性 5' 末端帽结构，该结构先于并允许核糖核酸酶降解 RNA 体。脱帽全酶是含有催化物质的核糖核蛋白组装体。核心由 Dcp2、激活剂和底物 RNA 组成，Dcp1 是 Dcp2 的重要相互作用伙伴，可直接增强酶活性。 Dcp1/2 复合物可以在溶液中以开放和闭合状态的整体存在，并且先前的研究表明，向闭合形式的转换可以在活性中产生类似开关的反应。灵活性阻碍了 Dcp1/2 复合物活性形式的晶体学解析；因此，在目标 1 中，我们将确定与不可水解帽结合的闭合活性形式 Dcp1/2 的结构。使用整合了结构和生化研究限制的集成建模平台进行类似物是一种质量控制途径，可以消除带有过早终止密码子的转录本，否则这些转录本会编码有害的、富含脯氨酸的核共受体。 2 (PNRC2) 是一种与脂肪生成和肥胖相关的蛋白质，在 NMD 过程中促进无义转录物的脱帽，并在诗道芬介导的 mRNA 衰减过程中促进正常转录物的降解。作为适配器，将 Dcp1 与识别 mRNA 的反式作用因子连接起来，也可刺激脱帽活性，并包含此作用所需的深度保守基序。在目标 2 中，我们将使用 NMD 期间研究 PNRC2 刺激脱帽的机制。在目标 3 中，我们将结合体外动力学测定和哺乳动物细胞功能研究，使用 X 射线确定 Dcp1/2 复合物的 PNRC2 激活的结构基础。这些研究将定义连接 Dcp1/2 与特定 mRNA 衰变途径的接头如何在招募和激活中发挥双重作用，更广泛地讲，该项目解决了 RNA 生物学中如何弱、非降解的新兴范例。特定的酶活性可以被招募到特定的 mRNA 上，然后由相关的蛋白质辅因子强力激活，以确保底物的正确靶向。