Structure and Function of the Decapping Enzyme Complex

脱帽酶复合物的结构和功能

• 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
• 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

 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、免疫反应非常重要，并确保基因表达的质量控制。真核 mRNA 降解的一个关键步骤是去除保护性 5' 末端帽结构，该结构先于核糖核酸外切酶降解 RNA 体，并允许其降解。脱帽全酶是一种核糖核蛋白组装体，含有由 Dcp2、激活剂和底物 RNA 组成的催化核心。 Dcp1 是 Dcp2 的重要相互作用伙伴，可直接增强酶活性。它还充当蛋白质相互作用平台，允许招募 Dcp1/2 特定的转录本，并通过一种知之甚少的机制促进帽水解。 Dcp1/2 复合物在溶液中可以以开放和闭合状态的整体形式存在，之前的研究表明，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，我们将使用整合了结构和生化研究限制的集成建模平台来确定与不可水解帽类似物结合的 Dcp1/2 的闭合活性形式的结构。无义介导的衰变 (NMD) 是一种质量控制途径，可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截短蛋白质。富含脯氨酸的核辅助受体 2 (PNRC2) 是一种与脂肪生成和肥胖相关的蛋白质，在 NMD 过程中促进含有无义转录物的脱帽，并在 Staufen 介导的 mRNA 衰减过程中促进正常转录物的降解。它充当适配器，将 Dcp1 与识别 mRNA 的反式作用因子连接起来。 PNRC2 还刺激脱帽活性，并包含此效果所需的深度保守基序。在目标 2 中，我们将结合体外动力学测定和哺乳动物细胞功能研究，研究 NMD 期间 PNRC2 刺激脱盖的机制。在目标 3 中，我们将使用 X 射线晶体学结合 SAXS 确定 PNRC2 激活 Dcp1/2 复合物的结构基础。这些研究将明确将 Dcp1/2 连接到特定 mRNA 衰减途径的接头如何在招募和激活中发挥双重作用。更广泛地说，该项目解决了RNA生物学中的一个新兴范例，即如何将弱的非特异性酶活性招募到特定的mRNA上，然后通过相关的蛋白质辅助因子强力激活，以确保底物的正确靶向。