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的降解是真核基因表达的关键步骤。它对于动物早期发育、分化、增殖、免疫应答和确保基因表达的质量控制是重要的。真核生物mRNA降解的关键步骤是去除保护性的5'端帽结构，其先于并允许RNA体被核糖核酸外切酶降解。脱帽全酶是一种核糖核蛋白组装体，含有由Dcp 2、激活剂和底物RNA组成的催化核心。Dcp 1是Dcp 2的重要相互作用伙伴，直接增强酶活性。它还作为一个蛋白质相互作用平台，允许Dcp 1/2被招募， 特异性转录物，并通过知之甚少的机制促进帽水解。Dcp 1/2复合物可以在溶液中以开放和封闭状态的集合存在，先前的工作表明转化为封闭形式可以在活动中产生开关样反应。灵活性阻碍了决议的活性形式的Dcp 1/2复杂的晶体学，因此，在目标1中，我们将确定的结构的封闭，活性形式的Dcp 1/2绑定到非水解帽类似物使用集成建模平台，结合结构和生化研究的限制。无义介导的衰变（NMD）是一种质量控制途径，其消除具有提前终止密码子的转录物，否则这些转录物将编码有害的截短蛋白质。富含脯氨酸的核辅助受体2（PNRC 2）是与脂肪形成和肥胖相关的蛋白质，其在NMD期间促进无义转录物的脱帽，并且在Staufen介导的mRNA衰变期间促进正常转录物的降解。它作为一个衔接子，将Dcp 1连接到识别mRNA的反式作用因子。PNRC 2还刺激脱帽活性，并含有这种效应所需的高度保守的基序。在目标2中，我们将使用体外动力学测定和哺乳动物细胞中的功能研究的组合来研究NMD期间PNRC 2刺激脱帽的机制。在目标3中，我们将使用X射线晶体学结合SAXS确定Dcp 1/2复合物的PNRC 2激活的结构基础。这些研究将确定连接Dcp 1/2与特定mRNA衰变途径的衔接子如何在募集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式，即弱的非特异性酶活性如何被招募到特定的mRNA中，然后被相关的蛋白质辅因子稳健地激活，以确保正确靶向底物。