Molecular Mechanisms that Control mRNA Decapping in Biological Condensates

控制生物浓缩物中 mRNA 脱帽的分子机制

• 批准号: 10577994
• 负责人: John D Gross
• 金额: $ 31.82万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-15 至 2027-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10577994
• 关键词: 7-methylguanosine; Active Sites; Address; Affect; Binding; Binding Sites; Biochemical; Biochemical Pathway; Biochemical Reaction; Biological; Biological Assay; Bypass; C-terminal; Cells; Complex; Conceptions; Coupled; Couples; Cytoplasm; Data; Digestion; Dimerization; Disease; Enhancers; Enzymes; Eukaryota; Excision; Exhibits; Exoribonucleases; Fission Yeast; Fluorescence; Gene Expression; Genes; Geometry; Human; In Vitro; Intellectual functioning disability; Lesion; Licensing; Liquid substance; Major Core Protein; Malignant Neoplasms; Mediating; Membrane; Messenger RNA; Modeling; Molecular; Molecular Conformation; Molecular Genetics; Multienzyme Complexes; Organelles; Outcome; Pathway interactions; Phase; Physical condensation; Physiologic pulse; Physiological; Property; Proteins; Publishing; RNA Binding; RNA Degradation; Regulation; Reporting; Repression; Signal Transduction; Structure; Testing; Transcript; Work; Yeasts; decapping enzyme; enzyme activity; human disease; mRNA Decay; mRNA Transcript Degradation; mRNA decapping; monomer; mutant; novel; reconstitution; targeted treatment; therapy design

PROJECT SUMMARY Cells organize biochemical reactions into biological condensates. P-bodies are conserved cytoplasmic condensates enriched in factors important for mRNA storage or degradation, but how these opposing outcomes may be achieved in condensates is unclear. A critical step in mRNA degradation is the removal of the 5'-7- methylguanosine cap by the decapping enzyme complex (Dcp1/Dcp2) that precedes and permits digestion of the mRNA body by conserved exoribonucleases. We have reconstituted biological condensates containing fission yeast Dcp1/Dcp2 and an enhancer of decapping protein 3 (Edc3), which are major core proteins of P- bodies. Using novel, activity-based fluorescence probes we have made two significant discoveries. First, contrary to the popular model that condensates enhance enzymatic reactions due to local concentration effects, we find that phase separation represses the activity of Dcp1/Dcp2 100-fold compared to dilute solution. Second, the decapping activity of these condensates can be rescued by Edc3. Our data suggest the protein interaction platform Dcp1 is an integrator of short-linear protein interaction motifs that couples phase separation to inactivation of decapping by promoting a conformational change in Dcp1/Dcp2 to an autoinhibited conformation. In Aim 1, we will determine the structure of the autoinhibited conformation of Dcp1/Dcp2 and test the hypothesis that short-linear motifs in Dcp2 directly interact with Dcp1 to promote a transient inactive conformation of the decapping complex. In Aim 2, we will study how condensates provide an additional layer for decapping repression, testing the hypothesis interactions that promote phase separation further stabilize the inactive conformation of Dcp1/Dcp2 in condensates. In Aim 3, we will determine the mechanism for activation of decapping in condensates, testing the hypothesis that Edc3 opposes the inhibitory action of short-linear inaction motifs in Dcp2 and promotes a conformational change that opens the RNA binding channel in Dcp1/Dcp2 to promote efficient decapping within condensates. Lesions important for repression of decapping in vitro will be tested for their function in EDC3-mediated mRNA decay in fission yeast. The proposed studies are poised to provide a paradigmatic example of how biological condensation is coupled to conformational control of enzyme activity that affects the fidelity of gene expression at the level of mRNA decay.

项目摘要 细胞将生化反应组织成生物凝聚物。P体是胞质内保守的 浓缩物富含对mRNA储存或降解重要的因子，但这些相反的结果是如何产生的？ 可能在冷凝物中实现，目前尚不清楚。mRNA降解中的关键步骤是去除5 '-7- 甲基鸟苷被脱帽酶复合物（Dcp 1/Dcp 2）帽化，该酶复合物先于并允许甲基鸟苷的消化， mRNA体被保守的核糖核酸外切酶切割。我们重组了生物浓缩物， 裂殖酵母Dcp 1/Dcp 2和脱帽蛋白增强子3（Edc 3），它们是P- 尸体使用新型的基于活性的荧光探针，我们有两个重大发现。第一，相反 对于缩合物由于局部浓度效应而增强酶促反应的流行模型，我们发现 相分离抑制Dcp 1/Dcp 2的活性100倍于稀释溶液。二是 这些缩合物的脱帽活性可以被Edc 3拯救。我们的数据表明蛋白质相互作用 平台Dcp 1是短线性蛋白质相互作用基序的整合剂，其将相分离耦合到 通过促进Dcp 1/Dcp 2的构象变化至自抑制构象来失活去帽。 在目的1中，我们将确定Dcp 1/Dcp 2的自抑制构象的结构并验证假设 Dcp 2中的短线性基序直接与Dcp 1相互作用，以促进Dcp 1的瞬时失活构象。 开盖复合物。在目标2中，我们将研究冷凝物如何为开盖提供额外的层 抑制，测试促进相分离的假设相互作用进一步稳定了非活性的 Dcp 1/Dcp 2在缩合物中的构象。在目标3中，我们将确定激活 在冷凝物中开盖，测试Edc 3反对短线性不活动的抑制作用的假设 Dcp 2中的基序并促进构象变化，打开Dcp 1/Dcp 2中的RNA结合通道， 促进冷凝物内的有效开盖。在体外对抑制脱帽重要的病变将是 测试它们在分裂酵母中EDC 3介导的mRNA衰变中的功能。拟议中的研究准备 提供了一个典型的例子，生物缩合是如何耦合到构象控制的酶 在mRNA衰变水平上影响基因表达保真度的活性。