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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要细胞将生化反应组织成生物凝聚物。 P-体是保守的细胞质浓缩物富含对 mRNA 储存或降解重要的因子，但这些相反的结果如何可能在凝结物中实现还不清楚。 mRNA 降解的关键步骤是去除 5'-7- 脱帽酶复合物 (Dcp1/Dcp2) 为甲基鸟苷加帽，该复合物先于并允许消化通过保守的核糖核酸外切酶形成 mRNA 体。我们重构了含有以下成分的生物浓缩物：裂殖酵母 Dcp1/Dcp2 和脱帽蛋白 3 (Edc3) 的增强子，它们是 P-的主要核心蛋白尸体。使用新颖的、基于活性的荧光探针，我们取得了两项重大发现。一、相反对于冷凝物由于局部浓度效应而增强酶反应的流行模型，我们发现与稀溶液相比，相分离将 Dcp1/Dcp2 的活性抑制 100 倍。其次，这些冷凝物的脱盖活性可以通过 Edc3 来挽救。我们的数据表明蛋白质相互作用平台 Dcp1 是短线性蛋白质相互作用基序的整合器，将相分离耦合到通过促进 Dcp1/Dcp2 构象变化至自抑制构象来失活脱帽。在目标 1 中，我们将确定 Dcp1/Dcp2 自抑制构象的结构并检验假设 Dcp2 中的短线性基序直接与 Dcp1 相互作用，促进 Dcp1 的短暂失活构象脱盖复杂。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非活性冷凝物中Dcp1/Dcp2的构象。在目标 3 中，我们将确定激活机制在冷凝物中脱盖，检验 Edc3 反对短线性不作为的抑制作用的假设 Dcp2 中的基序并促进构象变化，从而打开 Dcp1/Dcp2 中的 RNA 结合通道促进凝结水中有效的脱盖。对于抑制体外开盖很重要的损伤将是测试了它们在裂殖酵母中 EDC3 介导的 mRNA 衰减中的功能。拟议的研究旨在提供了生物缩合如何与酶的构象控制耦合的典型例子在 mRNA 衰减水平上影响基因表达保真度的活性。