Structural and functional studies of mRNA processing, stability and quality control

mRNA 加工、稳定性和质量控制的结构和功能研究

• 批准号: 10204562
• 负责人: LIANG TONG
• 金额: $ 79.28万
• 依托单位: COLUMBIA UNIV NEW YORK MORNINGSIDE
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-05-01 至 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10204562
• 关键词: Biochemical; Cell Nucleus; Cells; Cleavage And Polyadenylation Specificity Factor; Cleavage Stimulation Factor; Coenzyme A; Complex; Cryoelectron Microscopy; Crystallography; Cytoplasm; Defect; Enzymes; Event; Family; Fibrinogen; Foundations; Funding; Genetic Transcription; Goals; Histones; Human; Knowledge; Link; Mammals; Messenger RNA; Molecular; Molecular Mechanisms of Action; Nucleotides; Polyadenylation; Polynucleotide Adenylyltransferase; Protein Family; Proteins; Prothrombin; Quality Control; RNA; RNA Polymerase II; RNA Splicing; Reporting; Small Nuclear RNA; Structure; Yeasts; anti-cancer; cleavage factor; endonuclease; experimental study; human disease; mRNA Precursor; mRNA capping; reconstitution; stem; success

Project Summary Most eukaryotic messenger RNA precursors (pre-mRNAs) must undergo extensive co- transcriptional processing in the nucleus before they can be exported to the cytoplasm and function as mRNAs. The processing events include 5¢-end capping, splicing, and 3¢- end cleavage and polyadenylation. The 3’-end processing of most pre-mRNAs requires a large number of protein factors for its execution, including cleavage and polyadenylation specificity factor (CPSF), cleavage stimulation factor (CstF), cleavage factors I and II, and poly(A) polymerase (PAP). The 3’-end processing machinery in yeast has similarity to that in mammals, although there are also significant differences. Replication-dependent histone pre-mRNAs contain a conserved stem-loop near their 3¢- end and employ a distinct machinery for its processing, although it shares some key protein factors with the canonical pre-mRNA 3¢-end processing machinery. mRNA 5¢-end capping occurs early during transcription by RNA polymerase II, and it was generally believed that capping always proceeds to completion. We discovered earlier that the DXO/Rai1 family of proteins are part of a mRNA capping quality surveillance mechanism. They can possess RNA 5¢-end pyrophosphohydrolase (PPH) and decapping activities, and help remove incompletely capped mRNAs from cells. Despite the extensive studies on these mRNA processing and quality control factors, significant gaps remain in our knowledge on their molecular mechanisms of action. During the previous funding period, we determined the structure by cryo-EM of an active, fully-reconstituted human histone pre-mRNA 3¢-end processing machinery, the first structure of an active processing machinery. We have also shown that the DXO/Rai1 and Nudix family of enzymes can remove nucleotide metabolite caps on RNAs, such as NAD (deNADding), FAD (deFADding) and dephospho-CoA (deCoAping). These successes provide an excellent foundation for the proposed project. Our main goals for the current funding period are to produce new structural information on pre-mRNA and snRNA 3¢-end processing machineries, and to understand the molecular basis for the diverse decapping activities of the DXO/Rai1 and Nudix enzymes. We will carry out structural studies on the protein factors and their complexes by both cryo-EM and crystallography, and assess the structural observations by careful biochemical and functional experiments. The proposed project will greatly enhance our understanding of these important events in the mRNA lifecycle.

项目概要 大多数真核生物信使 RNA 前体 (pre-mRNA) 必须经历广泛的共同作用 在被输出到细胞质之前在细胞核中进行转录加工 并发挥 mRNA 的作用。加工事件包括5美分封端、拼接和3美分 末端裂解和聚腺苷酸化。大多数前体 mRNA 的 3' 端加工需要 大量蛋白质因子用于其执行，包括切割和 多聚腺苷酸化特异性因子 (CPSF)、裂解刺激因子 (CstF)、裂解 因子 I 和 II，以及聚 (A) 聚合酶 (PAP)。 3’端加工机械 酵母与哺乳动物的酵母有相似之处，但也存在显着差异。 复制依赖性组蛋白前体 mRNA 在其 3′- 附近含有保守的茎环 结束并使用不同的机器进行处理，尽管它有一些共同的关键 蛋白质因子与典型的前 mRNA 3 末端加工机制。 mRNA 5′端加帽发生在 RNA 聚合酶 II 转录过程的早期，并且它 人们普遍认为封顶总是会完成。我们发现 早些时候，DXO/Rai1 蛋白家族是 mRNA 封盖质量的一部分 监督机制。它们可以拥有RNA 5′端焦磷酸水解酶（PPH） 和脱帽活动，并帮助从细胞中去除不完全加帽的 mRNA。 尽管对这些 mRNA 加工和质量控制因素进行了广泛的研究， 我们对其分子作用机制的了解仍然存在重大差距。 在上一个资助期间，我们通过冷冻电镜确定了活性、 全重构的人类组蛋白前体mRNA 3′端加工机器，第一个 主动加工机械的结构。我们还表明 DXO/Rai1 Nudix 家族的酶可以去除 RNA 上的核苷酸代谢物帽，例如 NAD (deNADding)、FAD (deFADding) 和去磷酸辅酶 A (deCoAping)。这些 成功为拟议项目奠定了良好的基础。 我们当前资助期间的主要目标是产生新的结构信息 关于 pre-mRNA 和 snRNA 3 末端加工机制，并了解 DXO/Rai1 和 Nudix 多种脱盖活性的分子基础 酶。我们将对蛋白质因子及其复合物进行结构研究 通过冷冻电镜和晶体学，并通过仔细评估结构观察 生化和功能实验。拟议的项目将大大增强我们的 了解 mRNA 生命周期中的这些重要事件。