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: 10393669
• 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/10393669
• 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.

项目摘要 大多数真核信使RNA前体（前MRNA）必须进行广泛的共同 在将其导出到细胞质之前，核中的转录处理 并充当mRNA。处理事件包括5¢ - 端封，拼接和3¢ - 末端切割和聚腺苷酸化。大多数MRNA的3'-End处理需要 其执行的大量蛋白质因素，包括乳沟和 多腺苷酸化特异性因子（CPSF），切割刺激因子（CSTF），裂解 因子I和II，以及聚（A）聚合酶（PAP）。 3'-End处理机械 酵母与哺乳动物的相似性，尽管存在显着差异。 复制依赖性组蛋白PREMRNA在其3美分附近包含一个保守的茎环 结束和员工的处理方式独特的机器，尽管它有一些密钥 具有规范前MRNA的蛋白质因子3¢ - 末端处理机制。 mRNA 5¢ - 末端上限发生在RNA聚合酶II转录期间的早期，IT 人们普遍认为，封盖总是可以完成的。我们发现了 早些时 监视机制。它们可以拥有RNA 5¢ - 末端的焦磷酸化酶（PPH） 并拆除活动，并有助于清除细胞中未完全封闭的mRNA。 尽管对这些mRNA处理和质量控制因素进行了广泛的研究，但 我们对它们的分子作用机理的了解仍然存在很大的差距。 在上一个资金期间，我们确定了通过活动的冻结EM确定结构 完全取代的人类组蛋白前MRNA 3¢ - 末端处理机制，第一个 主动处理机械的结构。我们还显示了DXO/RAI1 Nudix酶家族可以去除RNA上的核苷酸代谢物盖，例如 nAD（剥皮），时尚（脱落）和dephospho-coa（破裂）。这些 成功为拟议项目提供了绝佳的基础。 我们当前资助期的主要目标是产生新的结构信息 在前mRNA和snRNA 3¢端处理机上，并了解 DXO/RAI1和NUDIX的潜水员推定活动的分子基础 酶。我们将对蛋白质因子及其复合物进行结构性研究 通过冷冻EM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。