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: 10797100
• 负责人: LIANG TONG
• 金额: $ 6.84万
• 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/10797100
• 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; Life Cycle Stages; Link; Mammals; Messenger RNA; Molecular; Molecular Mechanisms of Action; Nucleotides; Poly A; Polyadenylation; Polymerase; 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 (from the funded MIRA application) 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（来自 MIRA 资助申请） 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 在细胞核中进行转录加工，然后才能输出到细胞质并发挥作用 作为 mRNA。加工事件包括 5 美分末端封盖、拼接和 3 美分末端切割和 聚腺苷酸化。大多数前体 mRNA 的 3' 端加工需要大量蛋白质 其执行因素，包括切割和聚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’端 酵母的加工机制与哺乳动物的加工机制相似，尽管也存在显着的差异。 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 尽管它具有一些关键的蛋白质因子，但最终并采用独特的机械进行加工 使用规范的前 mRNA 3￠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，一般是 相信封顶总是会完成。我们之前发现 DXO/Rai1 蛋白质家族是 mRNA 封盖质量监控机制的一部分。他们可以拥有 RNA 5′端焦磷酸水解酶（PPH）和脱帽活性，并帮助去除不完全 来自细胞的加帽 mRNA。 尽管对这些 mRNA 加工和质量控制因素进行了广泛的研究，但仍存在显着差距 我们对其分子作用机制的了解仍然存在。在上一次融资期间 在此期间，我们通过冷冻电镜确定了活性的、完全重组的人类组蛋白预的结构 mRNA 3￠端加工机械，第一个结构的主动加工机械。我们有 还表明 DXO/Rai1 和 Nudix 酶家族可以去除核苷酸代谢物帽 RNA，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关于 pre-mRNA 的新结构信息 和 snRNA 3￠端加工机器，并了解不同的分子基础 DXO/Rai1 和 Nudix 酶的脱盖活性。我们将进行结构研究 通过冷冻电镜和晶体学分析蛋白质因子及其复合物，并评估其结构 通过仔细的生化和功能实验观察。拟议的项目将大大 增强我们对 mRNA 生命周期中这些重要事件的理解。

项目成果

Heme Oxygenase 2 Binds Myristate to Regulate Retrovirus Assembly and TLR4 Signaling.

• DOI: 10.1016/j.chom.2017.01.002
• 发表时间: 2017-02-08
• 期刊: Cell host & microbe
• 影响因子: 30.3
• 作者: Zhu Y;Luo S;Sabo Y;Wang C;Tong L;Goff SP
• 通讯作者: Goff SP

How Does Polymerization Regulate Human Acetyl-CoA Carboxylase 1?

• DOI: 10.1021/acs.biochem.8b00881
• 发表时间: 2018-09
• 期刊: Biochemistry
• 影响因子: 2.9
• 作者: Jia Wei;L. Tong

P-TEFb regulation of transcription termination factor Xrn2 revealed by a chemical genetic screen for Cdk9 substrates.

CDK9底物的化学遗传筛选显示了转录终止因子XRN2的P-TEFB调节。

• DOI: 10.1101/gad.269589.115
• 发表时间: 2016-01-01
• 期刊: Genes & development
• 影响因子: 10.5
• 作者: Sansó M;Levin RS;Lipp JJ;Wang VY;Greifenberg AK;Quezada EM;Ali A;Ghosh A;Larochelle S;Rana TM;Geyer M;Tong L;Shokat KM;Fisher RP
• 通讯作者: Fisher RP

Molecular mechanism for the regulation of yeast separase by securin.

• DOI: 10.1038/nature21061
• 发表时间: 2017-02-09
• 期刊: Nature
• 影响因子: 64.8
• 作者: Luo S;Tong L
• 通讯作者: Tong L

In vitro methylation of the U7 snRNP subunits Lsm11 and SmE by the PRMT5/MEP50/pICln methylosome.

• DOI: 10.1261/rna.079709.123
• 发表时间: 2023-11
• 期刊: RNA (New York, N.Y.)