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（来自资助的MIRA申请） 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和功能之前，在细胞核中的转录处理 作为mRNA。处理事件包括5¢端封，拼接和3¢ - 端裂解和 聚腺苷酸化。大多数前MRNA的3'-End处理需要大量蛋白质 其执行因素，包括裂解和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和聚（A）聚合酶（PAP）。 3'-end 酵母中的加工机械与哺乳动物中的加工机械相似，尽管也有重要的 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 结束和员工是其处理的独特机械，尽管它具有一些关键的蛋白质因素 使用规范前MRNA 3¢ - 端处理机械。 mRNA 5¢ - 末端上限发生在RNA聚合酶II转录期间的早期，通常是 认为封盖总是可以完成的。我们早些时候发现DXO/RAI1 蛋白质家族是mRNA限制质量监视机制的一部分。他们可以拥有 RNA 5¢ - 末端的焦磷酸水解酶（PPH）和划定活性，并有助于除去不完整的 从细胞上盖上mRNA。 尽管对这些mRNA处理和质量控制因素进行了广泛的研究，但差距很大 在我们的分子作用机理方面保持了解。在以前的资金中 时期，我们确定了活性，全面取代的人类组蛋白的冷冻EM结构 mRNA 3¢ - 端处理机械，这是主动处理机械的第一个结构。我们有 还表明DXO/RAI1和NUDIX酶家族可以去除核苷酸代谢物盖 在RNA上，例如NAD（NAD），FAD（Defadding）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生成有关MRNA的新结构信息 和snRNA 3¢末端处理机，并了解潜水员的分子基础 DXO/RAI1和NUDIX酶的替代活动。我们将进行有关 蛋白质因子及其复合物通过冷冻EM和晶体学，并评估结构 通过仔细的生化和功能实验进行观察。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.

• 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

Integrator subunit 4 is a 'Symplekin-like' scaffold that associates with INTS9/11 to form the Integrator cleavage module.

• DOI: 10.1093/nar/gky100
• 发表时间: 2018-05-04
• 期刊: Nucleic acids research
• 影响因子: 14.9
• 作者: Albrecht TR;Shevtsov SP;Wu Y;Mascibroda LG;Peart NJ;Huang KL;Sawyer IA;Tong L;Dundr M;Wagner EJ
• 通讯作者: Wagner EJ