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: 10580942
• 负责人: LIANG TONG
• 金额: $ 9.03万
• 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/10580942
• 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 (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. 1

项目摘要（来自获资助的MIRA申请）