Structural Studies of Messenger RNA Maturation and Decay

信使 RNA 成熟和衰变的结构研究

• 批准号: 7280376
• 负责人: CHRISTOPHER D. LIMA
• 金额: $ 39.48万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-09-01 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7280376
• 关键词: Affect; Biochemical Genetics; C-terminal; Characteristics; Complex; Consensus Sequence; Diphosphates; Enzymes; Eukaryota; Eukaryotic Cell; Excision; Funding; Genetic Structures; Genetic Transcription; Goals; Guanine; Guanosine Monophosphate; Hydrolysis; Mediating; Messenger RNA; Metabolism; Methyltransferase; Modification; Nucleotides; Pathway interactions; Phosphoric Monoester Hydrolases; Play; Polyadenylation; Positioning Attribute; Process; Proteins; RNA; RNA Caps; RNA Decay; RNA Degradation; RNA Polymerase II; RNA Polymerase III; RNA Processing; RNA Splicing; RNA polymerase II largest subunit; RNA triphosphatase; RNA, Messenger, Splicing; Recruitment Activity; Regulatory Element; Repression; Research Personnel; Research Proposals; Residual state; Role; Structure; System; Tertiary Protein Structure; Time; Transcription Elongation; Transcription Initiation; Transcriptional Regulation; Translations; Work; Yeasts; base; decapping enzyme; genetic analysis; guanylyltransferase; inhibitor/antagonist; inorganic phosphate; mRNA Precursor; mRNA guanylyltransferase; poly A specific exoribonuclease; polyadenylated messenger RNA; programs; tripolyphosphate

DESCRIPTION (provided by applicant): The stability of eukaryotic messenger RNA (mRNA) is regulated in part by co-transcriptional and posttranscriptional modifications that include capping, splicing, and polyadenylation. The 5' m7GpppN cap is the first co-transcriptional mRNA modification made during transcription and is required for efficient premRNA splicing, export, stability, and translation. Capping is catalyzed in three enzymatic steps by RNA triphosphatase, RNA guanylyltransferase, and RNA (guanine-N7) methyltransferase and the capping appartus is recruited to the transcription complex by direct interaction with the phosphorylated C-terminal domain of the largest subunit of RNA polymerase II (RNAPII). Interactions between the capping apparatus and RNAPII are also involved in activation and repression of transcription initiation and elongation, the details of which are poorly understood. We propose to illuminate important determinants and regulatory elements for eukarytic mRNA processing and transcription through Aim 1) structural and genetic analysis of cap forming enzymes in complex with one another, in complex with RNAPII, in complex with the phosphorylated RNAPII CTD; Aim 2) the structural, genetic, and biochemical characterization of CTD interacting proteins that include mammalian capping enzymes and yeast CTD phosphatase and methyltransferase enzymes; Aim 3) characterization of the Spt4/Spt5 transcriptional elongation complex. The RNA cap structure also plays a critical role in both major RNA decay pathways. After deadenylation of polyadenylated mRNA in the 5'-3' decay pathway, the Dcp1/Dcp2 decapping complex hydrolyzes the mRNA cap to expose the 5' RNA end to 5'-3' exoribonuclease activities. In the 3'-5' decay pathway, exosome-mediated degradation of RNA occurs from the 3' end after deadenylation, ultimately generating a cap structure that is hydrolyzed by enzymes in this pathway. Hydrolysis of the residual cap structure is predicted to eliminate m7G cap intermediates that might serve as potential inhibitors of mRNA translation, export, and processing factors that recognize messenger RNA via the 5' m7G cap. We propose in Aim 4) to illuminate the structural, mechanistic, and regulatory basis-for decapping enzymes in both 5'-3' and 3'-5 RNA decay pathways that mediate interactions with and degrade the 5' m7GpppN cap or capped RNA.

描述（由申请人提供）：真核信使RNA （mRNA）的稳定性部分由共转录和转录后修饰调节，包括盖层、剪接和聚腺苷化。5' m7GpppN帽是转录过程中第一个共转录mRNA修饰，是有效的premRNA剪接、输出、稳定性和翻译所必需的。旋盖由RNA三磷酸酶、RNA鸟嘌呤转移酶和RNA（鸟嘌呤- n7）甲基转移酶催化，旋盖装置通过与RNA聚合酶II （RNAPII）最大亚基的磷酸化c端结构域的直接相互作用被招募到转录复合体中。封盖装置和RNAPII之间的相互作用也参与转录起始和延伸的激活和抑制，其细节尚不清楚。我们建议通过Aim来阐明真核mRNA加工和转录的重要决定因素和调控元件1)相互复合物、与RNAPII复合物、与磷酸化RNAPII CTD复合物的帽形成酶的结构和遗传分析；目的2)CTD相互作用蛋白的结构、遗传和生化特性，包括哺乳动物封顶酶和酵母CTD磷酸酶和甲基转移酶；目的3)表征Spt4/Spt5转录延伸复合物。