Coupling of transcription with nascent pre-mRNA metabolism

转录与新生前 mRNA 代谢的耦合

• 批准号: 9267500
• 负责人: DAVID L BENTLEY
• 金额: $ 48.82万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 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 2021-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9267500
• 关键词: Address; Affect; Anabolism; C-terminal; Cells; Chromatin Structure; Complex; Coupled; Coupling; Cytoplasm; DNA; DNA Polymerase II; DNA-Directed RNA Polymerase; Disease; Event; Excision; Exons; Gene Expression; Gene Transfer; Genetic Transcription; Genomic approach; Goals; Introns; Kinetics; Lead; Malignant Neoplasms; Messenger RNA; Metabolism; Modeling; Pathway interactions; Phosphorylation; Polyadenylation; Process; Production; RNA; RNA Processing; RNA Splicing; RNA chemical synthesis; RNA-Binding Proteins; Recruitment Activity; Time; Transcript; Transcription Elongation; Transcription Process; Work; genetic approach; genetic information; mRNA Precursor; messenger ribonucleoprotein; particle; public health relevance

 DESCRIPTION (provided by applicant): The production of messenger RNA (mRNA) is the primary event in gene expression where genetic information is transferred from the gene's DNA into a disposable RNA copy. Corruption of this process is a hallmark of many diseases including cancer. mRNA synthesis requires not only making an RNA transcript but maturation of that transcript by 5' capping, excision of introns and splicing of exons and 3' end formation by cleavage/polyadenylation. The mRNA is also packaged with RNA binding proteins that facilitate its maturation and ultimate export to the cytoplasm as a messenger ribonucleoprotein particle (mRNP). The mRNA processing and packaging steps that radically transform the primary transcript occur largely co-transcriptionally; that is to say the substrate of mRNA processing and packaging factors is the growing nascent RNA that is extruded by an RNA polymerase II (pol II) molecule at rates of 0.5-4.5 kilobases/min. The goal of this proposal is to understand mRNP biosynthesis in its co- transcriptional context by focusing not on the mature mRNA products but on the nascent transcripts and how their transformation is affected by the process of transcription elongation that grows RNA chains. Our working model is that synthesis and processing of a mRNA precursor are carried out in an integrated fashion within a dynamic 'mRNA factory' complex that includes both RNA polymerase and processing factors some of which make direct contacts with the pol II C-terminal domain (CTD). We use genetic and genomic approaches to ask how ongoing transcription and mRNA maturation are coupled with one another in space through recruitment of factors to the `mRNA factory' and in time through kinetic coupling mechanisms to achieve efficient and accurate production of fully formed mRNP's. In this proposal we will address these questions: 1) How does pol II transcription elongation affect processing of the nascent transcript? 2) How are nascent transcripts folded, and how is folding affected by pol II transcription elongation? 3) How does transcription elongation affect RNA binding protein association with nascent transcripts? 4) How does elongation rate affect phosphorylation of the RNA pol II CTD and recruitment of processing factors to the transcription elongation complex? 5) How does co-transcriptional RNA processing affect elongation and chromatin structure?

 描述（由申请人提供）：信使RNA（mRNA）的产生是基因表达的主要事件，其中遗传信息从基因的DNA转移到一次性RNA拷贝中。这一过程的破坏是包括癌症在内的许多疾病的标志。mRNA合成不仅需要制备RNA转录物，而且需要通过5'加帽、内含子切除和外显子剪接以及通过切割/多腺苷酸化形成3'末端来使该转录物成熟。mRNA还与RNA结合蛋白一起包装，RNA结合蛋白促进其成熟并最终作为信使核糖核蛋白颗粒（mRNP）输出到细胞质。mRNA的加工和包装步骤，从根本上改变了初级转录发生在很大程度上共转录;也就是说，mRNA加工和包装因子的底物是生长中的新生RNA，其被RNA聚合酶II（pol II）分子以0.5-4.5个酶/分钟的速率挤出。通过不关注成熟的mRNA产物，而是关注新生的转录物，以及它们的转化如何受到生长RNA链的转录延伸过程的影响，来研究转录背景。我们的工作模型是，mRNA前体的合成和加工是在一个动态的“mRNA工厂”复合物内以集成的方式进行的，该复合物包括RNA聚合酶和加工因子，其中一些与pol II C-末端结构域（CTD）直接接触。我们使用遗传学和基因组学的方法来询问正在进行的转录和mRNA成熟是如何通过将因子募集到“mRNA工厂”中来在空间上相互耦合的，以及如何通过动力学耦合机制在时间上实现完全形成的mRNP的有效和准确的生产。在这个提议中，我们将解决这些问题：1）pol II转录延长如何影响新生转录本的加工？ 2)新生的转录本是如何折叠的，以及折叠是如何受到pol II转录延伸的影响的？ 3)转录延长如何影响RNA结合蛋白与新生转录本的结合？ 4)延伸率如何影响RNA pol II CTD的磷酸化和转录延伸复合物中加工因子的募集？ 5)共转录RNA加工如何影响延伸和染色质结构？