Coupling of transcription with nascent pre-mRNA metabolism

 描述(申请人提供)：信使核糖核酸(信使核糖核酸)的产生是基因表达中的主要事件，基因信息从基因的DNA转移到一次性的核糖核酸拷贝中。这一过程的腐败是包括癌症在内的许多疾病的标志。MRNA合成不仅需要RNA转录本，还需要通过5‘端封端、内含子切除和外显子剪接以及通过切割/多聚腺苷形成3’端来成熟该转录本。该mRNA还包装有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。从根本上改变初级转录本的mRNA加工和包装步骤在很大程度上是共转录的；也就是说，mRNA加工和包装因子的底物是生长的新生RNA，由RNA聚合酶II(PolII)分子以0.5-4.5千碱基/分钟的速度挤出。这一建议的目标是通过不关注成熟的mRNA产物而是新生的转录本，以及它们的转换如何受到生长RNA链的转录延伸过程的影响，来了解mRNP在其共转录背景下的生物合成。我们的工作模型是，信使核糖核酸前体的合成和加工在一个动态的“信使核糖核酸工厂”复合体中以一种集成的方式进行，该复合体包括RNA聚合酶和加工因子，其中一些因子与PolII的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研究正在进行的转录和mRNA的成熟是如何通过将因子招募到‘mRNA工厂’而在空间上相互耦合，并在时间上通过动力学耦合机制来实现高效和准确地生产完全形成的mRNP的。在这个建议中，我们将解决这些问题：1)PolII转录延长如何影响新生转录本的加工？2)新生转录本如何折叠，Pol II转录延伸如何影响折叠？3)转录延伸如何影响RNA结合蛋白与新生转录本的关联？4)延伸率如何影响RNA PolII CTD的磷酸化和加工因子向转录延伸复合体的募集？5)共转录RNA加工如何影响延伸和染色质结构？

 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?