Coupling of Transcription with Pre-mRNA Metabolism

描述（由申请人提供）：信使RNA的合成是基因表达的主要事件，对细胞的生命至关重要。 mRNA的产生需要通过pol II进行转录，并通过一组进行加帽、剪接和切割/聚腺苷酸化的蛋白质对初级转录物进行加工。由聚合酶而不是pol II产生的转录物不能正确地加工成成熟的mRNA。细胞核中存在蛋白质网络：蛋白质接触，将pol II的转录与负责mRNA成熟的特定RNA加工事件偶联。本提案的目的是阐明蛋白质如何沟通，以实现耦合的pol II转录与RNA包装和加工，使用生物化学和遗传学方法在哺乳动物细胞，青蛙卵母细胞和芽殖酵母。我们的研究正在测试这样一种想法，即mRNA是由一个包含RNA聚合酶II和RNA加工因子的“工厂”复合体制造的，RNA加工因子通过一个称为CTD的重复蛋白质结构域与mRNA接触。这个模型改变了我们对蛋白质的看法，这些蛋白质曾经被认为是独立运作的，但现在被认为在细胞核中相互协调。我们开始发现，转录和加工之间的整合的一个重要功能性后果是转录因子可以调节RNA加工的效率，相反，加工因子可以潜在地调节转录。这项工作可能有助于阐明mRNA的转录和加工在正常条件下是如何调节的，以及它们在疾病状态下是如何被错误调节的。前体mRNA的剪接缺陷导致所有遗传性疾病的大部分。本工作的具体目标是：1.比较pol Ⅱ转录与RNA加工包装偶联与非偶联的RNA加工包装机制.确定加帽酶如何调节pol II转录以及pol II转录机制的组成部分如何调节加帽。为了确定pol II延伸率、CTD七肽序列和CTD C-末端如何影响RNA包装、加帽、剪接和3'末端形成。确定哺乳动物加工因子是否与体内pol II三元复合物的延伸相关，以及三元复合物的加工因子组成在延伸过程中如何变化？

DESCRIPTION (provided by applicant): The synthesis of messenger RNA is the primary event in gene expression and is central to the life of cells. mRNA production requires transcription by pol II and processing of the primary transcript by a set of proteins which carry out capping, splicing and cleavage/polyadenylation. Transcripts made in by polymerases other than pol II are not processed correctly into mature mRNA. A network of protein:protein contacts in the cell nucleus exists to couple transcription by pol II with the specific RNA processing events responsible for maturation of mRNA. The objective of this proposal is to elucidate how proteins communicate to achieve coupling of pol II transcription with RNA packaging and processing using biochemical and genetic approaches in mammalian cells, frog oocytes and budding yeast. Our research is testing the idea that mRNA is made by a "factory" complex containing RNA pol II and RNA processing factors which contact it through a repetitive protein domain called the CTD. This model has changed the way we think about proteins which were once thought to operate independently but are now thought to be co-ordinated with one another in the nucleus. One important functional consequence of the integration between transcription and processing that we are beginning to uncover is that transcription factors can regulate the efficiency of RNA processing and conversely processing factors can potentially regulate transcription. This work may help elucidate how the transcription and processing of mRNAs are regulated under normal conditions and how they become mis-regulated in the disease state. Defects in splicing resulting of pre-mRNA's are responsible for a large fraction of all inherited diseases. The specific aims of this work are:1. To compare mechanisms of RNA processing and packaging that are coupled to pol II transcription with uncoupled processing and packaging of RNA in vivo.2. To determine how how capping enzymes regulate pol II transcription and how components of the pol II transcription machinery regulate capping.3. To determine how pol II elongation rate, CTD heptad sequences and the CTD C-terminus affect RNA packaging, capping, splicing and 3' end formation.4. To determine whether mammalian processing factors associate with elongating pol II ternary complexes in vivo and how the processing factor composition of ternary complexes changes during elongation?