Coupling of Transcription with Pre-mRNA Metabolism

描述(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PolII转录，并由一系列蛋白质处理初级转录物，这些蛋白质进行封端、剪接和切割/多聚腺苷化。由POLII以外的聚合酶产生的转录本不能正确地加工成成熟的mRNA。蛋白质网络：在细胞核中存在蛋白质接触，将PolII的转录与负责mRNA成熟的特定RNA处理事件结合在一起。该建议的目的是阐明在哺乳动物细胞、青蛙卵母细胞和发芽酵母中，蛋白质如何通过通讯实现PolII转录与RNA包装和处理的耦合，使用生化和遗传方法。我们的研究正在测试这样一种想法，即mRNA是由包含RNA PolII和RNA加工因子的“工厂”复合体组成的，这些复合体通过一个称为CTD的重复蛋白结构域与其联系。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行的，但现在被认为是在细胞核中相互协调的。我们开始发现转录和加工之间整合的一个重要功能结果是，转录因子可以调节RNA加工的效率，反过来，加工因子也可以潜在地调节转录。这项工作可能有助于阐明mRNAs的转录和处理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变得错误调节的。在所有遗传性疾病中，很大一部分是由前信使核糖核酸的剪接缺陷引起的。本工作的具体目的是：1.比较PolII转录偶联的RNA加工和包装机制与活体RNA的非偶联加工和包装。确定封端酶如何调控PolII转录，以及PolII转录机制的组成部分如何调控封端。为了确定PolII的延伸率、CTD七肽序列和CTD C末端如何影响RNA的包装、封端、剪接和3‘端的形成。为了确定哺乳动物的加工因素是否与体内Pol II三元复合体的延长有关，以及三元复合体的加工因子组成在延长过程中如何变化？

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?