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：蛋白质接触的网络，以将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，但现在被认为是在细胞核中相互协调的。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.确定哺乳动物加帽、剪接和切割多聚腺苷酸化因子如何与体内基因上的pol II延伸复合物相关。2.确定内含子共转录剪切的程度以及影响共转录剪切效率的因素。3.通过确定有助于共转录前mRNA加工、mRNA输出和hnRNP包装的CTD结构特征来检验CTD编码假说。

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 determine how mammalian capping, splicing and cleavage polyadenylation factors associate with pol II elongation complexes on genes in vivo. 2. To determine the extent to which introns are excised co-transcriptionally and what factors affect the efficiency of co-transcriptional splicing. 3. To test the CTD code hypothesis by determining the structural features of the CTD that contribute to co- transcriptional pre-mRNA processing, mRNA export and hnRNP packaging.