COUPLING OF TRANSCRIPTION WITH 5 AND 3 MRNA PROCESSING

mRNA的产生需要pol II的转录和蛋白质的加工。 初级转录物由一组进行加帽、剪接 和切割/聚腺苷酸化。聚合酶在体内转录其他 比pol II不能正确加工成成熟mRNA。一种机制 显然存在pol II与特定RNA的转录偶联 处理负责mRNA成熟的事件。虽然 耦合机制是至关重要的，它如何的细节 作品几乎完全不为人知。这项建议的目的是 了解耦合机制如何工作。这项工作可能有助于阐明 mRNA的剪接、3'端加工和稳定性受 正常情况以及它们在疾病状态下如何被错误调节。 所采取的一种方法是比较 RNA聚合酶的不同突变形式。这种做法导致了 识别偶联中的关键参与者：保守的C-末端 结构域（CTD）的pol II大亚基。后续战略， 是为了确定CTD和蛋白质之间的蛋白质相互作用， RNA加工和体外因子，然后测试它们的功能 在体内的意义。 这项工作是基于这样的假设，即处理之间的相互作用 因素和CTD负责之间的功能耦合 转录和处理。根据这一想法，“mRNA工厂” 含有pol II和RNA加工因子的复合物可以同时进行 在细胞核中转录和加工。一系列的生化和 基因实验被提议来测试这个想法。重点是 CTD在mRNA分子的5'和3'末端加工中的作用。 这项工作的具体目标是： 1.为了测试切割/多聚腺苷酸化因子之间的关联， CstF，CTD是体内mRNA 3'加工所必需的。 2.为了确定CTD是否足够以及必要， 高效的5 ′和3 ′加工。 3.为了确定3'处理是否依赖于CTD， 遗传上可接受的酿酒酵母 4.表征CTD和封端之间的相互作用 酶，并确定这些相互作用在体内的功能。

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 mae in vivo by polymerases other than pol II are not processed correctly into mature mRNA. A mechanism clearly exists to couple transcription by pol II with the specific RNA processing events responsible for maturation of mRNA. Although the coupling mechanism is of fundamental importance, the details of how it works are almost completely unknown. The objective of this proposal is to lean how the coupling mechanism works. This work may help elucidate how the splicing, 3' processing, and stability of mRNAs are regulated under normal conditions and how they become mis-regulated in the disease state. One approach taken is to compare processing of transcripts made by different mutated forms of RNA polymerase. This approach led to the identification of a key player in coupling: the conserved C-terminal domain (CTD) of the pol II large subunit. A follow-up strategy that has been taken is to identify protein-protein interactions between the CTD and RNA processing and factors in vitro and then to test their functional significance in vivo. This work is based on the hypothesis that interactions between processing factors and the CTD are responsible for the functional coupling between transcription and processing. According to this idea, "mRNA factory" complexes containing pol II and RNA processing factors may carry out both transcription and processing in the nucleus. A series of biochemical and genetic experiments is proposed to test this idea. The focus is on the role of the CTD in processing of the 5' and 3' ends of the mRNA molecule. Specific aims of this work are: 1. To test if the association between the cleavage/polyadenylation factor, CstF, and the CTD is necessary for mRNA 3' processing in vivo. 2. To determine if the CTD is sufficient as well as necessary for efficient 5' and 3' processing. 3. To determine if 3' processing is dependent on the CTD in the genetically amenable organism Saccharomyces cerevisiae. 4. To characterize the interactions between the CTD and the capping enzymes and to determine the functions of these interactions in vivo.