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.
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