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A characterization of the role of phosphorylation in regulating pre-mRNA splicing


Pre-mRNA splicing is a key process in gene regulation that involves the removal of noncoding sequences from nascent RNA transcripts to form a mature message that can be translated into a protein. The spliceosome is a large, dynamic macromolecular complex which is responsible for the identification and removal of these introns. To achieve its role in splicing, the Spliceosome undergoes a series of dynamic rearrangements involving protein-protein, protein-RNA, and RNA-RNA interactions. How these rearrangements are regulated is not well understood and may involve posttranslational modifications of the protein factors. The work presented in this thesis provides evidence that many splicing factors are in fact posttranslationally modified by phosphorylation, acetylation, ubiquitination, and glycosylation, although only a handful of these modifications have previously been described in detail. Our data indicate that phosphorylation of the yeast splicing factor Cus2 is important for its function in splicing. Specifically, we find that phosphorylation within a RNA binding domain (RRM) of Cus2 regulates its ability to associate with and mediate folding of its known substrate, the U2 snRNA. We discuss the potential implications of this finding, including the possibility that Cus2 is regulated in response to cellular conditions, including the cell cycle, in order to coordinate spicing with the events that occur during cell division. We further this investigation by exploring potential interactions between Cus2, its substrate U2 snRNA, and the Bur kinase complex. The mammalian homologs of these factors have been shown to physically interact in vitro to positively regulate transcription elongation. We investigated the possibility that a similar relationship exists between these factors in yeast. We were unable to find any evidence for a similar set of interactions in vivo or that the splicing factors have an obvious effect on transcription. Taken together, our data elucidate the role of posttranslational modifications in regulating Cus2's role in splicing and provide further insight into the connection between splicing and the transcription machinery

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