Gene expression is regulated in metazoans at both the transcriptional and post-transcriptional levels. This proposal is focused on the mechanisms of the regulation of pre-mRNA splicing, a post-transcription step essential for the production of functional messenger RNAs for protein synthesis. RNA splicing is a complex biochemical process that can be divided into three stages. The first is the recognition of functional splice sites, followed by a stage for spliceosome assembly and maturation. The final stage is the chemistr of cleavage to remove intron sequences and ligation to bring exons together. The proposed studies focus on the early stages of splice site recognition and spliceosome assembly, where most regulation may take place. The experiments, largely based on Dr. Fu's previous results, are divided into three specific aims.
The first aim i s to investigate how a family of splicing factors called SR proteins constitute a critical activity in committing specific pre-mRNAs to the splicing pathway in the mammalian system. Two SR proteins, SC35 and SF2/ASF, which have been shown to specifically commit human IgM and HIV tat pre-mRNA to splicing, respectively, will be studied. These studies are intende to test the hypothesis that SR proteins may cooperate with other splicing factors to form specific and functional splicing enhancer complexes at their binding sites. The second specific aim is to determine the function of a sequence specific RNA binding protein in splicing. PI's previous studies indicate that a splicing enhancer consisting of GAA repeats is not a binding site for any SR proteins tested. Rather, the repeats seem to be recognized by protein complex which includes a 37 kD GAA binding protein. This model system will be used to determine whether splicing commitment can be mediated by other sequence specific RNA binding proteins. The last aim is to examine how communication is established among 5' and 3' splice sites and splicing enhance complexes. The plan is to focus on a U2AF35-related protein, which has been implicated in playing a role in various bridging interactions. Together, the proposed experiments represents a step-wise approach to understand the mechanisms of splice site recognition and spliceosome assembly.
Showing the most recent 10 out of 65 publications
