The spliceosome is a complex and dynamic collection of RNA and proteins that removes introns from precursor mRNA transcripts. Alterations in the splicing machinery are associated with a diverse set of human diseases, ranging from cancer to retinitis pigmentosa. Insight into the mechanisms by which splicing leads to these pathological states requires an understanding of the functions of individual components within the spliceosome. In this proposal are plans to elucidate the role of the cyclophilin class of peptdyl-prolyl isomerases in splicing. Cyclophilins are highly conserved proteins and the target of the drug cyclosporin but their physiological functions remain enigmatic. I have solved the structures of several cyclophilins to atomic resolution as part of a structural genomics initiative and generated large numbers of soluble protein expression constructs. I also characterized these proteins in terms of their catalytic activities in solution and hypothesized potential substrate specificity based on in silico modeling. However, in vivo substrates for this enzyme class are not defined, making validation of my previous results difficult. Based on the finding that several nuclear cyclophilins are enriched in purified human splicing complexes, it is likely that the targets of spliceosomal cyclophilins will provide a great deal of information concerning cyclophilin:substrate specificity. Additionally, the sheer number of the cyclophilin family members found within the spliceosomal machinery and their unique distribution throughout splicing complexes indicate that these proteins are likely to be crucial for proper splicing activity. In order to test this hypothesis I will first be trained in the use of an in vitro splicing assay optimized in the Jurica lab to reconstitute spliceosomes with recombinant versions of potential splicing factors. I have already begun to use this assay to test for splicing activity in the presence of the cyclophilin PPIE, and can show that indeed this protein is necessary for proper splicing function. Next I will be trained in the Jurica lab in follow-up studies designed to find the stage of splicing at which PPIE exerts its effect, and learn how to purify spliceosomal complexes for use in mass spectrometric analysis. These studies will provide the first insight into the functional importance of the individual spliceosome-associated cyclophilins in pre-mRNA splicing and reveal the stage of spliceosome assembly that they target. After mastering these techniques I will carry on my work in spliceosomal cyclophilins in my own lab, where I will isolate individual components of the spliceosome found to associate with cyclophilins. I will then perform biophysical assays on the cyclophilin and protein of interest, and direct my efforts to solving complex structures of these protein complexes utilizing x-ray crystallography. The results of these structure/function studies of spliceosomal cyclophilins will expand our understanding of splicing mechanism to include the roles of cyclophilin specific protein:protein interactions and proline isomerization within the spliceosome.
Alterations in the splicing machinery are associated with a diverse set of human diseases ranging from cancer to retinitis pigmentosa. Insight into the mechanisms by which splicing leads to these pathological states requires an understanding of the functions of individual components within the spliceosome. This work will serve as a platform for developing small molecule reagents or protein mutants that specifically target key spliceosome components. These reagents will be used to elucidate the spliceosome's involvement in disease pathologies.