IMPACT OF 5FU ON THE STRUCTURE OF THE U4/U6 COMPLEX

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DESCRIPTION: One of the largest gaps in our knowledge concerning how the widely used anticancer drug 5-fluorouracil (5-FU) disrupts the RNA mediated processes is to what extent protein-RNA interactions are perturbed when uridine (s) (Urd) in the protein recognition site(s) of the RNA are substituted with FUrd. In this application the hypothesis will be tested that FUrd substitution in the Urd-rich Sm binding site of U4 snRNA disrupts recognition of U4 snRNA by the snRNP core proteins (Sm proteins: B, D1, D2, D3, E, F, and G), a collection of basic proteins named for their relative mobilities during SDS-PAGE. Each of the spliceosomal snRNAs (except U6 snRNA) contains an Sm binding site (consensus sequence in S. cerevisae = AAUUUUUGG). Failure of U4 snRNA to bind to the Sm complex would reduce the efficiency of U4 snRNA nuclear transport and inhibit formation of the spliceosome. Recent studies measuring the biological effects of mutations in U4 snRNA indicate virtually no tolerance for nucleotide substitution in the Sm binding site, suggesting adoption of a rigid recognition code between the Sm protein complex and the Sm binding site. It will be determined if FUrd substitution at and near the Sm binding site of U4 snRNA disrupts the structure and stability of this region of U4 snRNA or if it affects complexation of U4 snRNA with Sm proteins. This will be done by: 1) determining the three dimensional structure for the 3' region of U4 snRNA from S. cerevisae using multidimensional NMR spectroscopy in conjunction with molecular modeling; 2) assessing the effects of FUrd substitution on the structure, stability, and dynamics of the 3' region of U4 snRNA using NMR spectroscopy, nuclease probing, and UV hyperchromicity studies; 3) demonstrating that single nucleotide mutations in the Sm binding site for U4 snRNA decrease the affinity of the RNA for the Sm protein complex by immunoblotting 32P- labeled U4 snRNA that has been incubated with yeast cytosolic extract with Y12, an antibody specific for Sm protein from S. cerevisae; and 4) demonstrating that FUrd substitution in the Sm binding site of U4 snRNA disrupts RNA-protein complex formation also using immunoblotting procedures. These studies will provide novel information concerning the native structure of the 3' region of U4 snRNA, the effects of native nucleotide substitution on Sm complex formation and the effects of FUrd substitution on the structure and stability of U4 snRNA and its complex with Sm proteins.