The long-term goal of this project is to investigate the translational regulation of cellular gene expression. As his model system, the applicant will elucidate the translational regulation of thymidylate synthase (TS) expression, as TS is a critical target in cancer chemotherapy. This enzyme catalyzes the folate-dependent reductive methylation reaction which provides for the sole intracellular de novo source of thymidylate, a key precursor for DNA biosynthesis, and thus, TS plays a central role in maintaining the metabolic requirements of the cell. Studies from the applicant's laboratory have shown that TS also functions as an RNA binding protein in which it binds with high affinity (1-3 nM) to two different sites on its own TS mRNA. One site is located in the 5'-untranslated region and the second site in the protein-coding region. Binding of TS to either element results in translational repression of TS mRNA and inhibition of synthesis of TS protein. The model of TS translational autoregulation appears to have biological relevance in that it offers a rational mechanism for the tight control of TS expression within a given cell. However, treatment of TS protein with inhibitor compounds such as FdUMP or the antifolate inhibitor D1694 alters the synthesis of new TS protein. Disruption of this regulatory process would appear to provide an efficient adaptive mechanism for malignant cells to protect themselves in response to exposure to cytotoxic stress and thereby result in the development of cellular resistance. To further our understanding of the molecular elements underlying the translational regulation of TS, three specific aims are proposed in this application: (1) Characterize in further detail the critical cis-acting elements on TS mRNA that are required for the TS mRNA-TS protein for protein recognition of both the 5'-upstream and protein-coding region binding sites; (2) Characterize in further detail the critical domain or domains on the TS protein as well as identify the specific amino acid contact points directly involved in RNA binding; and (3) Characterize the intracellular localization of the TS protein-TS mRNA ribonucleoprotein complex. These studies will provide enhanced insight into the molecular elements mediating the interaction between TS protein and its cognate TS mRNA. Moreover, these molecular-based studies may provide the rational basis for the development of novel therapeutic strategies for human cancer.
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