Molecular Regulation of Translational Regulation

DESCRIPTION (provided by applicant): The long-term goal of this project is to characterize the regulation of gene expression at the translational level. As our model system, we will investigate the regulation of expression of dihydrofolate reductase (DHFR), a critical target in cancer chemotherapy. This enzyme catalyzes the NADPH-dependent reductive reaction, which gives rise to the reduced folate, tetrahydrofolate, a key intermediate in one-carbon transfer reactions. DHFR is, therefore, required for the de novo synthesis of purines and pyrimidines as well as for the synthesis of certain amino acids. Thus, DHFR plays a central role in maintaining the metabolic requirements of the cell. Previous studies from this lab have shown that in addition to its role in catalysis and cellular metabolism, DHFR also functions as an RNA binding protein. This protein binds with high affinity (5-6 nM) to its own DHFR mRNA, an interaction that results in the translational repression of DHFR mRNA with subsequent inhibition of synthesis of new DHFR protein. These studies demonstrate that the expression of DHFR is controlled at least, in part, by a translational autoregulatory feedback mechanism. This model of DHFR translational autoregulation has biological relevance in that it offers a rational mechanism for the tight control of DHFR expression within a given cell. However, treatment of DHFR protein with inhibitor compounds such as the antifolate analog, methotrexate (MTX), alters the normal DHFR protein-DHFR mRNA interaction, resulting in an enhanced translational efficiency of DHFR mRNA with an increased synthesis of new DHFR protein. Disruption of this normal regulatory process provides an efficient mechanism for malignant cells to protect themselves in response to exposure to cytotoxic stress and develop cellular drug resistance. To further our understanding of the molecular elements underlying the translational regulation of DHFR, three specific aims are proposed in this project: (1) Characterize the critical cis-acting elements on DHFR mRNA that are required for this RNA-protein interaction. In this aim, we propose to determine the critical nucleotide sequences and/or secondary structures required for protein binding; (2) Characterize the critical trans-acting elements on the DHFR protein that are necessary for RNA recognition. As part of this aim, we plan to identify the domain or domains on the DHFR protein as well as the critical amino acid contact points that mediate the process of RNA binding; and (3) Identify the cellular RNAs that interact with DHFR protein. In this aim, we propose to identify the cellular mRNAs in addition to DHFR mRNA whose expression and/or function may be under the control of human DHFR protein.