A number of antimetabolites (e.g. nucleoside analogs) have been shown to have a chemotherapeutic effect. Among these agents, 5-fluorouracil (FUra) is a widely used agent whose mechanism of action is still not clear. Numerous laboratories have demonstrated incorporation of FUra into RNA and this is considered to be one of its three major sites of action. The other two major effects are 1) conversion to the deoxyribonucleotide and inhibition of thymidylate synthetase and 2) conversion to the deoxyribonucleoside triphosphate and possible incorporation into DNA. This study will concentrate on the effects of FUra due to incorporation into RNA. Previously, the major RNA effect was considered to be inhibition of ribosomal RNA (rRNA) maturation. Our laboratory previously demonstrated that miscoding of messenger RNA (mRNA) appears to occur at concentrations of FUra where no effect on rRNA is evident. The study is based upon the preliminary observation that FUra causes an alteration in the properties of dihydrofolate reductase (DHFR) and several species of RNA. Specifically, we propose to study the following. 1) Further evaluate the effects of FUra incorporation into RNA on enzymes synthesized from the substituted mRNA (using DHFR and DHFR mRNA as a model enzyme and mRNA, respectively). 2) The effect of FUra on RNA processing (specifically DHFR RNA as a model) both in vivo and in vitro. 3) The effect of FUra on small nuclear RNA (using U1 RNA as a model). 4) rRNA. The relative importance of these four possible sites will be assessed. The results of this study will be able to provide means and models to study the RNA-mediated effects of other nucleoside analogs which are incorporated into RNA.
