Translation of thymidylate synthase (TS) mRNA is controlled by its own protein product, TS, in an autoregulatory manner. Direct binding of TS protein to two different cis-acting elements on the TS mRNA is associated with this translational regulation. In this study, an immunoprecipitation-reverse transcription-PCR technique was used to identify a TS ribonucleoprotein (RNP) complex in cultured human colon cancer cells. Using antibodies specific for TS protein, we show that TS is complexed in vivo with its own TS RNA. Furthermore, evidence demonstrating a direct interaction between the mRNA of the nuclear oncogene c-myc and TS protein is presented.Recently, there has been an increased interest in the characterization of translational regulatory mechanisms. There are a number of eukaryotic mRNAs whose expression is controlled at the level of translation (18). The regulated synthesis of the iron storage protein ferritin by iron represents one of the best-studied examples of this form of regulation (25). A stem-loop structure located within the 5' untranslated region (UTR) of ferritin mRNA, termed the iron-responsive element (IRE), represents the cis-acting element to which the IRE-binding protein binds (16,23). Recent studies have demonstrated that the redox state in the cell is an important determinant of the binding affinity of this protein to the IRE (15,17).Using an in vitro translation system, we showed that translation of human thymidylate synthase (TS) mRNA is regulated by its own protein product, TS, in a negative autoregulatory manner (7). Although translational autoregulation has been described in prokaryotic systems (2, 5), TS mRNA represents the first eukaryotic mRNA whose regulation is controlled in such a fashion. Furthermore, we demonstrated that incubation of TS protein with either the nucleotide substrate dUMP or the inhibitor 5-fluoro-dUMP repressed its inhibitory effect on TS mRNA translation. during the cell cycle is primarily regulated at the transcriptional level (1,19,30), there is now recent evidence suggesting control at the level of translation (22). These findings, taken together, offer supportive evidence for the model of TS translational autoregulation.The purpose of the present study was to identify a TS ribonucleoprotein (RNP) complex in a cultured cell system. With the use of specific antisera to TS, we show that TS protein is complexed with its corresponding TS RNA in human colon cancer cells. In addition, we present evidence demonstrating a specific interaction between the mRNA of the nuclear oncogene c-myc and TS protein.
MATERIALS AND METHODSCell culture. The characteristics of the human colon cancer cell line H630 have been previously described (32). The resistant H630-R1O subline was selected in vitro for resistance to 5-FU by exposure of the parent H630 cell line to stepwise increases in 5-FU and was maintained in medium containing 10 p,. Cell lines were grown in 75-cm2 plastic tissue culture flasks (Falcon Labware, Oxnard, Calif.) in growth medium containing RPMI 1640 with 1...