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Thymidylate synthase (TS) catalyzes the reductive methylation of dUMP by 5,10-methylenetetrahydrofolate to generate thymidylate and dihydrofolate. This enzymatic reaction provides for the sole intracellular de novo source of thymidylate, an essential precursor for DNA biosynthesis. As a result, TS remains a critical target enzyme in cancer chemotherapy (25, 60a).In addition to its role in enzyme catalysis, there is evidence that TS also functions as an RNA binding protein (5-7). Studies from this laboratory have demonstrated that the translation of human TS mRNA is regulated by its own protein product via a negative autoregulatory mechanism (5). The repression of TS mRNA translation by TS is mediated by specific binding of the protein to at least two distinct cis-acting sequences on its own mRNA (6). The first site corresponds to a 188-nucleotide (nt) sequence that includes the translational start site, while the second site is contained within a 100-nt sequence in the protein-coding region. However, in the presence of the nucleotide substrate dUMP or 5-fluoro-2Ј-deoxyuridine-5Ј-monophosphate (FdUMP), TS is unable to directly interact with its own mRNA, thus allowing for the synthesis of new protein to proceed (5, 7). Several in vitro studies have shown that shortterm exposure of human colon and breast cancer cells to TSinhibitory compounds, such as 5-fluorouracil (5-FU) or the antifolate analog ZD1694, is associated with an increased level of TS protein expression but no corresponding change in the levels of TS mRNA (8,9,35). These findings are consistent with earlier observations that both the RNA binding and the translational inhibition activities of TS are impaired in the presence of either nucleotide and/or folate substrates. Thus, the ability to regulate the expression of TS at the translational level in the setting of acute cytotoxic stress suggests that this regulatory event has biological relevance. Moreover, this process may represent an important mechanism by which normal cellular synthetic function can be controlled and a mechanism for the rapid development of cellular resistance in response to exposure to nucleotide inhibitors of TS, such as 5-FU, and antifolate inhibitors of TS, such as ZD1694, LY231514, and AG331.An immunoprecipitation-reverse transcription (RT)-PCR technique was recently developed to isolate from an intact cultured human colon cancer cell line a TS-ribonucleoprotein (RNP) complex made up of TS protein and TS mRNA (10). In addition to complexing with its own mRNA, TS formed an RNP complex with the mRNA of the c-myc transcription factor. Subsequent studies with RNA electrophoretic gel mobility shift assays (EMSAs) confirmed that the interaction between TS and c-myc mRNA was specific and identified the C-terminal coding region as being an important cis-acting regulatory element (11). Furthermore, in vitro translation experiments demonstrated that TS protein specifically repressed the translation of c-myc mRNA (11). Recent work has shown that TS is * Corresponding author. Mailing ad...

Using an immunoprecipitation-reverse transcription-PCR technique, we characterized a thymidylate synthase (TS) ribonucleoprotein complex in cultured human colon cancer cells that consists of TS protein and the mRNA of the nuclear oncogene c-myc. TS protein is complexed in intact cells with the C-terminal coding region of c-myc mRNA that includes nucleotide positions 1625 to 1790. RNA electrophoretic gel mobility shift assays confirm a specific interaction between TS protein and c-myc mRNA and provide additional evidence that the C-terminal coding region represents an important cis-acting regulatory element. Further evidence demonstrates that the in vitro translational efficiency of c-myc mRNA is inhibited as a result of its direct interaction with TS protein. In addition, the presence of exogenous c-myc mRNA specifically relieves the inhibitory effects of TS protein on TS mRNA translation.

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Previous studies have shown that the repressive effect of thymidylate synthase (TS) mRNA translation is mediated by direct binding of TS itself to two cis-acting elements on its cognate mRNA. To identify the optimal RNA nucleotides that interact with TS, we in vitro synthesized a completely degenerate, linear RNA pool of 25 nt and employed in vitro selection to isolate high affinity RNA ligands that bind human TS protein. After 10 rounds of selection and amplification, a single RNA molecule was selected that bound TS protein with nearly 20-fold greater affinity than native, wild-type TS RNA sequences. Secondary structure analysis of this RNA sequence predicted it to possess a stem-loop structure. Deletion and/or modification of the UGU loop element within the RNA sequence decreased binding to TS by up to 1000-fold. In vivo transfection experiments revealed that the presence of the selected RNA sequence resulted in a significant increase in the expression of a heterologous luciferase reporter construct in human colon cancer H630 and TS-overexpressing HCT-C:His-TS+ cells, but not in HCT-C18 cells expressing a functionally inactive TS. In addition, the presence of this element in H630 cells leads to induced expression of TS protein. An immunoprecipitation method using RT-PCR confirmed a direct interaction between human TS protein and the selected RNA sequence in transfected human cancer H630 cells. This study identified a novel RNA sequence from a degenerate RNA library that specifically interacts with TS.

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