Abstract
Thymidylate synthase (TS), which catalyzes the de novo synthesis of dUMP, is an important target for cancer therapy. In this report, the effects of 5-fluorouracil (5-FU) and ZD1694 on the regulation of TS gene expression were evaluated in zebrafish embryos. Our results revealed that the expression of TS was increased by about six-fold when embryos were treated with 1.0 µM 5-FU and there was a greater than 10-fold increase in the TS protein level after treatment with 0.4 µM ZD1694. Northern blot analysis confirmed that expression of TS mRNA was identical in treated or untreated embryos. Gel shift and immunoprecipitation assays revealed that zebrafish TS was specifically bound with its cognate mRNA in vitro and in vivo. We identified a 20 nt RNA sequence, TS:N20, localized to the 5′-UTR of TS mRNA, which corresponded to nt 13–32; TS:N20 bound to the TS protein with an affinity similar to that of the full-length TS mRNA. The MFold program predicted that TS:N20 formed a stable stem-loop structure similar to that of the cis-acting element found in human TS mRNA. Variant RNAs with either a deletion or mutation in the core motif of TS:N20 were unable to bind to the TS protein. In vitro translation experiments, using the rabbit lysate system, confirmed that zebrafish TS mRNA translation was significantly repressed when an excess amount of TS protein was included in the system. Additionally, a TS stability experiment confirmed that treatment of zebrafish embryos with 5-FU could increase the TS stability significantly, and the half life of TS protein was about 2.7 times longer than in untreated embryos. Our study revealed a structural requirement for the interaction of TS RNA with TS protein. These findings also demonstrated that the increase in TS protein induced by 5-FU occurs at the post-transcriptional level and that increased stability and translation efficiency both contributed to the increase in TS protein levels induced by TS inhibitors.
Highlights
Thymidylate synthase (TS) is a folate-dependent enzyme that catalyzes the reductive methylation of dUMP to dTMP using 5, 10-methylenetetrahydrofolate as a one-carbon donor
We demonstrated for the first time that, after short-term exposure of zebrafish to 5-FU or ZD1694, TS enzyme levels were elevated significantly
We found that human TS could bind to its cognate mRNA and repress the translation of TS mRNA
Summary
Thymidylate synthase (TS) is a folate-dependent enzyme that catalyzes the reductive methylation of dUMP to dTMP using 5, 10-methylenetetrahydrofolate as a one-carbon donor. A number of studies using cultured cell lines, tumor models and clinical specimens have shown that TS inhibitors induce about a 2- to 4-fold increase in TS levels [3,4]. Such an induction has been viewed as a potential barrier to successful therapeutic outcomes because the response to TS-directed chemotherapy is dependent on the enzyme concentration [5]. When 5-fluorouracil (5-FU) or other TS inhibitors bind to TS, the complex cannot interact with its cognate mRNA, which results in increased TS protein expression [10]
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