Abstract
5-fluorouracil (5-FU) is a chemotherapeutic agent that has been extensively studied since its initial development in the 1950s. It has been suggested that the mechanism of action of 5-FU involves both DNA- and RNA-directed processes, but this has remained controversial. In this study, using a series of in vivo reporter constructs capable of measuring translational recoding, we demonstrate that cells exposed to 5-FU display a reduced capacity to engage in a variety of translational recoding events, including +1 programmed frameshifting (PRF) and −1 PRF. In addition, 5-FU-treated cells are much less accurate at stop codon recognition, resulting in a significant increase in stop codon-readthrough. Remarkably, while the efficiency of cap-dependent translation appears to be unaffected by 5-FU, 5-FU-treated cells display a decreased ability to initiate cap-independent translation. We further show that knockdown of thymidylate synthase, an enzyme believed to be at the center of 5-FU-induced DNA damage, has no effect on the observed alterations in translational recoding. On the other hand, ribosomal RNA (rRNA) pseudouridylation, which plays an important role in translational recoding, is significantly inhibited. Taken together, our results suggest that the observed effect of 5-FU on recoding is an RNA-directed effect. Our results are the first to show definitely and quantitatively that translational recoding is affected by exposure to 5-FU. Thus, it is possible that a substantial portion of 5-FU cytotoxicity might possibly be the result of alterations in translational recoding efficiency.
Highlights
Fluoropyrimidine 5-fluorouracil (5-FU) was initially developed in the 1950’s following the observation that rat hepatomas metabolized uracil more rapidly than other nucleotides, implicating uracil metabolism as a potential antimetabolic target [1]
5-FU, which enters cells through the same facilitated transport mechanism used by uracil, is intracellularly converted into several active metabolites, including fluorodeoxyuridine monophosphate (FdUMP), fluorodeoxyuridine triphosphate (FdUTP), and fluorouridine triphosphate (FUTP) [3,4]
We show that while 5-FU treatment did not affect the efficiency of cap-dependent translation, significant changes in the efficiency of various translational recoding events, including programmed frameshifting, nonsense suppression or stop-codon readthrough, and cap-independent translation, were observed
Summary
Fluoropyrimidine 5-fluorouracil (5-FU) was initially developed in the 1950’s following the observation that rat hepatomas metabolized uracil more rapidly than other nucleotides, implicating uracil metabolism as a potential antimetabolic target [1]. Whether 5-FU treatment of mammalian cells results in a reduction in translational accuracy has yet to be extensively investigated In this regard, translational accuracy has been primarily analyzed in vitro using either mRNAs purified from 5-FU treated cells or with in vitro transcribed mRNAs that are fully substituted with 5-FU. When in vitro transcribed TS mRNA, which is 100% substituted with 5-FU, was translated in rabbit reticulocyte lysate, fully functional TS was generated, and found to be indistinguishable from TS generated from an in vitro transcript synthesized with uridine [32] While these studies would suggest that translational accuracy is not affected in 5-FU treated mammalian cells, this relies on the assumption that only the mRNA template is responsible for accurate mRNA decoding. Our results provide novel insights into the mechanism of 5-FU cytotoxicity
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