Identification of unique tRNA-specific effects of tRNA modifying enzymes on 5-fluorouracil toxicity in Saccharomyces cerevisiae
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Date
2025-05
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The Ohio State University
Abstract
tRNA acetyltransferase 1 (Tan1), methyltransferase 1 (Trm1), and pseudouridine synthase 1 (Pus1) are three highly conserved tRNA-modifying enzymes that lack phenotypes when deleted on their own in Saccharomyces cerevisiae under normal growth conditions. However, each of these S. cerevisiae knockout strains exhibits heightened sensitivity to stress induced by growth in the presence of the chemotherapeutic drug 5-fluorouracil (5FU), resulting in a growth defect. Since each of these enzymes modifies substrate tRNAs at locations outside of the anticodon loop, we hypothesized that destabilization of one or more hypomodified tRNAs in these deletion strains is the reason for increased 5FU cytotoxicity and that certain tRNA species may be more affected than others by the missing modifications. This hypothesis is consistent with the trend followed by the previously characterized 5FU sensitivity of m1G9 methyltransferase Trm10, which modifies a range of tRNAs in yeast but plays a biologically important role for one of these substrates, tRNATrp. Here, I show that in tan1Δ strains lacking the 12th position N-4-acetylcytidine (ac4C12) modification, individual overexpression of only two of the six hypomodified Tan1 substrates, tRNASer(CGA) and tRNASer(UGA), suppresses hypersensitivity to 5FU, indicating another example of a modification present in several tRNAs but only biologically essential for a subset of these species. Correspondingly, the same two tRNAs (tRNASer(CGA) and tRNASer(UGA)) also rescued growth hypersensitivity of the trm1Δ strain to 5FU, while the 16 other Trm1 substrate tRNAs for m22G26 dimethylation in S. cerevisiae did not. However, overexpressing any individual tRNA substrate did not suppress the toxicity of 5FU in pus1Δ strains lacking pseudouridylation by Pus1. To identify the quality control pathway encountered by hypomodified tRNASer(CGA) and tRNASer(UGA), an enzyme related to sensing several such pathways was deleted in combination with Tan1 and arrested the 5FU-induced growth defect. The effects of modification by Tan1 on tRNA stability were also examined with northern blotting, which revealed that these tRNAs are subject to a distinct mechanism of quality control from that which had previously been implicated after the loss of the m1G9 modification. Moreover, the specific genes related to decay of these hypomodified tRNAs remain undetermined, as well as how 5FU toxicity specifically impacts different small non-coding RNAs. Such studies have important implications for elucidating the biological impact of these highly conserved tRNA modifications.