AbstractEfficient protein synthesis in eukaryotes requires diphthamide modification of translation elongation factor eEF2 and wobble uridine modifications of tRNAs. In higher eukaryotes, these processes are important for preventing neurological and developmental defects and cancer. In this study, we usedSchizosaccharomyces pombeas a model to analyse mutants defective in eEF2 modification (dph1Δ), in tRNA modifications (elp3Δ), or both (dph3Δ) for sensitivity to cytotoxic agents and thermal stress. Thedph3Δandelp3Δmutants were sensitive to a range of drugs and had growth defects at low temperature.dph3Δwas epistatic withdph1Δfor sensitivity to hydroxyurea and methyl methanesulfonate, and withelp3Δfor methyl methanesulfonate and growth at 16 °C. Thedph1Δanddph3Δdeletions rescued growth defects ofelp3Δin response to thiabendazole and at 37 °C. Elevated tRNALysUUUlevels suppressed theelp3Δphenotypes and some of thedph3Δphenotypes, indicating that lack of tRNALysUUUmodifications were responsible. Furthermore, we found positive genetic interactions ofelp3Δanddph3Δwithsty1Δandatf1Δ, indicating that Elp3/Dph3-dependent tRNA modifications are important for efficient biosynthesis of key factors required for accurate responses to cytotoxic stress conditions.