Functional importance of Ψ(38) and Ψ(39) in distinct tRNAs, amplified for tRNA(Gln(UUG)) by unexpected temperature sensitivity of the s(2)U modification in yeast

The numerous modifications of tRNA play central roles in controlling tRNA structure and translation. Modifications in and around the anticodon loop often have critical roles in decoding mRNA and in maintaining its reading frame. Residues U(38) and U(39) in the anticodon stem–loop are frequently modified to pseudouridine (Ψ) by members of the widely conserved TruA/Pus3 family of pseudouridylases. We investigate here the cause of the temperature sensitivity of pus3Δ mutants of the yeast Saccharomyces cerevisiae and find that, although Ψ(38) or Ψ(39) is found on at least 19 characterized cytoplasmic tRNA species, the temperature sensitivity is primarily due to poor function of tRNA(Gln(UUG)), which normally has Ψ(38). Further investigation reveals that at elevated temperatures there are substantially reduced levels of the s(2)U moiety of mcm(5)s(2)U(34) of tRNA(Gln(UUG)) and the other two cytoplasmic species with mcm(5)s(2)U(34), that the reduced s(2)U levels occur in the parent strain BY4741 and in the widely used strain W303, and that reduced levels of the s(2)U moiety are detectable in BY4741 at temperatures as low as 33°C. Additional examination of the role of Ψ(38,39) provides evidence that Ψ(38) is important for function of tRNA(Gln(UUG)) at permissive temperature, and indicates that Ψ(39) is important for the function of tRNA(Trp(CCA)) in trm10Δ pus3Δ mutants and of tRNA(Leu(CAA)) as a UAG nonsense suppressor. These results provide evidence for important roles of both Ψ(38) and Ψ(39) in specific tRNAs, and establish that modification of the wobble position is subject to change under relatively mild growth conditions.