Impact of Pus1 Pseudouridine Synthase on Specific Decoding Events in .

Pus1-dependent pseudouridylation occurs in many tRNAs and at multiple positions, yet the functional impact of this modification is incompletely understood. We analyzed the consequences of deletion on the essential decoding of CAG (Gln) codons by tRNACUG in yeast. Synthetic lethality was observed upon combining the modification defect with destabilized variants of tRNACUG, pointing to a severe CAG-decoding defect of the hypomodified tRNA. In addition, we demonstrated that misreading of UAG stop codons by a tRNACUG variant is positively affected by Pus1. Genetic approaches further indicated that mildly elevated temperature decreases the decoding efficiency of CAG and UAG via destabilized tRNACAG variants. We also determined the misreading of CGC (Arg) codons by tRNAGUG, where the CGC decoder tRNAICG contains Pus1-dependent pseudouridine, but not the mistranslating tRNA. We found that the absence of Pus1 increased CGC misreading by tRNA, demonstrating a positive role of the modification in the competition against non-synonymous near-cognate tRNA. Part of the in vivo decoding defects and phenotypes in mutants and strains carrying destabilized tRNACAG were suppressible by additional deletion of the rapid tRNA decay (RTD)-relevant , suggesting the involvement of RTD-mediated tRNA destabilization.