Schizosaccharomyces pombe pus1 mutants are temperature sensitive due to decay of tRNA^Ile(UAU) by the 5′-3′ exonuclease Dhp1, primarily targeting the unspliced pre-tRNA

S. pombe pus1Δ strains are temperature sensitive and lack Ψ at anticipated sites in tRNAs. (A) S. pombe pus1Δ strains are temperature sensitive and are complemented by overexpression of pus1⁺. Independent [pus1⁺ leu2⁺] or [leu2⁺] transformants of WT and pus1Δ cells were grown overnight in EMMC-Leu media at 30°C. Then cells were diluted to an OD₆₀₀ of 0.5, serially diluted 10-fold, and spotted for growth analysis at the indicated temperatures on EMMC-Leu and YES media for 4 days. (B) tRNA^Lys(CUU) purified from S. pombe pus1Δ strains has reduced Ψ and commensurately increased uridine, relative to that from WT strains. WT and pus1Δ strains were grown in biological triplicates, and tRNA^Lys(CUU) was purified as described in Materials and Methods. Then RNA was digested to nucleosides, which were analyzed by HPLC as described in Materials and Methods. Levels of nucleosides in WT and pus1Δ strains are shown in blue and green, respectively. Standard deviations are indicated. The statistical significance was evaluated using a one-tailed Student's t-test assuming equal variance, indicated by (****) for P < 0.0001.