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

Spontaneous pus1Δ suppressors carrying mutations in genes of the RTD pathway lead to restoration of growth and tRNA^Ile(UAU) levels in S. pombe pus1Δ strains. (A) Spontaneous suppressors carrying mutations in dhp1 or tol1 suppress the temperature-sensitive phenotype of S. pombe pus1Δ strains. Strains were cultivated in YES media at 30°C, serially diluted, and analyzed for growth on EMMC-His, YES, and YES + 5-FU media. Cells on EMMC-His are shown after 4 days of incubation, cells on YES and YES + 5-FU are shown after 6 days of incubation. (B) Spontaneous pus1Δ suppressors carrying mutations in dhp1 or tol1 restore tRNA^Ile(UAU) levels in S. pombe pus1Δ strains at 30°C and 38.5°C. Strains were grown in biological replicates in YES media at 30°C and shifted to 38.5°C for 6 h, and then RNA was isolated and analyzed by Northern blotting. The bar chart shows relative levels of tRNA species. Strains are analyzed at 30°C (dark shades) and 38.5°C (light shades). Levels of tRNA^Ile(UAU) and tRNA^Ala(AGC) are depicted 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 < 0.05, (**) for P < 0.01, (***) for P < 0.001, (****) for P < 0.0001.