S. pombe pus1Δ strains are temperature sensitive due to reduced levels of tRNAIle(UAU). (A) S. pombe pus1Δ strains have reduced levels of tRNAIle(UAU) at 30°C, which are further reduced at 38.5°C, relative to levels in WT cells. S. pombe WT and pus1Δ strains were grown in biological triplicate in YES media at 30°C and shifted to 38.5°C. Bulk RNA was prepared from cells at time points as indicated and analyzed by northern blotting with probes as shown. (B) Quantification of tRNA levels in S. pombe WT and pus1Δ strains at 30°C and 38.5°C. The bar chart shows relative levels of tRNA species, each normalized to levels of the control tRNAGly(GCC) followed by normalization to the normalized level in the WT strain at 30°C. Levels of tRNAIle(UAU), tRNAIle(AAU), and tRNAiMet(CAU) are depicted in blue, green, and yellow, respectively, with dark shades at 30°C, and progressively lighter shades after 3 and 6 h at 38.5°C for WT cells, and after 0.5, 1.5, 3, and 6 h for pus1Δ mutants, as indicated. Standard deviations are shown. (C) Overexpression of tRNAIle(UAU) suppresses the temperature-sensitive growth defects of S. pombe pus1Δ strains. WT and pus1Δ cells were transformed with [leu2+], [pus1+ leu2+], or [tI(UAU) leu2+] as indicated. Independent transformants were grown overnight in EMMC-Leu media at 30°C, and cells were diluted and spotted as described in Figure 1A on EMMC-Leu and YES media, and incubated for 3 days at the indicated temperatures.
