Fig 2. S. pombe trm6Δ temperature sensitivity is associated with reduced tRNA_i^Met(CAU) levels.

(A) Northern analysis of tRNAs in S. pombe trm6Δ and WT cells before and after shift from 30°C to 38.5°C. Strains were grown in YES media at 30°C, shifted to 38.5°C for 9 hours as described in Materials and Methods, and RNA was isolated at the indicated times, and analyzed by northern blotting, with probes as indicated. tMi(CAU), tRNA_i^Met(CAU); tF(GAA), tRNA^Phe(GAA); tP(AGG), tRNA^{Pro (AGG)}; tA(AGC), tRNA^Ala(AGC); tG(GCC), tRNA^Gly(GCC). (B) Quantification of tRNA levels in S. pombe trm6Δ and WT cells at 30°C and 38.5°C. The bar chart depicts relative levels of tRNA species at each temperature, relative to their levels in the WT strain at 30°C (each itself first normalized to levels of the control tG(GCC)). For each tRNA, the dark shade indicates 30°C, and progressively lighter shades indicate time points (3, 6, 9 hours) Standard deviations for each tRNA measurement are indicated. The statistical significance of tRNA levels was evaluated using a two-tailed Student’s t-test assuming equal variance. ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p<0.0001. tMi(CAU), blue; tF(GAA), green; tP(AGG), orange; tA(AGC), yellow. (C) Overproduction of tRNA_i^Met(CAU) suppresses the ts growth defects of S. pombe trm6Δ mutants. Strains with plasmids as indicated were grown overnight in EMMC-Leu media at 30°C and analyzed for growth as in Fig 1A on indicated plates and temperatures. (D) Overproduction of tRNA_i^Met(CAU) restores tRNA_i^Met(CAU) levels in S. pombe trm6Δ mutants. Strains containing plasmids as indicated were grown in EMMC-Leu at 30°C and shifted to 38.5°C for 8 hours as described in Materials and Methods, and RNA from cells grown at 38.5°C was isolated and analyzed by northern blotting as in Fig 2A. (E) Quantification of tRNA levels in S. pombe trm6Δ mutants and WT strains overproducing tRNA_i^Met(CAU). tRNA levels were quantified as in Fig 2B.