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. 2020 Aug 25;16(8):e1008893. doi: 10.1371/journal.pgen.1008893

Fig 5. The temperature sensitivity of S. pombe trm8Δ mutants is associated with induction of the GAAC pathway due to tY(GUA) decay.

Fig 5

(A) S. pombe trm8Δ mutants induced lys4+ mRNA expression at 38.5°C but not at 36.5°C or 37.5°C. Strains as indicated were grown in YES media at 30°C and shifted to 36.5°C, 37.5°C, or 38.5°C for 8 hours (S15 Fig), and bulk RNA was isolated and analyzed by RT-qPCR as described in Materials and Methods. The mRNA levels of lys4+ were normalized to levels of act1+, a non-regulated control mRNA. WT, green; Sp trm8Δ, brown; Sp trm8Δ gcn2-1, light blue. Right side: GAAC induction of WT cells grown at 30°C in EMMC-His and treated with 10 mM 3-AT for 4 hours, evaluated in parallel. (B) An S. pombe trm8Δ gcn2-1 mutant had restored levels of tY(GUA) and tP(AGG) at 38.5°C. Bulk RNA from the growth done for Fig 5A was used for the northern analysis. (C) Quantification of tY(GUA) and tP(AGG) levels in WT, trm8Δ, and trm8Δ gcn2-1 mutants at different temperatures. tRNA levels were quantified as described in Fig 2B. (D) tY(GUA) overproduction repressed the GAAC induction of trm8Δ mutants at 38.5°C. Strains as indicated with plasmids expressing tY(GUA) and/or tP(AGG) were grown in EMMC-Leu media at 30°C and shifted to 38.5°C for 8 hours, and then RNA was isolated and lys4+ mRNA levels were analyzed by RT-qPCR as described in Fig 5A. Right side: GAAC induction of WT cells grown at 30°C in EMMC-His media, and treated with 10 mM of 3-AT for 4 hours, evaluated in parallel to other samples.