Fig 1. S. pombe trm6Δ mutants and trm61Δ mutants are temperature sensitive and lack m¹A₅₈.

(A) S. pombe trm6Δ and trm61Δ mutants are temperature sensitive on YES and EMMC-his media. S. pombe trm6Δ mutants, trm61Δ mutants, and WT cells were grown overnight in YES media at 30°C, diluted to OD₆₀₀ ~0.5, serially diluted 10-fold in YES media, and then 2 μL were spotted onto plates containing YES or EMMC-his media and incubated at the indicated temperatures for 3 days. (B) tRNA^Tyr(GUA) from S. pombe trm6Δ and trm61Δ mutants has no detectable m¹A, as measured by HPLC separation of nucleosides. S. pombe trm6Δ mutants, trm61Δ mutants, and WT cells were grown in biological triplicate in YES media at 30°C and tRNA^Tyr(GUA) was purified, and digested to nucleosides, and then nucleosides were separated by HPLC as described in Materials and Methods. (C) Quantification of levels of modified nucleosides of purified tRNA^Tyr(GUA) in S. pombe trm6Δ, trm61Δ, and WT strains. The chart shows average moles/mol of nucleosides with associated standard deviations; WT, blue; trm6Δ, orange; trm61Δ, gray. (D) tRNA^Tyr(GUA) and tRNA_i^Met(CAU) from S. pombe trm6Δ and trm61Δ mutants have little or no detectable m¹A₅₈. Bulk RNA from the growth for Fig 1B was analyzed by poison primer extension assay, as described in Materials and Methods, with primer OMT 775 (complementary to tRNA_i^Met(CAU) nt 76–61) and primer OMT 477 (complementary to tRNA^Tyr(GUA) 76–61) in the presence of ddGTP. The poison primer extension produces a stop at C₅₆ for both tRNA_i^Met(CAU) and tRNA^Tyr(GUA), and the presence of m¹A₅₈ results in a stop at N₅₉. A sequencing ladder is shown at the left. (E) Quantification of poison primer extension of tRNA^Tyr(GUA) and tRNA_i^Met(CAU). For each primer extension, the signals at N₅₉ and C₅₆ were first corrected by subtraction of the signals at A₅₈ and N₅₇ respectively.