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

S. pombe Pus1 pseudouridylates tRNA^Ile(UAU) at U₂₇, U₃₄, and U₃₆. (A) tRNA^Ile(UAU) purified from S. pombe pus1Δ strains has reduced Ψ and commensurately increased uridine and ncm⁵U, relative to WT strains. WT and pus1Δ strains were transformed with a [leu2⁺ tI(UAU)] plasmid, and transformants were grown in triplicate in EMMC-Leu media at 30°C to mid-log phase, and harvested. Then tRNA^Ile(UAU) was purified from bulk RNA and digested to nucleosides, which were analyzed by HPLC. Levels of nucleosides in WT and pus1Δ strains are shown 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 < 0.0001. (B) tRNA^Ile(UAU) purified from S. pombe pus1Δ strains lacks Ψ₂₇, Ψ₃₄, and Ψ₃₆, compared to WT strains. Purified tRNA was treated with CMCT and analyzed by primer extension, as described in Materials and Methods, with primer OFS049 (complementary to tRNA^Ile(UAU) nt 57–39). The primer extension stops in tRNA^Ile(UAU) from WT cells occur one residue 3′ of Ψ₂₇, Ψ₃₄, and Ψ₃₆, but are absent in tRNA^Ile(UAU) from pus1Δ strains, demonstrating the lack of Ψ₂₇, Ψ₃₄, and Ψ₃₆ in tRNA^Ile(UAU) from pus1Δ strains. A sequencing ladder is shown on the left. (C) Secondary structure of mature S. pombe tRNA^Ile(UAU). The anticodon is orange. (D) Secondary structure of intron-containing S. pombe tRNA^Ile(UAU). The anticodon is highlighted in orange, the intron is green.