Schizosaccharomyces pombe pus1 mutants are temperature sensitive due to decay of tRNAIle(UAU) by the 5'-3' exonuclease Dhp1, primarily targeting the unspliced pre-tRNA.

Abstract

The pseudouridylase Pus1 catalyzes pseudouridine (Ψ) formation at multiple uridine residues in tRNAs, and in some snRNAs and mRNAs. Although Pus1 is highly conserved, and mutations are associated with human disease, little is known about eukaryotic Pus1 biology. Here, we show that Schizosaccharomyces pombe pus1Δ mutants are temperature sensitive due to decay of tRNAIle(UAU), as tRNAIle(UAU) levels are reduced, and its overexpression suppresses the defect. We show that tRNAIle(UAU) is degraded by the 5'-3' exonuclease Dhp1 (ortholog of Saccharomyces cerevisiae Rat1), as each of four spontaneous pus1Δ suppressors had dhp1 mutations and restored tRNAIle(UAU) levels, and two suppressors that also restored tRNAIle(UAU) levels had mutations in tol1 (S. cerevisiae MET22 ortholog), predicted to inhibit Dhp1. We show that Pus1 modifies U27, U34, and U36 of tRNAIle(UAU), raising the question about how these modifications prevent decay. Our results suggests that Dhp1 targets unspliced pre-tRNAIle(UAU), as a pus1Δ strain in which the only copy of tRNAIle(UAU) has no intron (tI(UAU)-iΔ) is temperature resistant and undergoes no detectable decay, and the corresponding pus1Δ tI(UAU)-WT strain accumulates unspliced pre-tRNAIle(UAU). Moreover, the predicted exon-intron structure of pre-tRNAIle(UAU) differs from the canonical bulge-helix-loop structure compatible with tRNA splicing, and a pus1Δ tI(UAU)i-var strain with intron mutations predicted to improve exon-intron structure is temperature resistant and undergoes little decay. These results suggest that decay of tRNAIle(UAU) by Dhp1 in pus1Δ strains occurs at the level of unspliced pre-tRNAIle(UAU), implying a substantial role for one or more of the Ψ residues in stabilizing the pre-tRNA structure for splicing.