Loss of Anticodon Wobble Uridine Modifications Affects tRNA(Lys) Function and Protein Levels in Saccharomyces cerevisiae

In eukaryotes, wobble uridines in the anticodons of tRNA(Lys) (UUU), tRNA(Glu) (UUC) and tRNA(Gln) (UUG) are modified to 5-methoxy-carbonyl-methyl-2-thio-uridine (mcm(5)s(2)U). While mutations in subunits of the Elongator complex (Elp1-Elp6), which disable mcm(5) side chain formation, or removal of components of the thiolation pathway (Ncs2/Ncs6, Urm1, Uba4) are individually tolerated, the combination of both modification defects has been reported to have lethal effects on Saccharomyces cerevisiae. Contrary to such absolute requirement of mcm(5)s(2)U for viability, we demonstrate here that in the S. cerevisiae S288C-derived background, both pathways can be simultaneously inactivated, resulting in combined loss of tRNA anticodon modifications (mcm(5)U and s(2)U) without a lethal effect. However, an elp3 disruption strain displays synthetic sick interaction and synergistic temperature sensitivity when combined with either uba4 or urm1 mutations, suggesting major translational defects in the absence of mcm(5)s(2)U modifications. Consistent with this notion, we find cellular protein levels drastically decreased in an elp3uba4 double mutant and show that this effect as well as growth phenotypes can be partially rescued by excess of tRNA(Lys) (UUU). These results may indicate a global translational or protein homeostasis defect in cells simultaneously lacking mcm(5) and s(2) wobble uridine modification that could account for growth impairment and mainly originates from tRNA(Lys) (UUU) hypomodification and malfunction.