Dynamic modulation of Dnmt2-dependent tRNA methylation by the micronutrient queuine.

Dnmt2 enzymes are cytosine-5 methyltransferases that methylate C38 of several tRNAs. We report here that the activities of two Dnmt2 homologs, Pmt1 from Schizosaccharomyces pombe and DnmA from Dictyostelium discoideum, are strongly stimulated by prior queuosine (Q) modification of the substrate tRNA. In vivo tRNA methylation levels were stimulated by growth of cells in queuine-containing medium; in vitro Pmt1 activity was enhanced on Q-containing RNA; and queuine-stimulated in vivo methylation was abrogated by the absence of the enzyme that inserts queuine into tRNA, eukaryotic tRNA-guanine transglycosylase. Global analysis of tRNA methylation in S. pombe showed a striking selectivity of Pmt1 for tRNA(Asp) methylation, which distinguishes Pmt1 from other Dnmt2 homologs. The present analysis also revealed a novel Pmt1- and Q-independent tRNA methylation site in S. pombe, C34 of tRNA(Pro). Notably, queuine is a micronutrient that is scavenged by higher eukaryotes from the diet and gut microflora. This work therefore reveals an unanticipated route by which the environment can modulate tRNA modification in an organism.

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 (mcm5s2U). While mutations in subunits of the Elongator complex (Elp1-Elp6), which disable mcm5 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 mcm5s2U 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 (mcm5U and s2U) 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 mcm5s2U 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 mcm5 and s2 wobble uridine modification that could account for growth impairment and mainly originates from tRNA(Lys)UUU hypomodification and malfunction.

Urmylation and tRNA thiolation functions of ubiquitin-like Uba4·Urm1 systems are conserved from yeast to man.

The ubiquitin-like protein Urm1 from budding yeast and its E1-like activator Uba4 have dual roles in protein urmylation and tRNA thiolation pathways. To study whether these are conserved among eukaryotes, we used gene shuffles to replace the yeast proteins by their human counterparts, hURM1 and hUBA4/MOCS3. As judged from biochemical and genetical assays, hURM1 and hUBA4 are functional in yeast, albeit at reduced efficiencies. They mediate urmylation of the peroxiredoxin Ahp1, a known urmylation target in yeast, and support tRNA thiolation. Similar to hUBA4, yeast Uba4 itself is modified by Urm1 and hURM1 suggesting target overlap between eukaryal urmylation pathways. In sum, our study shows that dual-function ubiquitin-like Urm1·Uba4 systems are conserved and exchangeable between human and yeast cells.

Phosphorylation of Elp1 by Hrr25 is required for elongator-dependent tRNA modification in yeast.

Elongator is a conserved protein complex comprising six different polypeptides that has been ascribed a wide range of functions, but which is now known to be required for modification of uridine residues in the wobble position of a subset of tRNAs in yeast, plants, worms and mammals. In previous work, we showed that Elongator's largest subunit (Elp1; also known as Iki3) was phosphorylated and implicated the yeast casein kinase I Hrr25 in Elongator function. Here we report identification of nine in vivo phosphorylation sites within Elp1 and show that four of these, clustered close to the Elp1 C-terminus and adjacent to a region that binds tRNA, are important for Elongator's tRNA modification function. Hrr25 protein kinase directly modifies Elp1 on two sites (Ser-1198 and Ser-1202) and through analyzing non-phosphorylatable (alanine) and acidic, phosphomimic substitutions at Ser-1198, Ser-1202 and Ser-1209, we provide evidence that phosphorylation plays a positive role in the tRNA modification function of Elongator and may regulate the interaction of Elongator both with its accessory protein Kti12 and with Hrr25 kinase.