The conserved Wobble uridine tRNA thiolase Ctu1–Ctu2 is required to maintain genome integrity

Abstract

Modified nucleosides close to the anticodon are important for the proper decoding of mRNA by the ribosome. Particularly, the uridine at the first anticodon position (U34) of glutamate, lysine, and glutamine tRNAs is universally thiolated (S 2 U34), which is proposed to be crucial for both restriction of wobble in the corresponding split codon box and efficient codon-anticodon interaction. Here we show that the highly conserved complex Ctu1-Ctu2 (cytosolic thiouridylase) is responsible for the 2-thiolation of cytosolic tRNAs in the nematode and fission yeast. In both species, inactivation of the complex leads to loss of thiolation on tRNAs and to a thermosensitive decrease of viability associated with marked ploidy abnormalities and aberrant development. Increased level of the corresponding tRNAs suppresses the fission yeast defects, and our data suggest that these defects could result from both misreading and frame shifting during translation. Thus, a translation defect due to unmodified tRNAs results in severe genome instability.thiolation ͉ yeast

Role of the ubiquitin-like protein Urm1 as a noncanonical lysine-directed protein modifier

“…The role of thiomodification of tRNAs remains unclear. Thiolation of tRNAs may stabilize codon-anticodon interactions on the ribosome to improve translational efficiency (17,32,33). However, whether the Urm1 pathway contributes to efficient translation of endogenous proteins has not been determined.…”

“…Next, MOCS3 adenylates the C terminus of Urm1, followed by the transfer of sulfur to the terminal glycine of Urm1 (15). Urm1 associates with the thiouridylases ATPBD3 (also known as CTU1 in Homo sapiens and Ncs6p in S. cerevisiae) and CTU2 (Ncs2p in S. cerevisiae), which mediate the thiolation of wobble uridines in tRNA Lys(UUU) , tRNA Gln(UUG) , and tRNA Glu(UUC) (9)(10)(11)(12)(13)(16)(17)(18).…”

ALKBH8-mediated formation of a novel diastereomeric pair of wobble nucleosides in mammalian tRNA

“…In yeast, inactivation of wobble uridine modification enzymes can impair translational decoding, as measured by decreased translation of relevant 'codon-runs' 26,27 . To investigate the possible effects of the extensive wobble U mis-modification observed in the Alkbh8 − / − mice, we generated reporter plasmids containing both Renilla (Rluc; internal reference) and firefly (Fluc) luciferase genes, in which Fluc, but not Rluc, translation depends on in-frame translation with a codon-run of ten identical codons (Fig.…”

A functional genome-wide genetic screening identifies new pathways controlling the G1/S transcriptional wave

“…In fact, Rep2 is one of the proteins that is supposed to be not well translated in some Elongator mutants. 20 However, we do not observe any decrease in the amount of Rep2 that is present in cells of Elongator or tRNA methylation mutants (Fig. 3D).…”

“…To confirm that the effect measured with the reporter strain was on MBF-dependent transcription, we isolated RNA from wild type, Dyox1 and Drep2 cells as well as from Diki3/Delp1 and Dctu1 cells. We used Dctu1 cells since the Ctu1-Ctu2 complex catalyzes the thiolation at carbon 2 of U 34 (s 2 U 34 ) 20 and the modification of specific tRNAs is sequentially made by Trm112 complex and the Ctu1-Ctu2 complex. In fact, although Dctu1 is not included in Table 1 containing the lowest YFP/ FSC strains from our screening, Dctu1 strain had also a low YFP/FSC ratio (0.66).…”