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.

Fig. 1.

The conserved F29C4.6 (Ctu1) protein is required for embryogenesis at high temperatures. (A) Synchronized L1 worms from wild type or the tm1297 mutant were grown at 20°C, and samples were collected and stained with DAPI at the indicated time points. Developing eggs are magnified. (B) Same as in A, except worms were grown at 25°C. (C) The number of eggs laid and their viability at 25°C were determined for 10 individuals for both the wild type and the mutant.

Fig. 2.

Ctu1 forms a functional complex with Ctu2. (A) TAP purification and MS analysis of polypeptides associated with SPBC2G5.03 (Ctu1). The SPAC23A1.10 is a translation elongation factor often found in TAP purifications and was therefore not further investigated in this study. (B) RNAi knockdown of F29F11.3 (CTU-2) at 25°C. A representative adult worm 75 h after hatching is shown.

Fig. 3.

Deletion of the fission yeast ctu1 and ctu2 leads to genome instability. (A) Serial dilutions of indicated single or double deleted strains at 32°C (Left) and 36°C (Right). (B) Nomarski pictures of live indicated strains and DAPI/Calcofluor pictures of fixed cells from indicated strains grown at 36°C. Arrows point to aneuploid cells. (C) FACS analysis of indicated strains grown at 25°C or 36°C for 12 or 24 h. (D) Suppression of the growth defect of strains mutated for ctu1 by multicopy expression of tRNA^LYS, tRNA^GLU, or a combination of both. Serial dilutions at 32°C or 36°C are presented.

Fig. 4.

The thiolated uridine-34 is absent in a strain deleted for ctu1. Mass chromatograms for mcm⁵S²U (Upper) and mcm⁵ (Lower) of purified tRNAs from wild type (Left) and a strain deleted for ctu1 (Right) with relative abundance (y axis) and time in minutes (x axis). In each Inset, the mass spectrum of the protonated molecule and the protonated free base corresponding to the observed peak is presented with the chemical structure of the nucleoside detected.

Fig. 5.

The Ctu1–Ctu2 complex is required for thiolation of uridine-34 in tRNAs from yeast and nematode. (A) Purified tRNA from indicated strains was separated on 8 M urea containing polyacrylamide gels in the presence (Left) or absence (Right) of APM. Northern hybridization was performed with multiprimed probes corresponding to either tRNA^LYS or tRNA^MET as indicated. (B) The KADA (K62A D63A) and the AXXA (C142A C145A) mutations were introduced in Ctu1-TAP expressed from pREP-4, and the resulting plasmids were transformed in a ctu1-deleted strain together with an empty vector as indicated. Growth of corresponding strains was tested at 32°C and 36°C (Top), and the expression level of Ctu1-TAP was tested by Western blot (Middle). (Bottom) Purified tRNA from indicated strains was analyzed by APM gel as in A.

Fig. 6.

Ctu1 binds tRNA and is required for efficient translation. (A) Extracts from Ctu1-TAP or untagged control were incubated with in vitro synthesized, radiolabeled tRNA^LYS or tRNA^MET. After cross-linking, precipitation on IgG beads was performed and the elution product was analyzed on a denaturing gel. Western blot was performed on 10% of the precipitated product to check for the presence of Ctu1-TAP. The untreated transcribed tRNAs are shown in Right. (B) In vitro thiolation assay using purified Ctu1-TAP and Nfs1-TAP or untagged control. Precipitated proteins on beads were incubated with cysteine, ATP, and in vitro synthesized tRNA^LYS_UUU ortRNA^LYS_CUU. Resulting supernatants were separated on 8 M urea-containing polyacrylamide gels in the presence of APM. Northern hybridization was performed with a multiprimed probe corresponding to either tRNA^LYS. (C) A plasmid expressing a KQ-TAP protein (see Materials and Methods) under the control of the thiamine-repressed nmt promoter was transformed in the indicated strains. Induction was performed in medium lacking thiamine for the indicated time, and total protein extracts were analyzed by Western blot. (D) Indicated strains were plated on rich medium, on which a filter containing 0.2 mg or 1 mg of hygromycin B was placed. The size of the halo reflects the sensitivity of the strain toward the drug.