The cytosolic thiouridylase CTU2 of Arabidopsis thaliana is essential for posttranscriptional thiolation of tRNAs and influences root development

Abstract

Background: A large number of post-transcriptional modifications of transfer RNAs (tRNAs) have been described in prokaryotes and eukaryotes. They are known to influence their stability, turnover, and chemical/physical properties. A specific subset of tRNAs contains a thiolated uridine residue at the wobble position to improve the codon-anticodon interaction and translational accuracy. The proteins involved in tRNA thiolation are reminiscent of prokaryotic sulfur transfer reactions and of the ubiquitylation process in eukaryotes. In plants, some of the proteins involved in this process have been identified and show a high degree of homology to their non-plant equivalents. For other proteins, the identification of the plant homologs is much less clear, due to the low conservation in protein sequence.

Results: This manuscript describes the identification of CTU2, the second CYTOPLASMIC THIOURIDYLASE protein of Arabidopsis thaliana. CTU2 is essential for tRNA thiolation and interacts with ROL5, the previously identified CTU1 homolog of Arabidopsis. CTU2 is ubiquitously expressed, yet its activity seems to be particularly important in root tissue. A ctu2 knock-out mutant shows an alteration in root development.

Conclusions: The analysis of CTU2 adds a new component to the so far characterized protein network involved in tRNA thiolation in Arabidopsis. CTU2 is essential for tRNA thiolation as a ctu2 mutant fails to perform this tRNA modification. The identified Arabidopsis CTU2 is the first CTU2-type protein from plants to be experimentally verified, which is important considering the limited conservation of these proteins between plant and non-plant species. Based on the Arabidopsis protein sequence, CTU2-type proteins of other plant species can now be readily identified.

Figure 1

Alignment of CTU2-homologs. The CTU2-homologs of Arabidopsis (Arab) [TAIR: At4g35910], potato (Solanum tuberosum; Sola [UniProtKB: M4D5F7]), rice (Oryza sativa; Oryza [UniProtKB: Q2QMW0]), and humans (Homo sapiens; human [UniProtKB: Q2VPK5] were aligned using ClustalW software. Identical positions among the plant proteins or all proteins are indicated in black. Domains largely conserved between human and plant proteins [7] are framed red. Identical, conserved, and similar positions in the alignment are indicated by asterisks, colons, or single dots, respectively. The PP-loop important for ATP binding is underlined with a bold line.

Figure 2

The ctu2-2 mutant is affected in tRNA thiolation. A) Two ctu2 insertion mutants were identified. The T-DNA inserted in the 3′ untranslated region (ctu2-1) and in the third exon (ctu2-2). The insertion sites are indicated with arrowheads. B) RT-PCR experiments were performed on 900 ng of reverse transcribed total RNA from wild-type Columbia (WT), ctu2-1, ctu2-2, and complemented lines using CTU2- and ACTIN2-specific primers. Contaminating genomic DNA in the RNA preparation could be excluded by the length polymorphism to genomic DNA due to intronic sequence. The ACTIN2 PCR was performed to confirm that comparable amounts of RNA were used for the RT-reaction. C) Thiolated tRNAs (arrowhead) show reduced mobility in acrylamide gels in the presence of N-acryloylamino phenyl mercuric chloride (APM; left gel). tRNA of wild-type Columbia (WT) show a retarded band that is absent in the ctu2-2 mutant. The faint bands in the ctu2-2 lane are present also in the gel lacking APM (right gel) and thus do not represent thiolated tRNAs. D) Complementation of ctu2-2 with a wild-type clone of CTU2 reconstituted tRNA thiolation. Three independent transgenic lines are shown which are identical to those shown in B).

Figure 3

CTU2 interacts with ROL5. Protein-protein interaction between CTU2 and ROL5 (CTU1-type protein of Arabidopsis) was investigated by a yeast-two-hybrid experiment. In the presence of both proteins (left lane), yeast cells were able to grow under selective conditions and showed strong β-galactosidase activity, indicative of the interaction of the two proteins. No growing yeast cells were observed when transformation was done with the pGAD-CTU2 or pLEX-ROL5 constructs together with the empty second plasmid (pLEX and pGAD, respectively), indicating absence of self-activation function of either of the two proteins.

Figure 4

CTU2 is ubiquitously expressed.CTU2 expression was investigated with a CTU2:GUS fusion construct in transgenic Arabidopsis. Strong GUS activity was observed throughout plant development, in young seedlings (A, B), and rosette leaves (C), siliques/stem (D), flowers (E), and cauline leaves (F). Bars = 2.5 mm

Figure 5

The ctu2-2 mutant is affected in root development. A) The ctu2-2 mutant shows a reduction in lateral root formation compared to wild-type Columbia (WT) and the complemented ctu2-2 mutants (indicated by an asterisk; p = 0.01; n ≥ 15), determined after 8 days of growth in a vertical orientation. The complementation lines are the same as shown in Figure  2. The tRNA thiolation-defective rol5 mutant also shows a reduction, which is significantly stronger than in ctu2-2 (indicated by two asterisks; p = 0.05; n ≥ 15). Error bars represent the standard error of the mean. B) In contrast to wild-type Columbia, 7 days-old lrx1 mutant seedlings develop aberrant root hairs. This phenotype is suppressed by ctu2-2, resulting in wild type-like root hair formation in the lrx1 ctu2-2 double mutant.