Schematic organization of MnmE, GTPBP3, and Ras proteins. The four motifs (G1, G2, G3, and G4) of the consensus GTP-binding domain are indicated by black bars. In the diagram of MnmE and isoforms of GTPBP3, the shaded area represents the G domain. The sequences of the G1 to G4 and C-terminal motifs of these proteins are also depicted, with the invariant residues of the G motifs indicated in bold. The G motifs are generally involved in binding of guanine nucleotides and Mg²⁺, hydrolysis of GTP, or controlling the conformational change that regulates the functional state of GTPases. Note that the G4 motif of GTPBP3(Ins8A) and GTPBP3(Del8A) starts at position 374 and 353, respectively, due to the deletion in GTPBP3(Del8A) of a 21-amino-acid sequence (encoded by exon 8A) that extends between the G3 and G4 motifs. In the diagram of MnmE, the black area represents the N-terminal domain involved in dimerization, whereas the empty areas represent the regions that form part of the helical domain. In the diagrams of the GTPBP3 isoforms, the black area represents the N terminal domain, whereas the small empty area at the N terminus represents the mitochondrial targeting signal.

Direct PCRs to detect alternative splicing of human GTPBP3 exon 8A. Primers used are represented by arrows on the diagrams above the gels. PCR analysis was performed on cDNAs from eight different human tissues (Human I MTC panel; Clontech, California) and cDNA synthesized on DNase-treated mRNA isolated from human fibroblasts and lymphocytes and HEK-293 cells. M₁, GeneRuler 100-bp DNA Ladder (Fermentas); M₂, pUC19 DNA/MspI Marker, 23 (Fermentas).

Expression of GTPBP3 in HEK-293 cells. (A) Immunoblotting using anti-GTPBP3 of extracts from HEK-293 cells nontransfected (lanes 3, 5, and 7) or permanently transfected with plasmid pIC1055 (lanes 4, 6, and 8) or from E. coli strain DH5α/pIC1296 grown in the absence (lane 1) or in the presence (lane 2) of inducer (arabinose). Samples were separated by 10% SDS-PAGE, and the amounts of bulk protein which were analyzed in each lane were as follows: 100 μg (lanes 1 and 2), 200 μg (lanes 3 and 4), 300 μg (lanes 5 and 6), and 400 μg (lanes 7 and 8). Signals were visualized by ECL (lanes 1 and 2) and ECL Advance (lanes 3 and 8) from GE Healthcare, and during film exposure each pair of lanes (1 and 2, 3 and 4, 5 and 6, and 7 and 8) was independently handled until optimal visualization of GTPBP3 bands was achieved. Nonspecific bands show equal loading in each pair of lanes (paired as above). Position and size (in kDa) of FLAG-(ΔN)GTPBP3 (detected in lane 2) are indicated on the left, and the arrow marks GTPBP3(Ins8A) position (detected in lanes 4, 6, and 8). (B) Extracts from HEK-293 cells permanently transfected or not with pIC1055 (approximately 2 or 5 mg of protein, respectively) were immunoprecipitated using anti-GTPBP3 as described in Materials and Methods. Immunoprecipitates and total extracts were resolved in a NuPAGE 4 to 12% Bis-Tris gel (Invitrogen) and immunoblotted with anti-GTPBP3 and rabbit True-blot (eBioscience, Inc.). Lane 1, total extract of nontransfected HEK-293 cells (100 μg); lane 2, total extract of HEK-293 cells permanently transfected with plasmid pIC1055 (50 μg); lanes 3 and 4, material bound to anti-rabbit IgG agarose beads (no antibody); lanes 5 and 6, immunoprecipitates of nontransfected HEK-293 cells; lanes 7 and 8, immunoprecipitates of HEK-293 cells transfected with plasmid pIC1055. Positions and sizes (in kDa) of mass markers are indicated on the left, and the arrow (on the right) marks GTPBP3 position.

Gel filtration analysis of the multimerization behavior of the GTPBP3 G domain. Experiments were performed in 50 mM Tris, pH 7.5, 5 mM MgCl₂, 5 mM DTT, and 100 mM KCl or NaCl. This buffer was supplemented with 1 mM GTP or GDP as indicated. In all, a 150 μM concentration of the GTPBP3 G domain was incubated for 30 min on ice, under the conditions being investigated, before application onto the Superdex 75 10/300 GL column. Apparent molecular masses corresponding to elution volumes are indicated. AU, arbitrary units.

Knockdown of human GTPBP3 results in mitochondrial dysfunction. HEK-293 cells were transfected with GTPBP3 siRNAs or with negative control siRNAs, and oxygen consumption (A), superoxide detection in mitochondria with MitoSOX (B), mitochondrial membrane potential (C), and cellular ATP (D) were determined as specified in Materials and Methods. Bars indicate standard deviations of at least three different experiments (*, P < 0.05; **, P < 0.005; ***, P < 0.0005).

Alternative splicing events in human and mouse GTPBP3. The consensus splicing sequences in intron 7, the alternative acceptor site in exon 8A, and the polypyrimidine tracks upstream from exon 8A and 8B are shown. The exon/intron boundaries are indicated by vertical sticks. Splice donor and acceptor sites are indicated in bold uppercase letters. Note that the first nucleotide of exon 8B is a pyrimidine and not the more frequent guanine, which is indeed present as the first nucleotide of exon 8A. Sequences including putative branch sites (underlined) and polypyrimidine tracts are denoted in lowercase letters. Purines interrupting polypyrimidine tracts are indicated in bold italics. Amino acid sequences resulting from each splicing variant are denoted by one-letter amino acid code below the corresponding nucleotide sequence.

Working model of the U₃₄ modification pathway. Only steps relevant to this work are shown. The first stage in the modification of U₃₄ at position 5 is controlled by MnmE and GidA in bacteria and by their homologous proteins in yeast (MSS1 and MTO1) and human (GTPBP3 and MTO1). MnmE binds 5-formyl-THF, which might be the one-carbon-unit donor in the first step of the modification reaction. MnmE and GidA form a functional complex that would catalyze the production of a still unknown intermediate group (X) at position 5. Taurine (in human) or glycine (in yeast and E. coli) would be subsequently incorporated into tRNA by unidentified transferases to build τm⁵ or cmnm⁵, respectively. U₃₄ may undergo thiolation at position 2 mediated by MnmA (in E. coli) or its homologue MTU1 (in eukaryotes), together with other proteins.

Structure of human GTPBP3 gene and its products. (A) Diagram of human GTPBP3 gene structure as proposed by Li and Guan (23). Boxes represent exons, with the positions of initiation and termination codons indicated. Two poly(A) sites are indicated. The nine exons and eight introns are numbered. (B) Schematic representation of six GTPBP3 transcripts. Transcripts I to V were proposed to result from the alternative splicing of exons 1A, 7B, 8A, and 9B and intron 5 (23). Transcript VI is the new variant found in this work. Locations of the G motifs G1 to G4 are indicated by vertical arrows on splicing variant I. Locations of some the primers used in this work are indicated by horizontal arrows below variants II to V.

Direct PCRs to detect mRNA variants of human GTPBP3 that include exon 9B but differ in the splicing of exon 8A. Primers used are represented by arrows on the diagram and are indicated over each gel. Primer CNT1 spans the junction region between exons 7 and 8A, whereas CNT33 spans that between exons 7 and 8B. PCR analysis was performed on cDNAs from the indicated human tissues (Human I MTC panel; Clontech, California). M, GeneRuler 100-bp DNA Ladder (Fermentas).

Binding of GST-GTPBP3 proteins to mGDP and AlF_x. Fluorescence spectra (excited at 360 nm) include GTPase buffer (a) and the same solution after subsequent addition of 2 μM mGDP only (b), 10 μM protein (c), 10 mM NaF (d), and 70 μM AlCl₃ (e). The table underneath the figure summarizes the conditions under which the spectra were obtained. Note that AlF_x is formed by mixing an aluminum salt (AlCl₃) with NaF. Fluorescence emission spectra were recorded 15 min after AlCl₃ addition. λ, wavelength; I_F, fluorescence intensity; au, arbitrary units. (A) MnmE. (B) GST-MnmE. (C) GST-GTPBP3(Ins8A). (D) GST-GTPBP3(Del8A). (E) GST-GTPBP3(Ins8A) (KCl was replaced by NaCl in the assay buffer). (F) GST-GTPBP3(Del8A) (KCl was replaced by NaCl in the assay buffer).

The GTPBP3 N-terminal domain induces a GTP- and potassium-independent dimerization. (A) Native PAGE analysis of FLAG-(ΔN)GTPBP3(Ins8A). Lanes 1 and 3, FLAG-(ΔN)GTPBP3(Ins8A); lanes 2 and 4, MnmE. Positions and sizes (in kDa) of mass markers are indicated on the left. Position and approximate size (in kDa) of MnmE and FLAG-(ΔN)GTPBP3(Ins8A) dimers are indicated on the right. ME, β-mercaptoethanol. (B) Gel filtration of FLAG-(ΔN)GTPBP3(Ins8A). Experiments were performed as described in Materials and Methods. The peak at an apparent molecular mass of 120 kDa (indicated by an arrow) was collected and analyzed by Western blotting (inset). Lanes 1 and 3, fraction corresponding to the ∼120-kDa peak collected from gel filtration; lanes 2 and 4, input. The Western blot was developed with anti-FLAG (lanes 1 and 2) and anti-GTPBP3 (lanes 3 and 4). The size of a molecular mass marker is indicated in kDa on the right. (C) Gel filtration analysis of the GTPBP3 N-terminal domain. The gel filtration buffer was 50 mM Tris, pH 7.5, 5 mM MgCl₂, 5 mM DTT, and 250 mM KCl. Before application onto the column, the protein was incubated overnight at 4°C in the same buffer (filled triangles) or in a buffer containing 50 mM Tris, pH 7.5, 5 mM MgCl₂, 5 mM DTT, and 100 mM KCl (open circles). Apparent molecular masses corresponding to elution volumes are indicated. AU, arbitrary units.

Effect of GTPBP3 silencing on intracellular protein degradation. HEK-293 cells, untreated or treated as indicated, were incubated for 1 h with [³H]leucine in the absence (A) or in the presence (B) of cycloheximide (200 μg/ml), and protein degradation was measured at 0.5, 1, 2, and 4 h as described in Materials and Methods. Results, which are expressed as the percentage of radioactive protein remaining in the cells, are mean ± standard deviation of three different experiments with duplicated samples. Some standard deviation bars are hidden by the symbols. At the 4-h time point, differences between GTPBP3 siRNA and control values were significant at P values of <0.05 (A) and <0.0005 (B).

PCR analysis to detect alternative splicing of mouse GTPBP3 exon 8A. Primers used are represented by arrows on the diagrams and indicated over each gel. Exon 8A is denoted in gray. Primer RS5 spans the junction region between exons 7 and 8A, whereas RS7 spans that between exons 7 and 8B. PCR analysis was performed on cDNAs from 12 different mouse tissues (Mouse MTC panel I; Clontech, California). For the gels in panels A to E, the lanes are as follows: lanes 1, heart; lanes 2, brain; lanes 3, spleen; lanes 4, lung; lanes 5, liver; lanes 6, skeletal muscle; lanes 7, kidney; lanes 8, testis; lanes 9, 7-day embryo; lanes 10, 11-day embryo; lanes 11, 15-day embryo; lanes 12, 17-day embryo. M₁, GeneRuler 100-bp DNA Ladder (Fermentas); M₂, pUC19 DNA/MspI Marker, 23 (Fermentas).