Biogenesis of glutaminyl-mt tRNAGln in human mitochondria - PubMed (original) (raw)
Biogenesis of glutaminyl-mt tRNAGln in human mitochondria
Asuteka Nagao et al. Proc Natl Acad Sci U S A. 2009.
Abstract
Mammalian mitochondrial (mt) tRNAs, which are required for mitochondrial protein synthesis, are all encoded in the mitochondrial genome, while mt aminoacyl-tRNA synthetases (aaRSs) are encoded in the nuclear genome. However, no mitochondrial homolog of glutaminyl-tRNA synthetase (GlnRS) has been identified in mammalian genomes, implying that Gln-tRNA(Gln) is synthesized via an indirect pathway in the mammalian mitochondria. We demonstrate here that human mt glutamyl-tRNA synthetase (mtGluRS) efficiently misaminoacylates mt tRNA(Gln) to form Glu-tRNA(Gln). In addition, we have identified a human homolog of the Glu-tRNA(Gln) amidotransferase, the hGatCAB heterotrimer. When any of the hGatCAB subunits were inactivated by siRNA-mediated knock down in human cells, the Glu-charged form of tRNA(Gln) accumulated and defects in respiration could be observed. We successfully reconstituted in vitro Gln-tRNA(Gln) formation catalyzed by the recombinant mtGluRS and hGatCAB. The misaminoacylated form of tRNA(Gln) has a weak binding affinity to the mt elongation factor Tu (mtEF-Tu), indicating that the misaminoacylated form of tRNA(Gln) is rejected from the translational apparatus to maintain the accuracy of mitochondrial protein synthesis.
Conflict of interest statement
The authors declare no conflict of interest.
Figures
Fig. 1.
Primary structures of mitochondrial tRNAGlu and tRNAGln, and subcellular localization of mtGluRS and hGatCAB. (A) Nucleotide sequences of bovine mt tRNAGlu (Left) and mt tRNAGln (Right). Modified nucleosides: τm5s2U, 5-taurinomethyl-2-thiouridine; Ψ, pseudouridine; m1A, 1-methyladenosine; m2G, N2-methylguanosine; m1G, 1-methylguanine; m5C, 5-methylcytidine. (B) Subcellular localization of mtGluRS and three subunits of hGatCAB. Confocal fluorescence microscopic image of HeLa cells transfected with pEGFP-mtGluRS (top row), pEGFP-hGatA (second row), pEGFP-hGatB (third row) and pEGFP-hGatC (bottom row). Left panels show EGFP fluorescence. Middle images show mitochondria stained by MitoTracker Red. The right column shows the merged images.
Fig. 2.
Growth of HeLa cells treated with siRNAs targeting hGatCAB and direct identification Glu-tRNAGln in the cells. (A) Growth curves for HeLa cells treated with siRNAs for hGatA (Upper Left), hGatB (Upper Right), hGatC (Lower Left), or luciferase (control, Lower Right). The siRNA-treated cells were cultured in glucose (solid line) or galactose (dotted line) containing medium. Each plot is the average of three independent cultures (bars, ± SD). (B) A schematic of the procedure for direct analysis of amino acids attached to an aa-tRNA is shown. Total aa-tRNAs obtained from the cells are acetylated and individual acetyl-aa-tRNAs are affinity-purified using the solid-phase DNA probe method, and are then digested by RNase I. The acetyl-aminoacyl-adenosine (Ac-aa-Ado) fragments originating from the 3′-terminus were analyzed by LC/MS. (C) Mass chromatograms detecting Ac-Gln-Ado (m/z 438, solid line) and Ac-Glu-Ado (m/z 439, dotted line); authentic Ac-Gln-Ado and Ac-Glu-Ado (Top Left) separately prepared from mouse liver, HeLa cells (no treatment, Middle Left) and HeLa cells treated by siRNAs targeting for all subunits of hGatCAB (Bottom Left). The small peak at m/z 439 (dotted line) that overlaps with the Ac-Gln-Ado (m/z 438) peak was assigned to the isotopic ion of Ac-Gln-Ado. Collision-induced dissociation (CID) spectra of Ac-Gln-Ado (Upper Right) and Ac-Glu-Ado (Lower Right) are shown. Product ions for the adenine base (BH2+, m/z 136), AcGln moiety (m/z 171), and AcGlu moiety (m/z 172) were clearly detected.
Fig. 3.
In vitro reconstitution of Gln-tRNAGln formation by hGatCAB. (A) Gel filtration chromatography (Superdex 200) of hGatCA (gray line), hGatB (dotted line), and a mixture of hGatCA and hGatB (black line) detected by UV absorption at 220 nm. Elution profiles of hGatB alone (Upper) and hGatB in the hGatCAB complex (Lower) were analyzed by Western blotting using an anti-His-tag antibody. (B) In vitro reconstitution of Gln-tRNAGln formation by the recombinant hGatCAB. Phosphor-image of the TLC analysis of [14C]-labeled Gln and Glu deacylated from aa-tRNAs in transamidation experiments. The assays were performed in the presence (+) or absence (−) of hGatB or hGatCA. Gln or NH4+ was used as an amide donor. [14C]-labeled Glu-tRNAGlu or Glu-tRNAGln was used as a substrate, as indicated below the panel. Positions of Gln and Glu on TLC were determined using [14C]Gln and [14C]Glu as markers (right lane).
Fig. 4.
Status of Gln-tRNAGln in cells treated with siRNAs targeting hGatCAB and measuring the binding affinity of mtEF-Tu with Glu-tRNAGln. (A) Total aa-tRNAs were separated by acid-urea PAGE, and mt tRNAGln (Left) and mt tRNAGlu (Right) from HeLa cells treated with the siRNAs targeting luciferase (lane 2), hGatA (lane 3) or all three subunits of hGatCAB (lane 4) were detected by northern blotting. The lane 1 signal in each panel represents the deacylated tRNAs prepared by alkaline-treatment of aa-tRNAs. Positions of the aa-tRNA and deacyl-tRNA are shown by the closed and open arrowheads, respectively. (B) (Left) Time course experiments of the hydrolysis protection assay for Glu-tRNAGlu (square), Glu-tRNAGln (circle), and Gln-tRNAGln (triangle) in the presence (closed symbols) or absence (open symbols) of bovine mtEF-Tu. The remaining aa-tRNAs were measured and plotted. (Right) The rates of hydrolysis of aa-tRNAs measured in the presence of different concentrations of mtEF-Tu.
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