Codon-specific translational defect caused by a wobble modification deficiency in mutant tRNA from a human mitochondrial disease - PubMed (original) (raw)

Codon-specific translational defect caused by a wobble modification deficiency in mutant tRNA from a human mitochondrial disease

Yohei Kirino et al. Proc Natl Acad Sci U S A. 2004.

Abstract

Point mutations in the mitochondrial (mt) tRNA(Leu(UUR)) gene are responsible for mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS), a subgroup of mitochondrial encephalomyopathic diseases. We previously showed that mt tRNA(Leu(UUR)) with an A3243G or T3271C mutation derived from patients with MELAS are deficient in a normal taurine-containing modification (taum5U; 5-taurinomethyluridine) at the anticodon wobble position. To examine decoding disorder of the mutant tRNA due to the wobble modification deficiency independent of the pathogenic point mutation itself, we used a molecular surgery technique to construct an mt tRNA(Leu(UUR)) molecule lacking the taurine modification but without the pathogenic mutation. This "operated" mt tRNA(Leu(UUR)) without the taurine modification showed severely reduced UUG translation but no decrease in UUA translation. We thus concluded that the UUG codon-specific translational defect of the mutant mt tRNAs(Leu(UUR)) is the primary cause of MELAS at the molecular level. This result could explain the complex I deficiency observed clinically in MELAS.

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Figures

Fig. 1.

Fig. 1.

Cloverleaf structures of human mt tRNAsLeu(UUR) from WT cells (Left) and from MELAS cybrid cells with the A3243G mutation (Center) or T3271C mutation (Right). “U” on a round black background indicates unmodified wobble uridine. White letters on a square black background represent the respective point mutations. Symbols for the modifications are 5-taurinomethyluridine (τm5U), 1-methyladenosine (m1A), 1-methylguanosine (m1G), 2-methylguanosine (m2G), pseudouridine (Ψ), ribothymidine (T), dihydrouridine (D), and 5-methylcytidine (m5C) (17).

Fig. 2.

Fig. 2.

Construction of mt tRNALeu(UUR) with an unmodified uridine at position 34 using the molecular surgery technique. (a) Purification of mt tRNALeu(UUR) from human placenta. (Left) Denaturing gel electrophoresis of the total RNA fraction extracted from human placenta. (Right) Purified human mt tRNALeu(UUR) (indicated by an arrow). (b) Schematic depiction of the molecular surgery procedure used to construct mt tRNALeu(UUR) with an unmodified wobble uridine. The details of each step are described in Materials and Methods. BAP and PNK represent bacterial alkaline phosphatase and polynucleotide kinase, respectively. (c) RNA sequence ladders for the 5′ end-labeled WT and operated tRNALeu(UUR) were obtained by the Donis-Keller method (27). –E, without enzyme; Al, treatment with alkali; T1, RNase T1 (specific for G); U2, RNase U2 (for A > G); PM, RNase Phy M (for A and U); and CL3, RNase CL3 (for C). Arrows indicate the bands that were cleaved by RNase PhyM digestion. There is no band for τm5U34 in the WT tRNA because the modification prevents PhyM digestion, but there is a cleaved band for U34 in the operated tRNA. Arrowheads show the wobble positions in the alkaline ladders. The band corresponding to τm5U34 is shifted up due to its bulky substituent, whereas U34 shows a normal band in the ladder.

Fig. 3.

Fig. 3.

Translational activity of the operated tRNALeu(UUR) without the wobble modification and of the MELAS mutant tRNAsLeu(UUR). In vitro mitochondrial translation of test mRNAs containing the UUA (Left), UUG (Center), or UUC (Right) (negative control) codons was performed with WT tRNALeu(UUR)(WT), operated tRNALeu(UUR) with an unmodified wobble uridine (OP), and two MELAS mutant tRNAsLeu(UUR) that bear the A3243G (3243) or U3271C (3271) mutations and an unmodified wobble uridine. The radioactivity of the [3H]leucyl-tRNA input to the reaction mixture was defined arbitrarily as 100. The averages of three independent experiments with SD values are shown.

Fig. 4.

Fig. 4.

Ability of the operated tRNALeu(UUR) without the wobble modification to bind to UUR codons. Shown is binding of the WT tRNALeu(UUR) (filled circles) and the operated tRNALeu(UUR) that lacks the wobble modification (filled squares) to ribosomal A-sites containing UUA (Left) or UUG (Right). Three independent experiments were performed, and the average values are plotted with SD values.

Fig. 5.

Fig. 5.

Possible mechanism of molecular pathogenesis caused by the wobble modification deficiency of the mutant tRNAs in MELAS (Left) and MERRF (Right) patients. Pathogenic point mutation (A3243G or U3271C) in mutant tRNALeu(UUR) from MELAS patients causes a τm5U-modification deficiency, which results in a UUG codon–specific translational defect. The MERRF 8344 mutation in tRNALys causes a τm5s2U-modification deficiency that results in a translational defect for both cognate codons (AAA and AAG) (21).

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