Modification Defect at Anticodon Wobble Nucleotide of Mitochondrial tRNAsLeu(UUR) with Pathogenic Mutations of Mitochondrial Myopathy, Encephalopathy, Lactic Acidosis, and Stroke-like Episodes*

The mitochondrial tRNALeu(UUR) (R = A or G) gene possesses several hot spots for pathogenic mutations. A point mutation at nucleotide position 3243 or 3271 is associated with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes and maternally inherited diabetes with deafness. Detailed studies on two tRNAsLeu(UUR) with the 3243 or 3271 mutation revealed some common characteristics in cybrid cells: (i) a decreased life span, resulting in a 70% decrease in the amounts of the tRNAs in the steady state, (ii) a slight decrease in the ratios of aminoacyl-tRNAsLeu(UUR) versusuncharged tRNAsLeu(UUR), and (iii) accurate aminoacylation with leucine without any misacylation. As a marked result, both of the mutant tRNA molecules were deficient in a modification of uridine that occurs in the normal tRNALeu(UUR) at the first position of the anticodon. The lack of this modification may lead to the mistranslation of leucine into non-cognate phenylalanine codons by mutant tRNAsLeu(UUR), according to the mitochondrial wobble rule, and/or a decrease in the rate of mitochondrial protein synthesis. This finding could explain why two different mutations (3243 and 3271) manifest indistinguishable clinical features.

Point mutations in mitochondrial tRNA genes are frequently found in mitochondrial diseases leading to neuromuscular disorders (1). One of the major clinical subgroups of the mitochondrial encephalomyopathies, mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS), 1 is caused by a single base replacement in the tRNA Leu gene corresponding to the UUR (R ϭ A or G) leucine codons (tRNA Leu (UUR)) (2). The majority (80%) of MELAS patients have an A to G transition at nucleotide position (np) 3243 (3,4), whereas in about 10%, a T to C transition is observed at np 3271 (5). Mutations at np 3243 and 3271 have also been found to be associated with a diabetes subgroup named maternally inherited diabetes with deafness (6,7). Since approximately 1% of patients with diabetes carry 3243 mutant mitochondrial DNAs (mtDNA) in a heteroplasmic manner (8), the mitochondrial tRNA Leu (UUR) gene appears to be critically involved not only in neuromuscular disorders but also in some more generalized diseases.
Despite the fact that the two mutations occur in different regions of the tRNA Leu (UUR) gene (np 3243 is in the D-loop at G-14, and np 3271 is in the anticodon stem at C-40), the clinical symptoms they give rise to appear identical. In contrast, point mutations in the tRNA Lys gene are responsible for another subgroup of the mitochondrial encephalomyopathies, myoclonus epilepsy associated with ragged-red fibers (MERRF), which is distinguishable from MELAS by certain differences in its clinical presentation (9,10). Thus, the clinical features of mitochondrial encephalomyopathies depend on the species of the tRNA gene in which the mutation is located. It has yet to be clarified why point mutations at different locations in the same tRNA gene should bring about indistinguishable symptoms. It also remains unknown why the phenotypes are distinct, corresponding to particular tRNA genes with point mutations. To answer these questions, the effects arising from different mutations in the same tRNA gene need to be investigated at the molecular level.
Mutations in the mitochondrial genome are themselves directly responsible for decreased respiratory chain activity or oxygen consumption without nuclear gene involvement. This has been demonstrated by constructing cybrid cell lines in which mutant mtDNAs derived from patients are intercellularly transferred into human cells lacking mtDNA ( 0 cells) (11)(12)(13). Unusual RNA processing (14) or a termination of RNA synthesis (15) have been proposed as possible outcomes arising directly from mitochondrial mutations, which would in turn cause a decrease in respiratory activity. However, there is as yet no conclusive evidence to support these proposals.
In the case of MERRF, it has been shown that premature polypeptides are generated by the point mutation in the tRNA Lys gene (16). In MELAS, on the other hand, a decrease in protein synthesis was observed in cybrid cells containing more than 95% mtDNA mutated at np 3243; however, the extent of the deficiency in protein synthesis did not seem to parallel the decline in enzymatic activity (17,18). Similarly, in cybrid cells with homoplasmic mtDNA mutated at np 3271, complex I activity was severely reduced, but protein synthesis was only slightly lower than normal (19). Since the decreases in protein synthesis are relatively modest in both mutations, they cannot in themselves explain the marked reductions observed in respiratory enzymatic activities. That is, the modest decrease in protein synthesis observed in cells with exclusively mutant mtDNA does not appear to be the direct cause of the clinical symptoms presented by the disease.
The possibility remains that abnormalities in tRNA molecules with pathogenic mutations are the cause of mitochondrial diseases; it could be that these tRNAs induce the misincorporation of amino acids. However, this line of investigation has been little pursued because a chemical amount of the mutant tRNA has not been purified, probably due to technical difficulties. We have succeeded in purifying the mutant tRNA Leu (UUR) molecules in a chemical amount by taking advantage of the solid phase probing method we have developed (20), which revealed that a modification of the uridine at the first position of the anticodon is missing in both the mutants. This common abnormality might provide an insight into the pathogenesis of MELAS at the translational level.

EXPERIMENTAL PROCEDURES
Cybrid Cell Lines-Cybrid cell lines were constructed by intercellular transfer of patient mtDNA to 0 HeLa cells (EB8) and isolation of cybrid clones as described (13). Two cybrid cell lines were constructed by fusing EB8 cells with enucleated fibroblasts from a MELAS patient with the heteroplasmic T3271C mutation: the ML5-1-13 cell line exclusively containing mtDNA with the 3271 mutation and the ML5-15 cell line containing only normal mtDNA as a control cybrid cell line (19). Mitochondrial DNA with the A3243G mutation obtained from a MELAS patient with the 3243 mutation was transferred to EB8 cells, and the cybrid cell line ML2-2 possessing more than 90% of the A3243G-mutated mtDNA was obtained. The Ft2-11 cell line for use as a wild-type control was obtained by fusing EB8 cells with enucleated fetal human fibroblasts. The cybrid cells were cultured in normal medium (Dulbecco's modified Eagle's medium/F-12 (1:1) (Life Technologies, Inc.), 10% fetal calf serum).
Quantification of tRNAs Using Northern Blot Analysis-Total RNA from semiconfluent cultured cybrid cells was isolated by treatment with Isogen (Nippon Gene, Toyama, Japan). A 5-g sample of total RNA was subjected to Northern blotting using a 32 P 5Ј-end-labeled oligonucleotide probe specific for mitochondrial tRNA Leu (UUR): 5Ј-TGTTAAGAA-GAGGAATTGAACCTCTGACTG-3Ј (not including the mutation positions). The amount of tRNA Leu (UUR) was normalized by the amount of nuclear-encoded 5 S ribosomal RNA (the probe specific for 5 S rRNA was 5Ј-GGGTGGTATGGCCGTAGAC-3Ј complementary to the 3Ј region). RNAs were quantified by exposing the membrane to an imaging plate and examination with a BAS1000 bioimaging analyzer (Fuji Photo Film, Tokyo, Japan).
Analysis of tRNA Life Spans in Cybrids-Semiconfluent cultured cybrid cells were trypsinized, divided accurately into equal volumes, and spread on fresh dishes. After the cells had adhered to the dishes, the medium was changed with the one containing ethidium bromide (250 ng/ml), which is a potential inhibitor of mitochondrial transcription (21). Culture of the cells in each dish with ethidium bromide was continued for the indicated periods. Total RNA was then isolated from the dish and subjected to Northern blotting as described above.
Examination of tRNA Aminoacylation Levels in Cybrids-Total RNA from semiconfluent cultured cybrid cells was prepared under acidic conditions in a cold room and then electrophoresed in an acid polyacrylamide gel at 4°C according to the literature (22) to prevent deacylation. The subsequent Northern hybridization was carried out as described above. Aminoacyl-and nonacylated-tRNAs Leu (UUR) were quantified by exposing the membrane to an imaging plate, followed by analysis with the BAS1000 bioimaging analyzer.
Identification of Amino Acid Attached to Mutant tRNA Leu (UUR)-(A3243G) and tRNA Leu (UUR)(T3271C)-Total RNA (approximately 1000 A 260 units, with some variation depending on the cybrid line) was obtained by treatment with Isogen (pH 4.8) from 1 ϫ 10 9 to 3 ϫ 10 9 semiconfluent cultured cells (100 -300 dishes 9 cm in diameter) under acidic conditions at 4°C. In this analysis, HeLa cells were used to prepare the wild-type aminoacyl-tRNA Leu (UUR). Total RNA was dissolved in 50 mM sodium acetate (pH 5.0) and fractionated on a DEAE-Sepharose Fast Flow (Amersham Pharmacia Biotech) column (1 ϫ 45 cm) with a linear gradient of NaCl and MgCl 2 between 250 and 500 mM and between 8 and 16 mM, respectively, in a buffer consisting of 50 mM sodium acetate (pH 5.0) at 4°C. The fractions corresponding to aminoacyl-tRNA Leu (UUR) were monitored by dot hybridization and concentrated with Centriprep 30 (Amicon). Aminoacyl-tRNAs in the fraction enriched with aminoacyl-tRNA Leu (UUR) were acetylated with [l-14 C]acetic anhydride (American Radiolabeled Chemicals) at the amino terminus of the aminoacyl-tRNAs to stabilize the aminoacyl bond according to the method of Suzuki et al. (23). Then, [ 14 C]acetylaminoacyl-tRNA Leu (UUR) was extensively purified by the solid-phase probing method using a 3Ј-biotinylated oligonucleotide with a sequence identical to that used for the Northern hybridization, which was immobilized on streptavidin-agarose (Life Technologies, Inc.), as described previously (20,23). The [ 14 C]acetylaminoacyl-tRNA Leu (UUR) preparation was further purified by acid polyacrylamide gel electrophoresis. The [ 14 C]acetylamino acid released from the purified [ 14 C]acetylaminoacyl-tRNA Leu (UUR) by 0.1 M NH 4 OH treatment was analyzed by thinlayer chromatography (silica gel plate). The solvent system used was n-butanol/acetic acid/H 2 O (4:0.9:1 by volume). An acetylleucine marker was prepared by deacylation of the acetylleucyl-tRNA, which was aminoacylated with [ 14 C]leucine in vitro and acetylated with nonradioactive acetic anhydride. The TLC plate was exposed to an imaging plate, followed by analysis with the BAS1000 bioimaging analyzer. Twenty species of 14 C amino acids were also acetylated with nonradioactive acetic anhydride and used as markers.
Purification of tRNAs Leu (UUR) from Cybrid Cells Using the Solidphase Probing Method-About 300 -1000 A 260 units (with some variation depending on the cell line) of total RNA were extracted from approximately 10 9 semiconfluent cultured cybrid cells. Total RNA was incubated at 37°C for 3 h in 50 mM Tris-HCl (pH 9.5) to discharge amino acids from the tRNAs. The deacylated total RNA preparation was adjusted to a pH of 7.5 and fractionated on a DEAE-Sepharose Fast Flow column (1 ϫ 45 cm) with a linear gradient of NaCl and MgCl 2 between 250 and 500 mM and between 8 and 16 mM, respectively, in a buffer containing 20 mM Tris-HCl (pH 7.5). Fractions enriched with the tRNA Leu (UUR) were monitored by dot hybridization. tRNA Leu (UUR) was finally purified by the solid-phase probing method (20), followed by gel electrophoresis.
Sequence Determination, Including Modifications, of Mutant and Wild-type tRNAs Leu (UUR)-Purified tRNAs Leu (UUR) with or without the 3243 or 3271 mutation were sequenced by a combination of the methods of Donis-Keller (24) and Kuchino et al. (25). For the Donis-Keller (24) method, the homogeneous tRNA was labeled at the 5Ј terminus with [␥-32 P]ATP (110 TBq/mmol, Amersham Pharmacia Biotech) and T4 polynucleotide kinase (Toyobo, Osaka, Japan). The nucleotide-specific RNases used for restricted digestion of tRNA were RNase T 1 (Amersham Pharmacia Biotech), U 2 (Seikagaku Kogyo, Tokyo, Japan), PhyM (Amersham Pharmacia Biotech), and CL 3 (Roche Molecular Biochemicals). For the Kuchino et al. method (25), the homogeneous tRNA was heated at 95°C for 1.5 min in H 2 O for random cleavage at a single site. The 3Ј half fragments of the single-site-digested tRNA were 5Ј-labeled with [␥-32 P]ATP and T4 polynucleotide kinase and electrophoresed in 12 or 15% polyacrylamide denaturing gel. The ladders of 32 P 5Ј-labeled fragments were cut out one by one and eluted from the gel. Each of the 5Ј-labeled fragments was then digested completely by RNase P 1 (Seikagaku Kogyo). The resultant 5Ј-labeled nucleotides were analyzed one by one by two-dimensional thin-layer chromatography with two different solvent systems. Solvent system A consisted of isobutyric acid/concentrated ammonia/H 2 O (66:1:33 by volume) in the first dimension and 2-propanol/HCl/H 2 O (70:15:15 by volume) in the second dimension. In solvent system B, the first dimension was the same as that used for system A, but 0.1 M sodium phosphate (pH 6.8)/ammonium sulfate/1-propanol (100 ml:60 mg:2 ml) was used for the second dimension.
Nucleoside Analysis of tRNA Leu (UUR) by LC/MS Using Electrospray Ionization (ESI)/Ion Trap Mass Spectrometry-A Finnigan LCQ ion trap mass spectrometer (ThermoQuest) equipped with an ESI source and a Magic 2002 liquid chromatography system (Michrom BioResources) were used for nucleoside analysis of tRNA Leu (UUR). The wildtype and T3271C mutant tRNAs Leu (UUR) (0.05 and 0.02 A 260 units, respectively) were digested into nucleosides by nuclease P 1 (Seikagaku Kogyo) and bacterial alkaline phosphatase (Takara Shuzo, Kyoto, Japan) at 37°C for 1 h in 10 l of a reaction mixture containing 20 mM Tris-HCl (pH 8.0), 10 g/ml nuclease P 1 , and 9 units/ml bacterial alkaline phosphatase. Purified tRNAs were prepared as described above. The hydrolysate of tRNA Leu (UUR) was injected into a C18 column (2.1 ϫ 250 mm) with a 3 ϫ 10-mm precolumn cartridge (Inertsil ODS3; GL Science). The solvent system consisted of 5% methanol (Solvent A) and 100% methanol (Solvent B), developed by 2-step linear gradient from 0% B to 14% B in the first half-hour and from 14% B to 58% B in the second half-hour at a flow rate of 150 l/min. The chromatographic effluent was directly conducted into the ESI ion source without splitting. Positive and negative ions were scanned by turns throughout the separation under the following conditions: flow rate of sheath gas, 100 arbitrary units; capillary temperature, 250°C; spray voltage, 4.25 kV. Scans were acquired over an m/z range from 103 to 750.

Decreased Life Spans of Both Mutant tRNA Leu (UUR)-(A3243G) and tRNA Leu (UUR)(T3271C), Resulting in Lower
Steady-state Amounts-We first quantified the steady-state amounts of the mutant tRNAs Leu (UUR) by Northern blotting; the values were normalized by the amount of nuclear-encoded 5 S rRNA as an internal standard. The steady-state amounts of tRNA Leu (UUR) with the 3243 mutation (tRNA Leu (UUR)-(A3243G)) and of tRNA Leu (UUR) with the 3271 mutation (tRNA Leu (UUR)(T3271C)) in the respective cybrid clones (clones ML2-2 and ML-5-1-13) were about 30% that of the wild-type tRNA Leu (UUR) in the control cybrid clones with wildtype mtDNA (Fig. 1a). In contrast, the steady-state amounts of tRNA Phe and tRNA Ile (encoded upstream and downstream of the tRNA Leu (UUR) gene, respectively) remained unchanged in both the mutant and control cybrid cells (data not shown). We then examined the degradation rate of the mutant and wildtype tRNAs Leu (UUR) in the presence of a mitochondrial transcription inhibitor, ethidium bromide (21), and found the life spans of the mutant tRNAs Leu (UUR) to be significantly shortened (Fig. 1b). The half-life of the wild-type tRNA Leu (UUR) was estimated to be about 56 h, whereas those of the 3243 and 3271 mutants were only about 6 and 12 h, respectively (Fig. 1b). Therefore, it can be concluded that the reduced steady-state levels were due to the shortened life spans of the mutant tRNAs.
No Misaminoacylation in Mutant tRNA Leu (UUR)(A3243G) and tRNA Leu (UUR)(T3271C)-Next, the possibility of misaminoacylation in the mutant tRNAs Leu (UUR) in the cybrid cells was examined by identifying the amino acid(s) attached to the mutant tRNAs Leu (UUR). For this purpose, we purified aminoacyl-tRNAs Leu (UUR) with or without the mutations by acetylation with [ 14 C]acetic anhydride at the amino terminus as reported (23), and the [ 14 C]acetylamino acids released from the [ 14 C]acetylaminoacyl-tRNAs Leu (UUR) by alkaline treatment were identified. As shown in Fig. 2a, only acetylleucine was detected in either of the mutant tRNAs Leu (UUR) as well as in the wild-type tRNA, demonstrating that no misaminoacylation occurred in the mutants. No trace amount of any other acetylamino acid was detected, even after very long exposure (45 days) of the TLC plate to an imaging plate (not shown). On the other hand, acidic gel electrophoresis followed by Northern blotting revealed that the extent of aminoacylation in these two mutant tRNAs Leu (UUR) was slightly but significantly lower than that in the wild-type tRNA Leu (UUR) (Fig. 2b). The percentages of leucine acylated in the wild-type and in the 3243 and 3271 mutants were estimated to be 77, 68, and 65%, respectively. Since, as noted above, the mutant tRNAs Leu -(UUR) amount to approximately 30% of the wild-type tRNA, the total mutant aminoacyl-tRNAs were estimated to comprise less than 30% of the wild-type aminoacyl-tRNA. quences, including modified nucleotides, by combining the methods of Donis-Keller (24) and Kuchino et al. (25). Sequence analysis by the Donis-Keller method (24) of the wild-type and two mutant tRNAs Leu (UUR) labeled at their 5Ј-ends revealed that tRNA Leu (UUR) from the A3243G mutant had the A to G transition in the D-loop, whereas that from the T3271C mutant had the U to C transition in the anticodon stem (Fig. 3a, upper  panels), thereby confirming the accurate transcription of the mutant mitochondrial tRNA genes in the mitochondria of the cybrid cells. It is noted that the tRNA preparation with the 3271 mutation is essentially homogeneous, whereas tRNA Leu (UUR) with the 3243 mutation coexists with approximately 30% of the wild-type tRNA Leu (UUR) due to the heteroplasmy of mtDNA in the cybrid. Differences in the digestion patterns of tRNAs digested with nucleases and by alkaline treatment sometimes suggests the presence of modified nucleotides. In fact, the sequence ladders in the lower panels of Fig. 3a indicate that the first letter of the anticodon of the wild-type tRNA Leu (UUR) is resistant against RNase PhyM (A-or Uspecific) digestion. Also, an unusual upper shift of the band at this position was observed in the alkaline-treated lanes, suggesting that it is probably not a usual uridine but a modified one. Using two-dimensional thin-layer chromatography (TLC) with two different solvent systems as described by Kuchino et al. (25), we also sequenced the wild-type and mutant tRNAs Leu (UUR) by developing one by one size-selected labeled 5Ј-terminal residues derived from single-site digestion of the tRNAs. The spot obtained from the 5Ј-labeled nucleotide at the wobble position of the wild-type tRNA Leu (UUR) did not correspond to that for unmodified uridine nor to any spots for already known modified uridines (Fig. 3b) (26); it did, however, correspond exactly to the spot obtained from the wobble position of bovine mitochondrial tRNA Leu (UUR) (27). A minor spot (indicated by arrows), which might have been produced from the modified uridine during the analytical procedures, always appeared concomitantly (Fig. 3b). Taken together, these findings suggest that the uridine at the wobble position is modified -as has been reported for bovine and human tRNAs Leu (UUR) (27, 28) -but the chemical structure is unknown. Although no digestion was observed at position U33 with PhyM, the alkaline ladder was normal and this residue was confirmed to be unmodified uridine by two-dimensional TLC analysis (not shown). The resistance against PhyM digestion was probably due to the influence of the modification of U34, since such a phenomenon is sometimes observed around a sequence containing a modified nucleotide(s).
This novel modified uridine (U*) was characterized by nucleoside analysis of the wild-type tRNA Leu (UUR) by LC/MS using ESI/ion trap mass spectrometry. Positive and negative ions were scanned throughout the separation, since it has been reported that uridine derivatives are difficult to detect as positive ions using electrospray ionization (29). The modified uridine (U*) at the wobble position was detected at 22.95 min as a negative ion with an m/z of 380.1 (Fig. 4, a and c). From this, its molecular weight is estimated to be 381.1Da, which is identical to that found recently by our group at the wobble position of bovine mitochondrial tRNA Leu (UUR) (27), the largest among the modified uridines reported so far (27,30).
The fact that digestion of both the mutant tRNAs Leu (UUR) with PhyM resulted in discrete bands at the wobble position in the sequence ladders suggests that they have unmodified uridine at this position. Additionally, the spot observed at the wobble position of the mutant tRNA Leu (UUR)(T3271C) in the two-dimensional TLC analysis clearly corresponds to unmodified uridine (Fig. 3b) (25,26). These results lead us to conclude that the mutant tRNA Leu (UUR) with the 3271 mutation has no modification at the wobble position, leaving the anticodon as UAA. Absence of the modified uridine from the mutant tRNA Leu (UUR)(T3271C) was also confirmed by the LC/MS nucleoside analysis. As shown in Fig. 4, a and b, positive ions of 1-methylguanosine (m 1 G) and 2-methylguanosine (m 2 G) were respectively detected in both the wild-type and mutant tRNAs Leu (UUR) with the intensities expected from the amount of the tRNAs subjected to the analysis (0.05 and 0.02 A 260 units of the wild-type and mutant tRNAs, respectively). In contrast, the modified uridine (U*) observed in the wild-type tRNA Leu -(UUR) was not detected in the mutant tRNA Leu (UUR) (Fig. 4b,  bottom panel).
In the case of the ML2-2 (A3243G) mutant cells, which possess both the mutant tRNA and a small amount of the wildtype (Fig. 3a, upper panel), both unmodified and modified uridine were detected at the wobble position (Fig. 3a, lower panel  and 3b). This modified uridine was identical to that found at the wobble position of the wild-type tRNA Leu (UUR) (Fig. 3b). To evaluate the correlation between the mutation and the extent of the modification, fragments expanding the region from positions 1 to 34 of the tRNA with and without modification were isolated on the alkaline ladder of the sequencing gel (Fig. 3c) and their nucleotide sequences were further analyzed (24). The 5Ј-fragment with unmodified uridine at the wobble The aminoacyl-tRNA Leu (UUR) and uncharged tRNA Leu (UUR) were separated by acid gel electrophoresis and detected by Northern blotting using a 5Ј-end-labeled oligonucleotide (5Ј-TGTTAAGAAGAGGAATT-GAACCTCTGACTG-3Ј) as a probe. The upper and lower bands correspond to aminoacyl-tRNA Leu (UUR) and uncharged tRNAs Leu (UUR), respectively. Lanes 1, 2, and 3 depict samples from the wild-type cybrid, the 3271 mutant, and the 3243 mutant, respectively. position (U34) had only G14 at np 3243, while two bands corresponding to A and G were observed in the fragment with modified U34 (Fig. 3c). This finding demonstrates that the tRNA Leu (UUR) with the wild-type sequence (A14) had the fully modified U34 and that the unmodified U34 originated only from the mutant tRNA Leu (UUR). Thus, the minor spot corresponding to the modified uridine is mainly derived from the coexisting wild-type tRNA Leu (UUR) (Fig. 3b).
To compare other modified nucleotides of the wild-type and mutant tRNAs Leu (UUR), we further analyzed all the nucleo-  (UUR)(T3271C). a, sequence ladders obtained by the Donis-Keller method (24) for wild-type and mutant tRNAs Leu (UUR) labeled at their 5Ј-ends from each cybrid clone in the regions around the respective point mutations (upper panels) and anticodon loop (lower panels). ϪE, Al, T1, U2, PM, and CL3 indicate no treatment and treatment by alkaline digestion, RNase T 1 (specific for G), RNase U 2 (for A Ͼ G), RNase Phy M (for A and U), or RNase CL 3 (for C), respectively. In the lower panels, bands corresponding to both modified uridine (U*) and unmodified uridine (U) are present at the wobble position in the mutant tRNA Leu (UUR)(A3243G), whereas unmodified U alone is evident in tRNA Leu (UUR)(T3271C); both of the wild-type tRNAs have a band corresponding only to U*. b, two-dimensional thin-layer chromatographic patterns of a 32 P-labeled nucleotide at the anticodon first position derived from the three purified tRNAs Leu (UUR) according to the method of Kuchino et al. (25). The upper panels show the results with solvent system A, and the lower panels show those with solvent system B, which are described under "Experimental Procedures." pU, pA, pG, pC, and pU* indicate the position of each nucleotide. In the panels for the wild-type tRNA Leu (UUR), the major spot (pU*) does not correspond to any of the spots for unmodified uridine or already known modified uridines. In the panels for the tRNA Leu (UUR)(T3271C), the major spot clearly corresponds to unmodified uridine (pU), whereas in the case of tRNA Leu (UUR)(A3243G), both pU and pU* are observed. A minor spot, indicated by arrows, which might have been produced from U* during the analytical procedures, always appears concomitantly. c, further sequencing of the bands, including RNA fragments covering the region from the 5Ј-end up to the nucleotide at the wobble position. The bands were obtained from the alkaline ladder of tRNA Leu (UUR), purified from the cybrid cell line ML2-2. Only the mutant nucleotide G at np 3243 was clearly detected in the fragment with the putative unmodified uridine.

FIG. 4. Nucleoside analysis of the wild-type and mutant tRNAs Leu (UUR) by LC/MS using ESI/ion trap mass spectrometry.
Shown are UV absorption profiles and mass chromatograms of positive and negative ion scanning of the wild-type tRNA Leu (UUR) (a) and mutant tRNA Leu (UUR)(T3271C) (b). m 1 G and m 2 G were detected in a range (m/z) from 297.8 to 298.4 as positive ions from both the wild-type and mutant tRNAs Leu (UUR). The amount of each methylguanosine was well comparable to the amount of each tRNA molecule subjected to the analysis. The novel modified uridine (U*) was detected in a range (m/z) from 379.9 to 380.3 as a negative ion, and it resulted exclusively from the wild-type tRNA. The normalized (NL) value of the mass chromatogram in the range m/z 379.9 -380.3 for the mutant tRNA Leu (UUR)(T3271C) was automatically determined with the value of the wild-type tRNA according to the ratio of the m 1 G and m 2 G values of the wild-type and mutant tRNAs ((2.76 ϫ 10 4 (E4)) (9.65 ϫ 10 5 (E5))/(3.40ϫ 10 6 (E6))). No signal for a negative U* ion was detected from the mutant tRNA in the calculated normalized value (7.87 ϫ 10 3 (E3)), and no significant differences were observed in the intensities for the other modified nucleosides. c, mass spectrum for the novel modified uridine. A singly charged negative ion of U* with an m/z of 380.1 was detected from the wild-type tRNA Leu (UUR). tides by two-dimensional TLC using the method of Kuchino et al. (25). They were all identified as known nucleosides, namely, m 1 G, m 2 G, dihydrouridine (D), pseudouridine (⌿), 5-methylcytidine (m 5 C), ribothymidine (T), and 1-methyladenosine (m 1 A) (Fig. 5). As all the modified nucleotides in the wild-type and mutant tRNAs Leu (UUR) were the same except for the modified uridine (U*), it is concluded that the mutant tRNAs Leu (UUR) specifically lack the modification at the wobble position. DISCUSSION We found that both the mutant tRNA Leu (UUR)(A3243G) and tRNA Leu (UUR)(T3271C) were markedly unstable in the respective cybrid cells despite there being only one nucleotide substitution (Fig. 5), resulting in significantly decreased steady-state amounts of these tRNAs (Fig. 1). In addition, the extent of aminoacylation of the mutant tRNAs was relatively low (Fig.  2b). The total amounts of leucyl-tRNAs Leu (UUR) with the mutations were estimated to be less than 30% that of the wild-type counterpart. In the case of the mutant tRNA Lys in MERRF, it has been proposed that an approximately 50% reduction of aminoacyl-tRNA Lys contributes to severe impairment of protein synthesis and the production of premature polypeptides, resulting in reduced respiratory activity (16). Previous studies on the 3243 MELAS mutation (14,17) have proposed that the decline in enzymatic activity may be caused by the decrease in protein synthesis. When the ratio of mutant mtDNA to total mtDNA exceeded 95%, the protein synthesis activity suddenly decreased (17). As shown in this study, the reduced amount of aminoacyl-tRNA Leu (UUR) with the 3243 mutation could explain the reduction in protein synthesis. However, a number of reports suggest that a decrease in protein synthesis cannot explain the decline in respiratory activity or in oxygen consumption (18,19,32). Even when the mitochondrial protein synthesis rate was normal, the enzymatic activity of complex I was observed to be significantly affected in cybrid clones containing 60 to 95% mutant mtDNA (18,32). In the case of the 3271 mutation, no significant decrease in protein synthesis was observed even when the cells contained a homoplasmic mutation, although the enzymatic activity was nearly abolished (19). Thus, the decrease in protein synthesis may not in itself contribute directly to the pathogenesis caused by mitochondrial dysfunction. Some unusual mobilities of proteins in SDS-poly-acrylamide gel electrophoresis have been reported (18,19,31), and the ratios of [ 3 H]leucine and [ 35 S]methionine incorporated into polypeptides encoded in mtDNA were altered by the 3243 mutation (32), which strongly suggests that amino acids were misincorporated into the proteins synthesized in the mitochondria with the mutant mtDNA. To clarify whether or not the mutant tRNAs were misaminoacylated using the method of Suzuki et al. (23), we purified for the first time the aminoacyl-tRNAs Leu (UUR) from the two mutant cybrid cell lines homogeneously and, as shown in Fig. 2a, demonstrated clearly that no misaminoacylation occurred in the mutant tRNAs Leu (UUR). It should, however, be noted that although the method used has been demonstrated to be sufficiently sensitive to detect only 3% misincorporation of leucine into serine tRNA from Candida yeast (33), given the limited amounts of aminoacyl-tRNAs Leu (UUR) with the mutations available, the possibility that there could be a small percentage of misaminoacylation cannot be excluded.
Our study revealed that the wild-type tRNA Leu (UUR) contains an unknown modified uridine at the wobble position and that this modification occurs at the uracil base, not at 2Ј-OH in the ribose ring, because a fragment ion (m/z 247.8) without a ribose moiety was specifically detected from the modified uridine of bovine mitochondrial tRNA Leu (UUR) by collision-induced dissociation analysis. In contrast, this uridine modification is absent in the tRNA Leu (UUR) with a mutation at either np 3243 or 3271 (Fig. 5). Helm et al. (28) recently analyzed human tRNA Leu (UUR) and quantitatively compared the modified nucleotides by labeling tRNA with [ 32 P]phosphate in cybrid cells containing the 3243 mutation. They reported that a nonreproducible result was obtained for the quantification of the modified nucleotide at the wobble position in the cultured cells, although their sequence agrees with ours, including the other modified nucleotides. This discrepancy may stem from culturing the cells in a low concentration of phosphate in order to label the tRNAs isotopically. In contrast, we purified chemical amounts of tRNAs from mass cultures of the respective cybrid cells with a fresh and complete medium. It is interesting to note that both of the mutant tRNAs Leu (UUR) are deficient in the modification at the wobble position despite having mutations at different positions. This is the first observation of a common modification defect affected by different point mutations within a single tRNA gene.
The deficiency in uridine modification at the wobble position in the mutant tRNAs Leu (UUR) strongly suggests mistranslation by these mutant tRNAs according to the mitochondrial wobble rule. All or most family boxes of mitochondria (34 -38) and Mycoplasma spp. (39,40) contain only a single tRNA species with the anticodon sequence UNN, the first nucleotide U being unmodified. This suggests that the unmodified U at this position pairs with all four bases, A, G, C, and U, at the third position of the codons. Indeed, in vitro translation experiments have shown that Mycoplasma tRNAs with an unmodified uridine at the wobble position translate all the corresponding four synonymous codons (41,42). Thus, it is suggested that unmodified uridine at the wobble position of mutant tRNAs Leu (UUR) can recognize all four nucleotides at the third position of codons, giving rise to the translation of not only the usual UUR (R ϭ A or G) leucine codons but also the unusual UUY (Y ϭ C or U) phenylalanine codons in mitochondria of MELAS patients, which may eventually lead to the incorporation of leucine into phenylalanine sites at a certain frequency. Because the modification deficiency is observed for both the 3243 and 3271 mutations, it is likely that the lack of the modification at the wobble position leads to the particular MELAS phenotype through mistranslation. Results obtained with a MELAS patient provide evidence in support of our hypothesis; the phenylalanine residue at position 251, a highly conserved amino acid in the carboxyl terminus of cytochrome c oxidase subunit III, was converted to leucine, which was found to arise from a point mutation in the cytochrome c oxidase subunit III gene, regardless of the mutation in the tRNA Leu (UUR) gene (43,44). This finding strongly supports the idea that the replacement of a phenylalanine residue by leucine at a certain position of a mitochondrial respiratory protein(s), whatever the cause of the amino acid replacement is, could eventually give rise to the particular symptoms of MELAS, probably through impairment of protein assembly into a respiratory chain complex(es) or of the enzymatic activities of the complex(es). Therefore, the pathogenesis of MELAS is mainly considered due to the lack of the modification and the resultant mistranslation, which could explain why different point mutations in the same tRNA manifest indistinguishable clinical features. Work is now in progress on a detailed analysis to verify this proposition by purifying the proteins and directly determining the misincorporation by the mutant tRNAs Leu (UUR).