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Human Molecular Genetics Advance Access originally published online on January 30, 2006
Human Molecular Genetics 2006 15(6):897-904; doi:10.1093/hmg/ddl007
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Acquisition of the wobble modification in mitochondrial tRNALeu(CUN) bearing the G12300A mutation suppresses the MELAS molecular defect

Yohei Kirino1,2, Takehiro Yasukawa1,3, Sanna K. Marjavaara4,{dagger}, Howard T. Jacobs4,5, Ian J. Holt3, Kimitsuna Watanabe1,2,{ddagger} and Tsutomu Suzuki1,2,*

1Department of Chemistry and Biotechnology, Graduate School of Engineering and 2Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan, 3MRC-Dunn Human Nutrition Unit, Hills Road, Cambridge CB2 2XY, UK, 4Institute of Medical Technology and Tampere University Hospital, University of Tampere, Tampere FI-33014, Finland and 5Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, UK

* To whom correspondence should be addressed. Tel: +81 358418752; Fax: +81 338160106; Email: ts{at}chembio.t.u-tokyo.ac.jp

Received December 13, 2005; Accepted January 26, 2006


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
The A3243G mutation in the mitochondrial gene for human mitochondrial (mt) tRNALeu(UUR), responsible for decoding of UUR codons, is associated with mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS). We previously demonstrated that this mutation causes defects in 5-taurinomethyluridine ({tau}m5U) modification at the anticodon first (wobble) position of the mutant mt tRNALeu(UUR), leading to a UUG decoding deficiency and entraining severe respiratory defects. In addition, we previously identified a heteroplasmic mutation, G12300A, in the other mt leucine tRNA gene, mt tRNALeu(CUN), which functions as a suppressor of the A3243G respiratory defect in cybrid cells containing A3243G mutant mtDNA. Although the G12300A mutation converts the anticodon sequence of mt tRNALeu(CUN) from UAG to UAA, this tRNA carrying an unmodified wobble uridine still cannot decode the UUG codon. Mass spectrometric analysis of the suppressor mt tRNALeu(CUN) carrying the G12300A mutation from the phenotypically revertant cells revealed that the wobble uridine acquires de novo {tau}m5U modification. In vitro translation confirmed the functionality of the suppressor tRNA for decoding UUG codons. These results demonstrate that the acquisition of the wobble modification in another isoacceptor tRNA is critical for suppressing the MELAS mutation, and they highlight the primary role of the UUG decoding deficiency in the molecular pathogenesis of MELAS syndrome.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
Mitochondrial DNA (mtDNA) mutations are known to be associated with a wide spectrum of human diseases whose unifying characteristic is the impairment of mitochondrial function (1Go,2Go). Over 150 pathogenic mutations have been reported so far, more than half of which are located within mitochondrial (mt) tRNA genes (2Go). These mutations confer a variety of structural and functional defects that affect tRNAs at the post-transcriptional level, by impairing their maturation, modification, folding, aminoacylation or association with translation factors (3Go,4Go). Among these defects, those which impair post-transcriptional modification have been the subject of particular attention in recent years (5Go,6Go).

Mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS), which form a major clinical subgroup of the mitochondrial encephalomyopathies, are caused by any of several different single base replacements in the mt tRNALeu(UUR) gene, which is responsible for the translation of UUR (R=A or G) leucine codons in mitochondrial gene (Fig. 1A) (7Go–9Go). We previously showed that in cybrid cells carrying homoplasmic MELAS-causing mutations in mtDNA, the taurine-containing modified uridine ({tau}m5U; 5-taurinomethyluridine) (10Go) that is normally found at the anticodon first (wobble) position of mt tRNALeu(UUR) remains unmodified in mutant mt tRNALeu(UUR) molecules bearing either the A3243G or T3271C mutation (11Go). More recently, we reported that mutant mt tRNAsLeu(UUR) bearing one of five different point mutations (A3243G, G3244A, T3258C, T3271C or T3291C), as have been found in tissues from patients with MELAS symptoms, lack {tau}m5U modification (12Go). In contrast, mt tRNAsLeu(UUR), bearing different point mutations found in patients that have mitochondrial diseases lacking the defining features of MELAS syndrome, had the normal {tau}m5U modification (12Go). These results illustrate a clear correlation between the absence of wobble modification and the phenotypic features of MELAS. As modification of wobble uridines in tRNAs is responsible for specific and efficient codon recognition (13Go–15Go), the presence of the unmodified uridine in the mutant mt tRNALeu(UUR) is expected to induce a significant decoding disorder. Indeed, we previously demonstrated that an artificial mt tRNALeu(UUR) of the wild-type sequence, including all modified bases except for {tau}m5U at the wobble position, was severely defective in decoding UUG but not UUA codons (16Go). This result clearly indicates that the taurine-modification at the wobble position in mt tRNALeu(UUR) plays a crucial role in UUG decoding, presumably by stabilizing the U:G wobble base pairing. The UUG codon-specific translational defect caused by the wobble modification deficiency is thought to play a primary role in the molecular pathogenesis of MELAS.


Figure 0071
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  		Figure 1. Cloverleaf structures of the two human mitochondrial leucine tRNAs responsible for UUR and CUN codons. (A) The modified nucleosides of mt tRNALeu(UUR) were previously determined (11Go). The arrow shows the A to G MELAS mutation at nucleotide position 3243. The first nucleoside of the MELAS mutant tRNA anticodon is unmodified. (B) The modified nucleosides of mt tRNALeu(CUN) were determined in this study. The arrow shows the G to A suppressor mutation at nucleotide position 12300. The first anticodon nucleoside is unmodified in wild-type mt tRNALeu(CUN), whereas it is partially modified to {tau}m5U in the suppressor tRNA. Symbols for modifications are 1-methyladenosine (m1A), 1-methylguanosine (m1G), 2-methylguanosine (m2G), pseudouridine ({psi}), 5-taurinomethyluridine ({tau}m5U), ribothymidine (T), dihydrouridine (D) and 5-methylcytidine (m5C). Their positions are indicated according to the nucleotide numbering system of tRNAs (33Go).

 
In a separate study, we (17Go) isolated a phenotypically revertant, A549 lung carcinoma cybrid cell line bearing >99% MELAS A3243G mutant mtDNA, but with normal respiratory activity. This line was found to harbor a second, heteroplasmic mutation, G12300A, in the anticodon sequence of mt tRNALeu(CUN) gene, which is the other leucine isoacceptor tRNA responsible for decoding CUN (N=any base) codons. The severe impairment of both mitochondrial translation and respiratory activity in the A3243G cybrid was suppressed by the heteroplasmic mt tRNALeu(CUN) mutation across a wide variety of heteroplasmy levels for G12300A (18Go). The wild-type mt tRNALeu(CUN) has an unmodified uridine at its wobble position, which allows all four CUN codons to be decoded in accord with the mitochondrial four-way wobble rule (19Go). The G12300A mutation converts the anticodon sequence of mt tRNALeu(CUN) from UAG to UAA, which is identical to that of mt tRNALeu(UUR), suggesting that the suppressor mt tRNALeu(CUN) with the UAA anticodon could potentially translate UUR codons and thereby rescue the decoding disorder conferred by the MELAS mutation. However, given our finding that the MELAS mutant mt tRNALeu(UUR) with an unmodified wobble uridine is incapable of translating UUG codons (16Go), the suppressor mt tRNALeu(CUN) also with an unmodified wobble uridine should similarly be unable to translate the UUG codon efficiently, unless it is modified to {tau}m5U. Five different point mutations associated with MELAS were found to cause a wobble modification deficiency of mt tRNALeu(UUR) (12Go), strongly suggesting that the RNA-modifying enzyme responsible for {tau}m5U biogenesis must recognize the tertiary structure and/or primary sequence of mt tRNALeu(UUR). As the primary sequence of human mt tRNALeu(CUN) shares only 42% homology with that of mt tRNALeu(UUR) (Fig. 1B), it is a priori unlikely that mt tRNALeu(CUN) is recognized by this RNA-modifying enzyme and hence undergoes de novo {tau}m5U modification, even though its anticodon sequence, conferred by the G12300A suppressor mutation, is identical to that of mt tRNALeu(UUR).

In order to determine how MELAS A3243G cybrid cells with the suppressor G12300A mutation can revert to a wild-type phenotype at the molecular level, we undertook a detailed mass spectrometric analysis of the suppressor mt tRNALeu(CUN) with the G12300A mutation isolated from revertant cells and performed a functional analysis of its decoding properties.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
De novo {tau}m5U modification at the wobble position of the G12300A suppressor mt tRNALeu(CUN)
As the primary sequence of human mt tRNALeu(CUN) including modified bases had not been reported, we first purified wild-type mt tRNALeu(CUN) from total RNA extracted from human placenta (16Go) and determined its nucleotide sequence, including modified bases, by a combination of the Donis-Keller enzymatic digestion (20Go), Kuchino post-labeling (21Go) and liquid chromatography/mass spectrometric (LC/MS) techniques (22Go,23Go). As shown in Figure 1B, four species of modified bases were identified at six positions in wild-type mt tRNALeu(CUN): 1-methyladenosine (m1A) at position 9, 2-methylguanosine (m2G) at position 10, 1-methylguanosine (m1G) at position 37 and pseudouridine ({Psi}) at positions 27, 28 and 31. The wobble uridine was confirmed to be unmodified.

To analyze the suppressor mt tRNALeu(CUN) possessing the G12300A mutation, a lung carcinoma cybrid cell line, GT61, in which ~61% of the mtDNA has the G12300A mutation and 99% has the A3243G mutation (18Go), was used. Suppressor mt tRNALeu(CUN) carrying the G12300A mutation was isolated from GT61 cybrid cells by utilizing an improved solid-phase DNA probe method (10Go,22Go) (Fig. 2A). After purification by gel electrophoresis, the suppressor tRNA and control wild-type mt leucine tRNAs were then subjected to nucleoside analysis by LC/MS using electrospray ionization (ESI)/ion trap mass spectrometry. As shown in the mass chromatograms in Figure 2B, {tau}m5U at the wobble position in mt tRNALeu(UUR) purified from human placenta was detected as a positive ion with m/z 382, whereas {tau}m5U was not detected in wild-type mt tRNALeu(CUN). A low level of {tau}m5U was clearly detected in suppressor tRNA purified from GT61 cells (Fig. 2B). The mass spectrum for a proton adduct form of {tau}m5U is shown in Figure 2C. Judging from the areas of the peaks corresponding to {tau}m5U on the mass chromatograms (Fig. 2B), 29% of the suppressor tRNA contains the {tau}m5U modification. Considering the extent of heteroplasmy of mtDNA in the GT61 cybrids (39% of mtDNA has G12300 [GenBank] and 61% has A12300), this result suggests that there are three species of mt tRNALeu(CUN) in GT61 cell mitochondria: 39% is wild-type mt tRNALeu(CUN) with an unmodified wobble uridine, 32% is suppressor mt tRNALeu(CUN) with an unmodified wobble uridine and 29% is suppressor mt tRNALeu(CUN) with the {tau}m5U modification.


Figure 0072
Figure 0072
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  		Figure 2. Mass spectrometric analysis of the suppressor mt tRNALeu(CUN) with the G12300A point mutation. (A) Isolation of the G12300A suppressor mt tRNALeu(CUN) from GT61 cybrid cells. ‘Crude’ and ‘12300’ indicate the total RNA fraction extracted from GT61 cybrids and the isolated G12300A suppressor mt tRNALeu(CUN), respectively (indicated by an arrow). (B) Nucleoside analysis of wild-type mt tRNALeu(UUR) (left panels), wild-type mt tRNALeu(CUN) (middle panels) and the G12300A suppressor mt tRNALeu(CUN) (right panels) by LC/MS using ESI/ion-trap mass spectrometry. Top and bottom panels are mass chromatograms detecting proton adducts of m1A (m/z 282) and {tau}m5U (m/z 382), respectively. The arrow indicates the peak of {tau}m5U found in the suppressor tRNA. The mass chromatogram of {tau}m5U was normalized to the peak intensity of m1A. (C) Mass spectrum showing a singly charged positive ion of proton adduct of {tau}m5U in the isolated G12300A suppressor mt tRNALeu(CUN). (D) LC/MS analysis of RNase CL3-digested RNA fragments of wild-type mt tRNALeu(CUN) (left panels) and the G12300A suppressor mt tRNALeu(CUN) (right panels). Top panels show UV traces at 254 nm. Second, third and fourth panels are mass chromatograms produced by integration of doubly(–2) and triply(–3) charged ions of anticodon-containing fragments, U{tau}m5UAAm1GCp (m/z 1044.6+696.0), UUAAm1GCp (m/z 975.6+650.1) and UUAGm1GCp (m/z 983.6+655.4), respectively. Bottom panels are mass chromatograms detecting A{Psi}{Psi}GG{Psi}Cp (m/z 1129.7+752.8) in the D-arm of mt tRNALeu(CUN), which was employed to normalize both tRNAs for quantification of anticodon-containing fragments. The sequences of the respective anticodon fragments are represented at right. The arrowheads indicate RNase CL3-cleavage sites.

 
The isolated suppressor tRNA was digested by RNase CL3 (specific to C) and subjected to RNA fragment analysis by LC/MS. Figure 2D shows mass chromatograms of the anticodon-containing RNA fragments spanning from U33 to C38 produced by RNase CL3 digestion of the wild-type and the suppressor mt tRNAsLeu(CUN). Only one fragment, UUAGm1GCp (MW 1969), was detected for wild-type mt tRNALeu(CUN), as expected (left panels in Fig. 2D). In the case of the suppressor tRNA, three species of anticodon-containing fragment, UUAGm1GCp (MW 1969), U{tau}m5UAAm1GCp (MW 2091) and UUAAm1GCp (MW 1953), were clearly detected (right panels in Fig. 2D). Taken together with the nucleoside analysis shown in Figure 2B, this fragment analysis indicates the coexistence of three species of mt tRNAsLeu(CUN): wild-type mt tRNALeu(CUN) with the UAG anticodon, suppressor mt tRNALeu(CUN) with the UAA anticodon and suppressor mt tRNALeu(CUN) with the {tau}m5UAA anticodon. The presence of {tau}m5U in the suppressor mt tRNA was confirmed by a sensitive detection method involving primer extension (12Go) (Supplementary Material, Fig. S1).

G12300A suppressor tRNALeu(CUN) with partial {tau}m5U modification can decode UUR codons in a mitochondrial translation system
We examined whether the G12300A suppressor mt tRNALeu(CUN) translates UUR codons as a substitute for the A3243G mutant mt tRNALeu(UUR), which only poorly decodes the UUA codon and is virtually incapable of decoding the UUG codon (16Go). The translational activity of the isolated suppressor tRNA was measured in an in vitro mitochondrial translation system (16Go,24Go). Human mt tRNAs were purified from the respective cybrid cells and aminoacylated with radioisotope-labeled amino acids. Messenger RNAs containing 30 repeats of the leucine codons UUA or UUG were transcribed in vitro. As shown in Figure 3, wild-type mt tRNALeu(UUR) was efficient in decoding both UUA and UUG codons, whereas wild-type mt tRNALeu(CUN) could not translate either mRNA. The MELAS A3243G mutant mt tRNALeu(UUR) lacking the wobble modification showed a substantial reduction in UUA decoding (because of the point mutation) as well as a severe reduction in UUG decoding (because of the modification defect), as reported previously (16Go). The G12300A suppressor mt tRNALeu(CUN) exhibited UUA and UUG decoding activities more efficient than those of the A3243G mutant mt tRNALeu(UUR). Considering that the isolated suppressor tRNA consists of three species of mt tRNAsLeu(CUN), it is most likely that the 29% of the population that has the {tau}m5UAA anticodon and the 32% that has the unmodified UAA anticodon can both contribute to UUR codon translation, whereas the 39% of the population of molecules that are wild-type mt tRNALeu(CUN) cannot decode either codon. In particular, only the suppressor tRNA with {tau}m5UAA anticodon should be able to translate the UUG codon, as {tau}m5U was previously demonstrated to be essential to allow mt tRNALeu(UUR) to decode this codon (16Go). The observation that the G12300A suppressor tRNA decodes the UUG codon less efficiently than does the wild-type mt tRNALeu(UUR) can be explained by the low frequency (29%) of suppressor tRNA molecules bearing the modified {tau}m5UAA anticodon. In contrast, the efficient decoding of the UUA codon by G12300A suppressor tRNA is consistent with the earlier observation that the modification is non-essential for decoding of UUA (16Go) and that 61% of all tRNAs derived from the mt tRNALeu(CUN) gene in GT61 cells bear either a UAA or {tau}m5UAA anticodon.


Figure 0073
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  		Figure 3. Translational activity of the G12300A suppressor tRNALeu(CUN) with the wobble taurine modification. In vitro mitochondrial translation of poly(UUA)30 (left) and poly(UUG)30 (right) was performed with wild-type tRNALeu(UUR) (UUR), the A3243G mutant tRNALeu(UUR) with an unmodified wobble uridine (3243), wild-type tRNALeu(CUN) (CUN) and the G12300A suppressor mt tRNALeu(CUN) (12300). The radioactivity of the [3H]leucyl-tRNA input to the reaction mixture was defined arbitrarily as 100. The averages of three independent experiments are plotted; error bars indicate SD values.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 
We here provide evidence that partial modification of the anticodon of the G12300A suppressor tRNA occurs in vivo, enabling it to decode both UUA and UUG codons. Our findings suggest the following scenario to explain the mechanism by which the G12300A mutation suppresses the MELAS A3243G phenotype (Fig. 4). In wild-type mitochondria, mt tRNALeu(UUR), with anticodon {tau}m5UAA, and mt tRNALeu(CUN), with anticodon UAG, are responsible for decoding UUR and CUN codon groups, respectively. In contrast, in cells with the MELAS A3243G mtDNA mutation, the mutant mt tRNALeu(UUR) is incapable of decoding the UUG codon because of the absence of {tau}m5U modification (16Go). Phenotypically revertant cells still carrying >99% A3243G mutant mtDNA contain a suppressor mt tRNALeu(CUN) with the G12300A mutation. Approximately half of the suppressor mt tRNALeu(CUN) molecules have an unmodified UAA anticodon, which allows translation of only the UUA codon. The remaining suppressor mt tRNALeu(CUN) population exhibits {tau}m5U modification at the wobble position, which allows translation of the UUG codon in addition to the UUA codon. We propose that the emergence of the G12300A suppressor mt tRNALeu(CUN) with the {tau}m5UAA anticodon is essential for normal mitochondrial translation because this tRNA is the sole species that can decode the UUG codon in the mitochondria of revertant cells (Fig. 4). The ‘gain of wobble modification’ by mt tRNALeu(CUN), in combination with the alteration of anticodon sequence conferred by the G12300A mutation, suppresses the decoding disorder caused by the absence of wobble modification of the MELAS mutant mt tRNALeu(UUR).


Figure 0074
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  		Figure 4. Schematic depiction of the MELAS decoding disorder and rescue by G12300A suppressor tRNAs. In normal cells, mt tRNALeu(UUR) and tRNALeu(CUN) decode UUR and CUN codons, respectively (left). In pathogenic cells with the MELAS A3243G mutation, the severe reduction in decoding activity of the UUG codon is caused by the absence of {tau}m5U modification in mt tRNALeu(UUR). Revertant cells contain a suppressor mt tRNALeu(CUN) with the G12300A mutation. The suppressor tRNA with an unmodified wobble uridine translates only the UUA codon, whereas the modified version is the only species capable of decoding the UUG codon because of the stabilization of the U:G wobble base pair. The remaining wild-type mt tRNALeu(CUN) is still responsible for decoding CUN codons (right).

 
In mammalian mitochondrial genetic code system, there are eight family codon boxes [Leu(CUN), Val, Ser(UCN), Pro, Thr, Ala, Arg and Gly], each of which is deciphered by a single tRNA species having unmodified wobble uridine which is responsible for decoding all four codons in the family box according to the mitochondrial four-way wobble rule (19Go). This suggests that unmodified wobble uridine pairs with all four bases, A, G, C and U, at the third position of each codon. Indeed, unmodified wobble uridine in wild-type mt tRNALeu(CUN) (anticodon UAG) is responsible for decoding all four CUN codons. However, G12300A suppressor mt tRNALeu(CUN) bearing unmodified wobble uridine (anticodon UAA) does not follow this rule and only decodes UUA codon, similar to the case of mt tRNALeu(UUR) with unmodified wobble uridine (16Go). The sequences of these two mt tRNAsLeu(CUN) are identical except for the anticodon third letter. We consider that different property of the unmodified wobble uridine in these two tRNAs arises from thermodynamic stability in the codon–anticodon pairing. Namely, there is one G:C pair in CUN–UAG pairing of mt tRNALeu(CUN), and no G:C pair in UUR–UAA pairing of its suppressor tRNA. Thus, it is suggested that the G:C (or C:G) pair in the codon–anticodon interaction enables mt tRNA with unmodified wobble uridine to decode all four codons in family boxes. In fact, all eight mt tRNAs bearing unmodified wobble uridine, which are responsible for family boxes, have at least one G or C base at their anticodon second or third letter, forming at least one G:C (or C:G) pair with codons of mRNA. In addition, it is demonstrated that mutant mt tRNALeu(UUR) and mt tRNALys, both of which have no G or C in their anticodon, could never decode codons ending in pyrimidine (UUY or AAY, respectively) when lacking wobble modifications (16Go,25Go).

In the suppressor cybrid cells first isolated (17Go), the frequency of the G12300A mutation in mtDNA was ~10%. It was also found that the steady-state levels of mt tRNALeu(UUR) and tRNALeu(CUN) were similar (26Go). Taking into account that about half of the G12300A suppressor mt tRNALeu(CUN) exhibits the wobble modification, it would appear that only 5% of the total amount of mt tRNALeu(CUN), i.e. the low proportion with the {tau}m5UAA anticodon, is sufficient to rescue UUG decoding and to suppress the mitochondrial translation defect due to the MELAS A3243G mutation. It is also consistent with the fact that <10% heteroplasmy for mtDNA wild-type at nucleotide position 3243 is sufficient to restore a substantial level of mitochondrial protein synthesis. Both observations imply that there is normally a considerable excess of mt tRNAs in the mitochondrial translation system, although this may not be the case in all tissues. The absence of wobble modification, rather than a low steady-state level of mt tRNALeu(UUR), is thus the primary cause of the MELAS mitochondrial translation disorder.

Our first concern in studying this suppression mechanism was to determine whether the suppressor mt tRNALeu(CUN) is recognized by the RNA-modifying enzyme responsible for the {tau}m5U formation. Our experiments have shown that the biogenesis of {tau}m5U in mutant mt tRNALeu(UUR) is readily impaired by several point mutations involved in pathogenesis, indicating that overall tRNA architecture, as well as the primary sequence of mt tRNALeu(UUR), is required for {tau}m5U modification. However, in this study, we found that the G12300A suppressor mt tRNALeu(CUN) exhibits only partial {tau}m5U modification. As the primary sequences of the two leucine-specific tRNAs are dissimilar (42% homology) (Fig. 1), this finding demonstrates that the adenosine at the anticodon third position (A36) is a determinant for {tau}m5U modification of the suppressor mt tRNALeu(CUN). However, A36 is clearly not sufficient for modification, as the mutant forms of mt tRNALeu(UUR) found in patients with MELAS retain A36. Moreover, a set of cryptic determinants for {tau}m5U modification must be embedded in the primary sequence and/or structure of mt tRNALeu(CUN). In other words, G36 in wild-type mt tRNALeu(CUN) works as a negative determinant to prevent {tau}m5U modification, resulting in an unmodified wobble uridine. In human mitochondria, {tau}m5U or {tau}m5s2U is found in five mt tRNAs that translate Leu(UUR), Trp, Lys, Glu and Gln codons (10Go). The A36 residue is not conserved among these tRNAs, and it is difficult to identify common structures or candidate determinants for {tau}m5U biogenesis shared by this group. This observation indicates that the individual sequence and features of each mt tRNA define the determinants that are recognized by the RNA-modifying enzyme that generates {tau}m5U. If there is only one system for {tau}m5U biogenesis in human mitochondria, this implies that a complex system of positive and negative determinants in primary tRNA sequences must confer the necessary specificity.

As emphasized earlier, the biosynthesis of {tau}m5U is not fully understood. We previously reported that free taurine is a direct substrate for the second step of {tau}m5U modification (10Go), but the initial step remains to be ascertained. A series of pathogenic point mutations, as well as the G12300A suppressor mutation, are considered to influence the first step of {tau}m5U modification. However, we still need to identify a methyl donor for the C5-methylene group of {tau}m5U and its RNA-modifying enzyme(s), in order to understand properly the molecular pathology of the associated diseases. The {tau}m5U is classed as an xm5U modification, which includes 5-methylaminomethyluridine (mnm5U) in bacterial tRNAs and 5-carboxynethylaminomethyluridine (cmnm5U) in yeast and nematode mt tRNAs (13Go). In Escherichia coli, mnmE (trmE) and gidA are required for the initial step of mnm5U modification (27Go–29Go). In both human and yeast, MSS1 and MTO1 were found to be the respective homologs of the bacterial mnmE and gidA genes (30Go,31Go). Their protein products form heterodimers in mitochondria, and mutations affecting these genes are associated with respiratory defects (32Go). These observations suggest that the human MSS1 and MTO1 genes encode enzymes that are responsible for the initial step of {tau}m5U modification in mt tRNAs. Development of an in vitro {tau}m5U reconstitution system using a recombinant MSS1/MTO1 complex will provide a key to clarify {tau}m5U biosynthesis in mt tRNAs associated with mitochondrial diseases.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
SUPPLEMENTARY MATERIAL
 REFERENCES
 
Cybrid cell lines
A549 lung carcinoma cybrid cells containing wild-type mtDNA or mtDNA with the A3243G and G12300A mutations have been described (17Go). This study used the GT61 line, in which 99% of mtDNA has the A3243G mutation and 61% has the G12300A mutation (18Go). Cybrid cells were cultured in normal medium [Dulbecco's modified Eagle's medium/F-12 (1:1) (Gibco-BRL) and 10% fetal calf serum]. The mtDNA mutation profile was repeatedly monitored by PCR analysis as previously described (17Go).

Purification of human mitochondrial tRNALeu(CUN)
A crude RNA fraction was extracted from GT61 cells, and the tRNA fraction was obtained by anion exchange column chromatography using DEAE–Sepharose fast flow with a linear gradient of NaCl (200–500 mM) and MgCl2 (8–16 mM) in a buffer containing 20 mM HEPES–KOH (pH 7.5). The mt tRNALeu(CUN) fraction was purified to homogeneity by an improved solid-phase DNA probe method (22Go), termed chaplet column chromatography, which employs a synthetic 3'-biotinylated DNA probe complementary to tRNALeu(CUN) (5'-TACTTTTATTTGGAGTTGCACC-3'). Wild-type mt tRNALeu(CUN) and mt tRNALeu(UUR) were purified from total RNA extracted from human placenta as described (16Go). Mutant mt tRNALeu(UUR) bearing the A3243G mutation was purified from cybrid cells as described (11Go).

Sequence determination of wild-type mitochondrial tRNALeu(CUN)
Purified human mt tRNALeu(CUN) was sequenced by a combination of the methods of Donis-Keller (20Go) and Kuchino et al. (21Go). For the method of Donis-Keller (20Go), 5'- or 3'-32P-labeled mt tRNA was purified by gel electrophoresis. The nucleotide-specific RNases T1 (Amersham Pharmacia Biotech), U2 (Seikagaku Kogyo), PhyM (Amersham Pharmacia Biotech) and CL3 (Boehringer Mannheim) were used for restricted digestion of tRNA. Concerning the method of Kuchino et al. (21Go), 5'-32P-labeled nucleotides in the tRNA were analyzed by two-dimensional thin-layer chromatography. Solvent systems consisted of isobutyricacid/ammonia/H2O (66:1:33 by volume) in the first dimension and 2-propanol/HCl/H2O (70:15:15 by volume) in the second dimension were used.

Mass spectrometry
An LCQ ion-trap mass spectrometer (ThermoFinnigan) equipped with an ESI source and HP1100 liquid chromatography system (Agilent) was used to analyze nucleosides and RNA fragments. For nucleoside analysis, purified tRNA (0.04 A260 unit) was digested into nucleosides at 37°C for 3 h in a 10 µl reaction mixture containing 20 mM HEPES–KOH (pH 7.5), 10 µg/ml nuclease P1 and 0.5 U/ml bacterial alkaline phosphatase. The hydrolysates were analyzed by LC/MS as previously described (22Go). For RNA fragment analysis, purified tRNA (0.04 A260 unit) was digested with RNase CL3 (0.04 U, Boehringer Mannheim) in 20 mM Tris–HCl (pH 7.6) at 37°C for 3 h and treated with 0.1 N HCl at 0°C for 3 h to cleave the 2',3'-cyclic phosphate, and then analyzed by mass spectrometry as previously described (23Go).

In vitro mitochondrial translation
The in vitro translation assay was carried out essentially as described previously (16Go,24Go). The aminoacyl-tRNALeu was incubated at 37°C for 10 min in a buffer consisting of 50 mM Tris–HCl (pH 8.0), 15 mM MgCl2, 2 mM ATP, 3.3 µM [3H]L-leucine (1.85 MBq/mmol, American Radiolabeled Chemicals) and 20 µg human mt leucyl-tRNA synthetase. Poly(UUA)30 and Poly(UUG)30 were synthesized in vitro using T7 RNA polymerase. The reaction mixture (20 µl) contained 50 mM Tris–HCl (pH 8.6), 15 mM MgCl2, 5 mM KCl, 1 mM DTT, 0.5 mM spermine, 2.5 mM phosphoenolpyruvate, 2.5 U/ml pyruvate kinase, 0.5 mM GTP, 12 pmol mt EF-Tu, 8 pmol mt EF-G, 2 pmol mt ribosome, 4 µg of poly(UUR)30 and 0.1 pmol [3H]Leu-tRNALeu. The mixture was incubated at 37°C for 15 min and the radioactivity of the polymerized amino acids was measured by liquid scintillation (ALOKA).


   SUPPLEMENTARY MATERIAL
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
REFERENCES
 
Supplementary Material is available at HMG Online.


   ACKNOWLEDGEMENTS
 
We are grateful to Dr Takeo Suzuki and Mr Y. Ikeuchi for technical advice and helpful suggestions. This work was supported by grants-in-aid for scientific research on priority areas from the Ministry of Education, Science, Sports and Culture of Japan (to T.S. and K.W.), by a JSPS Fellowship for Japanese Junior Scientists (to Y.K.), by a grant from the New Energy and Industrial Technology Development Organization (NEDO, to T.S.), by the Human Frontier Science Program (grant RG0349 to T.S.) and by grants (to H.T.J) from Academy of Finland, Juselius Foundation and Tampere University Hospital Medical Research Fund.

Conflict of Interest statement. None declared.


   FOOTNOTES
 
{dagger} Present address: Neuroscience Research Programme, Biomedicum Helsinki, University of Helsinki, FI-00014 Helsinki, Finland. Back

{ddagger} Present address: Biological Information Research Center (BIRC), National Institute of Advanced Industrial Science and Technology (AIST), 2-41-6 Aomi, Koto-ku, Tokyo 135-0064, Japan. Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 REFERENCES
 

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