Human Molecular Genetics, 2000, Vol. 9, No. 4 463-465
© 2000 Oxford University Press
The np 3243 MELAS mutation: damned if you aminoacylate, damned if you don’t
1Institute of Medical Technology and Tampere University Hospital, University of Tampere, 33101 Tampere, Finland, 2Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK and 3MRC Dunn Human Nutrition Unit, Hills Road, Cambridge CB2 2XY, UK
Received 14 October 1999; Revised and Accepted 17 December 1999.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONLOSS OR GAIN OF...THE SAME MITOCHONDRIAL HAPLOTYPE...THE np 3243 MOLECULAR...WHERE DO WE GO...REFERENCES |
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The np 3243 MELAS mtDNA mutation in tRNAleu(UUR) has been variously proposed as a loss-of-function or as a gain-of-function mutation, based on apparently contradictory studies in cultured cell lines. A new report describing the molecular effects of the mutation in vivo now mirrors this variability. This should prompt a more systematic re-investigation of cells carrying the mutation, in order to separate primary from secondary and pathogenic from compensatory effects, all of which may contribute to disease phenotype. Nuclear genetic and developmental background, mitochondrial haplotype, and epigenetic effects may all influence the pathological outcome. Defects in both base-modification and aminoacylation of the mutant tRNA could play critical roles.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONLOSS OR GAIN OF...THE SAME MITOCHONDRIAL HAPLOTYPE...THE np 3243 MOLECULAR...WHERE DO WE GO...REFERENCES |
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The mitochondrial mutation at np 3243 (A3243G) in the gene for tRNAleu(UUR) has been the subject of intensive investigations over the past 10 years in many laboratories, in an effort to understand the molecular mechanism by which it brings about cellular dysfunction and disease. The mutation is associated with a particularly broad spectrum of clinical phenotypes. One important goal of current research is therefore to understand why some individuals with the mutation suffer only relatively mild symptoms, such as ocular myopathy, diabetes or deafness, whereas others suffer from the devastating MELAS syndrome (mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes). The consensus view is that these greatly variable phenotypes are not related simply to differences in dosage and tissue distribution of the mutant mitochondrial DNA (mtDNA). Other factors must be involved. Understanding the molecular mechanism of pathogenesis is a crucial first step towards the development of any rational strategy for future therapy and prevention.
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LOSS OR GAIN OF FUNCTION? |
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TOPABSTRACTINTRODUCTIONLOSS OR GAIN OF...THE SAME MITOCHONDRIAL HAPLOTYPE...THE np 3243 MOLECULAR...WHERE DO WE GO...REFERENCES |
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Much of the molecular analysis has been done in cybrid cells, where the properties of mitochondrial DNAs from diverse sources, including the two types of mtDNA found in heteroplasmic individuals, can in principle be compared in a ‘control’ nuclear background. Broadly speaking, two schools of thought have emerged from these studies. On the one hand, several groups, mainly working on osteosarcoma or lung carcinoma cell cybrids, have found evidence that the mutant tRNA may be functionally deficient. It is poorly aminoacylated (1,2), hypomodified at a site in the D-stem (3) and also expressed at a lower abundance than control tRNAleu(UUR) in some (4), but not all (5), cell lines. The view emerging from such studies is that the mutation is functionally recessive, in the sense that it represents a loss of function of a tRNA essential for the decoding of leucine UUR codons, neatly accounting for the threshold effect whereby very high relative levels (>95%) of mutant mtDNA are needed to bring about a severe respiratory impairment with loss of mitochondrial protein synthesis.
A quite different picture has emerged from a study in HeLa cell cybrids, and is also supported by findings from patient-derived lymphoblastoid cell lines and some other osteosarcoma cybrid studies. In the HeLa cell background, tRNAleu(UUR) amino- acylation seems to be only slightly affected (6), even though there is a clear drop in the steady-state level of the mutant tRNA compared with wild-type. Importantly, however, the mutant tRNA lacks the wobble-base U hypermodification that should restrict decoding to leucine UUR codons (i.e. by discriminating against phenylalanine UUY codons). The implication is that the mutation impairs mitochondrial protein synthesis via a gain of function that leads to frequent misreading of phenylalanine and perhaps some other codons, as illustrated in Figure 1.
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In patient-derived lymphoblastoid cells the abundance of tRNAleu(UUR) is unaffected by the mutation, as is the overall amount of mitochondrial protein synthesis, yet the relative amount of incorporation of leucine into some mitochondrial translation products is diminished (7). This suggests that the mutant tRNA might be mischarged with other amino acids, or that other tRNAs might compete effectively with the functionally impaired mutant tRNA, resulting also in misincorporation at UUR codons. In cells carrying, for example, only 70% mutant mtDNA, the result was a significant decrease in cytochrome c oxidase activity (7). Part of the attraction of this gain-of-function hypothesis is that it can account for overt disease in the majority of patients who carry much less than the threshold level of mtDNA for loss of protein synthesis inferred from cybrid studies. This applies even to single muscle fibres, where mitotic segregation cannot be invoked. However, it should be stated that direct measurements of protein synthesis have not been carried out in vivo. Another gain-of-function mechanism proposed for the mutation comes from observations that a precursor-like RNA accumulates in at least some mutant cell lines (4,8), prompting the suggestion that it may somehow interfere with ribosome function (8).
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THE SAME MITOCHONDRIAL HAPLOTYPE IN DIFFERENT NUCLEAR BACKGROUNDS |
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TOPABSTRACTINTRODUCTIONLOSS OR GAIN OF...THE SAME MITOCHONDRIAL HAPLOTYPE...THE np 3243 MOLECULAR...WHERE DO WE GO...REFERENCES |
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Our own work seems to support both views. In A549 lung carcinoma cell cybrids, we found that a heteroplasmic anticodon mutation in tRNAleu(CUN), that theoretically should create a novel tRNA capable of reading UUR codons, was able to suppress both the protein synthesis defect and respiratory impairment of cells carrying very high levels of np 3243 mutant mtDNA (4). Further cybridization experiments show that this phenotypic suppression can be serially passed on with the ‘suppressor mtDNA’ in the A549 background (N. Hance, S.K. Lehtinen, H.T. Jacobs and I.J. Holt, unpublished data). Broadly these observations are consistent with the loss-of-function hypothesis.
Other findings, especially in 143B osteosarcoma cybrids carrying np 3243 mutant mtDNA from the same source, would contradict this view. The aminoacylation defect may be less severe than in the A549 background (unpublished data), but at least one abnormal mitochondrial translation product is clearly detected in 143B cybrids (9), at levels of mutant mtDNA below the threshold for total loss of mitochondrial translation. Like in lymphoblastoid cells (7), there is clearly a respiratory deficiency under conditions where mitochondrial protein synthesis is apparently normal (9), recalling similar observations of others in the same background (2). This would be consistent with the idea that the mitochondrial translation products manufactured in such cells are somehow less ‘normal’ than they appear to be on SDS–PAGE. For example, if they contained a low but functionally significant proportion of misincorporated amino acids. This idea is also supported by the fact that serial passage of mtDNA from the suppressor cell line described above into the heterologous 143B background produces only an intermediate phenotype in which the respiration rate remains well below that of control cells (N. Hance, S.K. Lehtinen, H.T. Jacobs and I.J. Holt, unpublished data). We can also detect a clear structural change of tRNAleu(UUR) in mutant 143B cybrids, and also in A549 cybrids. This could be due to a similar defect in wobble-base hypermodification as has been found in HeLa cybrids.
A further observation that we have made is that an as yet unidentified structural modification of tRNAleu(CUN) seems to be associated with high levels of the np 3243 mutation in both A549 and 143B cells (1, and unpublished data). We have suggested that this may be a compensatory response to the mutation, and could, for example, represent a partial suppression mechanism, recruiting a proportion of tRNAleu(CUN) to read UUR codons. In all these cases it is by no means a straightforward matter to distinguish primary effects of the mutation and cellular responses that might mitigate it or modify its effects, or are secondary pathological consequences. In cultured cells there is, in addition, always a danger that one is observing the end result of processes of selection that are quite different from any that may operate in vivo.
A major problem in interpreting all these disparate observations in cultured cells is the fact that all the experiments referred to were carried out on immortalized, usually tumour-derived cell lines, which are known to be aneuploid. The nature of such aneuploidy is not, however, constant, even for a given cell line. Indeed, cells within an apparently uniform culture may have various different chromosome constitutions. Therefore, any argument as to which ‘behaviour’ is truly physiological may ultimately be meaningless. None may be so.
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THE np 3243 MOLECULAR PHENOTYPE IN VIVO |
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TOPABSTRACTINTRODUCTIONLOSS OR GAIN OF...THE SAME MITOCHONDRIAL HAPLOTYPE...THE np 3243 MOLECULAR...WHERE DO WE GO...REFERENCES |
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The accompanying Article (10) suggests a challenging alternative view of the disparate results described above, namely that they are all valid. Börner et al. (10) report analyses of mutant tRNAleu(UUR) representation and aminoacylation not in tumour-derived cybrids, but in biopsy samples obtained from patients, using the ingenious technique of RNA circularization by RNA ligase to distinguish and quantify aminoacylated versus non-acylated tRNA, following an oxidation and deacylation procedure. The results offer considerable support for the loss-of-function hypothesis, in that there was a clear deficiency of aminoacylated tRNAleu(UUR) in biopsy samples taken from most np 3243 patients, and tRNAleu(UUR) levels were in general low. Crucially, however, not all biopsy samples showed the same phenomenon. In some, the mutant tRNA appeared to be expressed as efficiently as wild-type, yet showed evidence of a specific defect in aminoacylation. In others, the mutant tRNA was under-expressed but aminoacylated efficiently. In still others both expression and aminoacylation seemed to be impaired, and in one sample no abnormality at all could be detected. In other words, the molecular consequences of the mutation can be as diverse in vivo as they are in cultured cell models, and there is hence no need to invoke aneuploidy or other artefacts of long-term cell culture to explain away the previously noted discrepancies.
One caveat to this conclusion is that no patients in the above study had mild phenotypes, such as isolated deafness or ocular myopathy, that are more commonly observed in np 3243 disease. The phenotypes of those patients included in the survey were all relatively severe, although not identical, but even those with the features of ‘classic MELAS’ fell into all categories as regards expression and aminoacylation levels. It is possible that more mildly affected patients would have yielded different, and perhaps more coherent results, since secondary effects might be expected to be more common in severely affected patients.
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WHERE DO WE GO NOW? |
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TOPABSTRACTINTRODUCTIONLOSS OR GAIN OF...THE SAME MITOCHONDRIAL HAPLOTYPE...THE np 3243 MOLECULAR...WHERE DO WE GO...REFERENCES |
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These results should nevertheless prompt a thorough and systematic re-investigation of the factors that may be influencing expression of the np 3243 phenotype: nuclear genetic and perhaps developmental background, mitochondrial haplotype and, importantly, epigenetic processes whereby cells may respond to the presence of a mutation that impairs tRNA function by a variety of compensatory mechanisms, leading to distinct pathophysio- logical outcomes both at the level of the cell and that of the organism.
Paradoxically, Börner et al.’s (10) recourse to an in vivo analysis strengthens the view that cell culture models of np 3243 disease may not be fundamentally flawed after all, and may even help us to elucidate exactly what those crucial modifying factors are. Rather than trusting (or hoping) that our favourite model system is more valid than those of others who obtain conflicting results, we should now begin to pool resources and, in particular, to exploit the wide armoury of tools in somatic cell genetics and comparative genomics to home in on what it is that makes one cybrid model behave differently from another.
Perhaps we are indeed looking at a plethora of secondary effects and compensatory mechanisms, but ultimately these may be very important in understanding the features and course of disease in patients. For example, it may be that in one nuclear background, a sufficient amount of the mutant tRNA is correctly aminoacylated but incorrectly modified, resulting in frequent misreading of phenylalanine codons. This might lead to a defined set of clinical features associated with the synthesis of specific, abnormal mitochondrial translation products. The level of expression of tRNAphe may be critical in the biological manifestation of such a defect, since it would compete with the mutant tRNA for translation of UUY codons. The biosynthesis of tRNAphe is believed to depend mainly or exclusively on the rRNA transcription unit, rather than the overlapping, full genome-length transcription unit of the heavy strand. Since the np 3243 mutation also impairs termination at the attenuator site for rRNA transcription (11), the level of tRNAphe could be influenced upwards or downwards by the mutation, via a variety of response mechanisms.
In other tissues or individuals, in which the mitochondrial leucyl-tRNA synthetase may be expressed at a lower level, or may fail to charge the unmodified tRNA, high levels of the mutation might produce a quite different set of clinical features, associated with a generalized loss of mitochondrial protein synthesis or stalling at UUR codons. However, we should bear in mind the possibility that the primary effect of the mutation on tRNA function may yet prove to be something that nobody has yet thought of.
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ACKNOWLEDGEMENTS |
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We thank J. Poulton, G. Attardi, C. Florentz, J.A. Enriquez, G.M.C. Janssen, J.M.W. van den Ouweland, K. Watanabe, S. Ohta, S. Pääbo and L.A. Bindoff for useful discussions, as well as N. Hance, S.K. Lehtinen, A. El Meziane, J.N. Spelbrink and Z.H. Shah, plus the other members of our laboratories, for their contributions to our ideas and permission to cite unpublished data. Our research on the np 3243 mutation is supported by the Finnish Academy, the Muscular Dystrophy Group of Great Britain and Tampere University Hospital Medical Research Fund.
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel/Fax: +358 3215 7731; Email: howy.jacobs@uta.fi
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REFERENCES |
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TOPABSTRACTINTRODUCTIONLOSS OR GAIN OF...THE SAME MITOCHONDRIAL HAPLOTYPE...THE np 3243 MOLECULAR...WHERE DO WE GO...REFERENCES |
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1 El Meziane, A., Lehtinen, S.K., Holt, I.J. and Jacobs, H.T. (1998) Mitochondrial tRNA(Leu) isoforms in lung carcinoma cybrid cells containing the np 3243 mtDNA mutation. Hum. Mol. Genet., 7, 2141–2147.
2 Janssen, G.M.C., Maassen, J.A. and van den Ouweland, J.M.W. (1999) The diabetes-associated 3243 mutation in the mitochondrial tRNALeu(UUR) gene causes severe mitochondrial dysfunction without a strong decrease in protein synthesis. J. Biol. Chem., 274, 29744–29748.
3 Helm, M., Florentz, C., Chomyn, A. and Attardi, G. (1999) Search for differences in post-transcriptional modification patterns of mitochondrial DNA-encoded wild-type and mutant human tRNA(Lys) and tRNA(Leu(UUR)). Nucleic Acids Res., 27, 756–763.
4 El Meziane, A., Lehtinen, S.K., Hance, N., Nijtmans, L.G.J., Dunbar, D., Holt, I.J. and Jacobs, H.T. (1998) A tRNA suppressor mutation in human mitochondria. Nature Genet., 18, 350–353.[Web of Science][Medline]
5 Koga, Y., Davidson, M., Schon, E.A. and King, M.P. (1993) Fine mapping of mitochondrial RNAs derived from the mtDNA region containing a point mutation associated with MELAS. Nucleic Acids Res., 21, 657–662.
6 Yasukawa, T., Suzuki, T., Ueda, T., Ohta, S. and Watanabe, K. (2000) Modification defect at anticodon wobble nucleotide of mitochondrial tRNALeu(UUR) with pathogenic mutations of mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes. J. Biol. Chem., 275, 4251–4257.
7 Flierl, A., Reichmann, H. and Seibel, P. (1997) Pathophysiology of the MELAS 3243 transition mutation. J. Biol. Chem., 272, 21789–27196.
8 Schon, E.A., Koga, Y., Davidson, M., Moraes, C.T. and King, M.P. (1992) The mitochondrial transfer RNA-leu(UUR) mutation in MELAS—a model for pathogenesis. Biochim. Biophys. Acta, 1101, 206–209.[Medline]
9 Dunbar, D.R., Moonie, P.A., Zeviani, M. and Holt, I.J. (1996) Complex I deficiency is associated with 3243G:C mitochondrial DNA in osteosarcoma cell cybrids. Hum. Mol. Genet., 5, 123–129.
10 Börner, G.V., Zeviani, M., Tiranti, V., Carrara, F., Hoffmann, S., Gerbitz, K.D., Lochmüller, H., Pongratz, D., Klopstock, T., Melberg, A., Holme, E. and Pääbo, S. (2000) Decreased aminoacylation of mutant tRNAs in MELAS but not in MERRF patients. Hum. Mol. Genet., 9, 467–475.
11 Hess, J.F., Parisi, M.A., Bennett, J.L. and Clayton, D.A. (1991) Impairment of mitochondrial transcription termination by a point mutation associated with the MELAS subgroup of mitochondrial encephalomyopathies. Nature, 351, 236–239.[Medline]
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