Wobble modification defect suppresses translational activity of tRNAs with MERRF and MELAS mutations
Introduction
Mitochondrial DNA (mtDNA) mutations are associated with a wide spectrum of human diseases caused by mitochondrial dysfunction (Schon et al., 1997). Among these, a pathogenic point mutation at nucleotide position (np) 8344 in the tRNALys gene is found in most patients with myoclonus epilepsy associated with ragged-red fibers (MERRF; Shoffner et al., 1990) and a mutation at either np 3243 or 3271 in the tRNALeu(UUR) gene is responsible for mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS; Goto et al., 1990, Kobayashi et al., 1990, Goto et al., 1991, Kobayashi et al., 1991), both of which are major subgroups of the mitochondrial encephalomyopathies. In the case of MELAS, symptoms arising from the two different mutation positions are clinically indistinguishable except for the age when the first episodic attack occurs (Sakuta et al., 1993). The 3243 mutation has also been observed in the diabetes subgroup maternally inherited diabetes with deafness (MIDD; van den Ouweland et al., 1992) and in progressive external ophthalmoplegia (PEO; Johns et al., 1991, Moraes et al., 1993). Even if patients harbor the same mutation, the phenotypes and severity of the diseases vary – presumably because the overall mutation load differs from one patient to another; the mutation load is heterogeneous within a tissue, and each patient has his or her own nuclear background. To unravel the molecular pathogenesis, it is indispensable that we understand these variations as well as how the point mutations in tRNA genes essentially cause abnormalities in the functioning of the tRNA molecules and of the mitochondria that have the pathogenic tRNAs, independently from the possible factors influencing clinical manifestation noted above.
For this purpose, more than a decade ago cybrid cells were constructed in which mitochondria and mtDNAs from patients were intercellularly transferred into human cells lacking mtDNA (ρ0 cells) (King and Attardi, 1989). Using the cybrid cell system, base substitutions at nps 8344, 3243, and 3271 were confirmed to be directly responsible for decreasing mitochondrial respiratory activity without nuclear gene involvement (Chomyn et al., 1991, Chomyn et al., 1992, Hayashi et al., 1993). Subsequently, there have been extensive investigations aimed at determining how these point mutations result in defective respiration at the molecular level. In the case of MERRF, mutant cybrid cells were found to severely impair mitochondrial protein synthesis and to produce abortive polypeptides which, it was proposed, could be accounted for by a reduction in aminoacyl-tRNALys (Enriquez et al., 1995). On the other hand, a biopsy analysis of MERRF patients revealed no significant decreases in either the abundance or the aminoacylation of mutant tRNALys (Börner et al., 2000). In the case of 3243 MELAS mutant cybrid cells, a mitochondrial translation defect was clearly observed in cybrid cells containing the mutant mtDNA in more than 95% of the total mtDNA (Chomyn et al., 1992, Dunbar et al., 1996), and aminoacylation and the steady-state amount of the mutant tRNALeu(UUR) were reported to be lowered (El Meziane et al., 1998a, El Meziane et al., 1998b, Janssen et al., 1999). However, no reduction in the steady-state amount was observed in other 3243 MELAS cybrid cells (Koga et al., 1993). In a 3271 MELAS homoplasmic cybrid, although mitochondrial protein synthesis was only mildly affected, the amount of aminoacyl-tRNALeu(UUR) was also reduced (Hayashi et al., 1993, Yasukawa et al., 2000a). Thus, a quantitative decrease in aminoacyl-tRNALeu(UUR) alone appears insufficient to explain the mitochondrial translational defect. Termination of RNA synthesis (Hess et al., 1991) or unusual RNA processing (Schon et al., 1992) have been proposed as possible outcomes arising from the 3243 mutation, which would in turn cause decreased respiratory activity. However, there is as yet no conclusive evidence to support these propositions. The observed features suggest that rather than the decrease in the amount of aminoacyl-tRNA, the likely primary cause of the translational defect resulting in mitochondrial respiratory dysfunction is a certain functional abnormality in the mutant tRNA.
To clarify how the point mutations directly influence the structure and function of the mutant tRNAs that give rise to the mitochondrial disorders, we have analyzed mitochondrial tRNA molecules harboring the point mutations in considerable detail. Sequence analyses of the mutant tRNAs, including modified nucleotides, revealed that both tRNALys with the 8344 mutation (tRNALys[A8344G]) and tRNAsLeu(UUR) with either the 3243 or 3271 mutation (tRNALeu(UUR)[A3243G] and tRNALeu(UUR)[U3271C], respectively) have a fatal qualitative abnormality – all three mutant tRNAs specifically lack the post-transcriptional modification of uridine at the anticodon first letter (the wobble position) due to the point mutations (Yasukawa et al., 2000a, Yasukawa et al., 2000b). Since modification at this position generally controls precise and efficient discrimination of codons, our findings implied that mutant tRNAs without the wobble modification are unable to fulfill their normal specific role as acceptor molecules in the translation process. To examine the quality of mutant tRNAs in the translational elongation step, we reconstituted the mitochondrial translation system in vitro and measured the tRNA to codon binding affinity. These experiments showed that the three mutant tRNAs actually did lose their translational activity due to the absence of the modification (Yasukawa et al., 2001). Additionally, our findings suggested that the 3243 mutation itself contributes to the tRNALeu(UUR) translational defect to a certain extent. The magnitude of the decline in mitochondrial translation in mutant cell lines carrying the mutant tRNAs corresponded well with the severity of loss of translational ability in each mutant. These results indicate that defective wobble modification is the primary cause of mitochondrial translational deficiency, resulting in mitochondrial dysfunction in MERRF and MELAS mutant cells.
Section snippets
Cybrid cell lines
The mutant cybrid cell lines used, ME1-4, ML2-2-2, and ML5-1-13, were described previously (Yasukawa et al., 2000a, Yasukawa et al., 2000b). These cell lines exclusively contain mtDNA with np A8344G (MERRF), A3243G (MELAS), and T3271C (MELAS) point mutations, respectively. As a control, we used a wild-type cell line, Ft2-11, which harbors mitochondria with wild-type mtDNA derived from fetal human fibroblasts. The cybrid lines were originally obtained by the intercellular transfer of patient
Post-transcriptional modification defect in MERRF- and MELAS-mutant tRNA molecules
The cybrid cell lines used were constructed previously by the intercellular transfer of mtDNA from MERRF and MELAS patients, or from fetal human fibroblasts as a control, to ρ0 HeLa cells (King and Attardi, 1989, Hayashi et al., 1991). To uncover any abnormal features in the structure and/or functioning of mitochondrial tRNA molecules with the pathogenic point mutations, a procedure for the purification of mutant mitochondrial tRNAs was first established. Since the amount of mitochondrial tRNA
Acknowledgements
We would like to express our thanks to Dr J.-I. Hayashi (Tsukuba University) for giving us an original mutant cybrid cell line of the ME1-4 clone and to Dr L.L. Spremulli (University of North Carolina) for kindly providing an EF-Tumt expression vector. We are also grateful to Dr Norie Ishii (Nippon Medical School), Dr T. Hanada (Tokyo University), and Mr T. Suzuki (Tokyo University) for their technical assistance and helpful advice. This work was supported by a Grant-in-Aid for Scientific
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