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Human Molecular Genetics Pages 2141-2147  

Mitochondrial tRNALeu isoforms in lung carcinoma cybrid cells containing the np 3243 mtDNA mutation
Introduction
Results
   np 3243 mutant tRNALeu(UUR) is not aminoacylated in lung carcinoma cybrids
   np 12300 suppressor mutant tRNALeu(CUN) is aminoacylated
   Deacylated mitochondrial tRNALeu(CUN) is present in two isoforms
Discussion
   Aminoacylation defect of np 3243 mutant tRNALeu(UUR)
   Indirect effects of the np 3243 mutation on the structure of tRNALeu(CUN)
   A natural suppression mechanism for the np 3243 mutation?
Materials And Methods
   Cell lines and culture
   Oligonucleotides
   DNA and RNA extraction
   Genotyping of mtDNA by RFLP analysis of PCR products
   Gel electrophoresis and northern blots
Acknowledgements
References

Mitochondrial tRNA<sup>Leu</sup> isoforms in lung carcinoma cybrid cells containing the np 3243 mtDNA mutation

Mitochondrial tRNALeu isoforms in lung carcinoma cybrid cells containing the np 3243 mtDNA mutation

Abdellatif El Meziane1,2, Sanna K. Lehtinen1, Ian J. Holt3 and Howard T. Jacobs1,4,*

1Institute of Medical Technology and Tampere University Hospital, University of Tampere, 33101 Tampere, Finland, 2Departement de Biologie, Faculté des Sciences et Techniques, Université Cadi-Ayyad, Marrakesh, Morocco, 3Department of Molecular and Cellular Pathology, University of Dundee, Dundee DD1 9SY, UK and 4Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK

Received August 3, 1998; Revised and Accepted September 17, 1998

We have investigated the representation of structural isoforms of the two mitochondrial leucyl tRNAs in lung carcinoma cybrid cell lines containing the np 3243 (MELAS) mtDNA mutation, alone or in combination with the np 12300 suppressor mutation. The mutant tRNALeu(UUR) is aminoacylated very poorly or not at all, whereas the suppressor tRNALeu(CUN) is efficiently aminoacylated. Deacylated mitochondrial tRNALeu(CUN) is present, in all human cells tested, in two structural isoforms that are separable on denaturing gels, indicating a difference in primary structure. The ratio of the two isoforms differs between cell types and is strongly biased towards one isoform in lung carcinoma cybrids containing high levels of the np 3243 mutation, compared with control cybrids. We propose that structural modification of tRNALeu(CUN) could be a natural suppression mechanism for the np 3243 and other mitochondrial tRNALeu(UUR) mutations and could underlie some of the phenotypic variability of np 3243 disease.

INTRODUCTION

Mammalian mitochondrial DNAs encode a minimal set of 22 transfer RNAs capable of decoding all 60 sense codons found in mitochondrial protein coding genes (1-3). The ability to use a single tRNA to decode up to four synonymous codons is held to be an example of expanded wobble (Fig. 1). Transfer RNAs for codon groups such as arginine (CGN) (N, any nucleotide; R, purine, i.e. A or G; Y, pyrimidine, i.e. C or U), leucine (CUN) or glycine (GGN) contain a U in the wobble base position (U34 in the conventional nomenclature). Some other tRNAs also with a U in the wobble base position are restricted to decoding two-codon sets ending in a purine, e.g. lysine (AAR) or leucine (UUR), and in these cases U34 is believed to be hypermodified (4). The currently accepted model relating U34 modification and mitochondrial tRNA decoding properties is summarized in Table 1 and illustrated in Figure 1. Hypermodification at the wobble base U also defines, and in some cases restricts, tRNA decoding properties in bacteria (4,5). In yeast mitochondria (6) and also in Mycoplasma (7) the modified U34 wobble base is 5-carboxy-methylaminomethyluridine, although the specific modification is not known in mammalian mitochondria. The latter modification is also found in tRNAMet in ascidian mitochondria (8). U34 is believed to be unmodified in mammalian mitochondrial tRNAs that have unrestricted decoding capacity (Fig. 1 and Table 1), as in fungi (9). Restriction of decoding specificity by wobble base U modification has been demonstrated directly in bacteria (10).

Mitochondrial tRNA mutations in humans are associated with a variety of disease states (11), tRNALeu(UUR) being frequently involved. A heteroplasmic A->G transition at np 3243 is found in association with diverse pathological phenotypes (12-14), ranging from purely ocular myopathy through diabetes with deafness to the full MELAS syndrome (mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes). Although some of this diversity is accountable for by mutant gene dosage and tissue distribution (15,16), much of it is unexplained (17,18). Cellular phenotypes associated with the np 3243 and other mitochondrial mutations have been studied in cybrid cells in which patient-derived mtDNA has been transferred to [rho]0 cells of a defined nuclear background (19-22). This has enabled clear demonstration of a mutant gene dosage effect for the np 3243 mutation in each of two nuclear backgrounds (143B osteosarcoma and A549 lung carcinoma cells). Cybrids with 60-90% mutant mtDNA exhibit only a mild complex I defect (22), but at >90% mutant DNA a general impairment of respiratory function is found, with almost complete loss of mitochondrial protein synthesis at a mutant level of 98% or greater. The mutation is associated with a drop in the steady-state level of tRNALeu(UUR) (21). In 143B cells this has been proposed to be associated with impaired aminoacylation (23; J.M.W. van den Ouweland and J.A. Enriquez, personal communication). An aminoacylation defect is also found for the np 8344 tRNALys mutation, associated with the MERRF syndrome (24).

Mitochondrial protein synthesis and respiration can be restored to wild-type levels in lung carcinoma cybrids containing 99% np 3243 mutant mtDNA by the presence of a heteroplasmic, second site mutation (at np 12300) in the anticodon of the tRNALeu(CUN) gene (21). The mutation is predicted to create a suppressor tRNA capable of decoding UUR leucine codons. This observation supports the hypothesis that the primary molecular defect in the case of the np 3243 mutation is the inability to translate the UUR codon group.

Table 1. Generally accepted decoding specificities of human mitochondrial tRNAs with wobble base U34a
U34 unmodified U34 hypermodified
tRNA/anticodon Codon group tRNA/anticodon Codon group
Thr/UGU ACN Lys/U*UU AAR
Pro/UGG CCN Gln/U*UG CAR
Arg/UCG CGN Glu/U*UC GAR
Leu/UAG CUN Trp/U*CA UGR
Ala/UGC GCN Leu/U*AA UUR
Gly/UCC GGN    
Val/UAC GUN    
Ser/UGA UCN    
aOnly the 13 tRNAs with wobble base U34 are shown: all others except tRNAMet have G in this position and are restricted to decoding two-codon groups ending in a pyrimidine. tRNAMet (AUR codons plus initiation) has C in the wobble position.

The discovery of the np 12300 suppressor mutation raises a number of questions regarding tRNA function in human mitochondria, notably those connected with wobble base modification and decoding specificity. If U34 in the suppressor tRNA remains unmodified, the question arises why it does not also recognize UUY phenylalanine codons, inserting the wrong amino acid at these positions with consequences presumably as severe as failure to decode UUR. If, on the other hand, U34 is modified to restrict its decoding properties, the question arises how the base modification machinery recognizes the suppressor tRNA as a substrate, given that it has only a single base difference from wild-type tRNALeu(CUN).

In this paper we report an analysis of tRNALeu aminoacylation and structural isoforms in lung carcinoma cybrids containing the np 3243 mutation, alone or in combination with the np 12300 suppressor mutation at various levels. The results indicate that the suppressor tRNA is aminoacylated, almost certainly with leucine, whereas the mutant tRNALeu(UUR) is uncharged, at least in this cell background. Furthermore, in all cells tested tRNALeu(CUN) was found in two structural isoforms separable on denaturing gels, hence differing in primary structure. The two isoforms are found at different relative levels in different cell types, but one isoform is found in great preponderance in lung carcinoma cybrids containing high levels of the np 3243 mutant.


Figure 1. Wobble base U modification in human mitochondrial tRNAs. Transfer RNAs dedicated to decoding all four codons of a synonymous group, such as leucine (CUN, where N is any nucleotide), are believed to have an unmodified U in the wobble base (first anticodon) position. The left panel shows the anticodon stem and loop of human mitochondrial tRNALeu(CUN), with non-Watson-Crick base pairs indicated by a (+), plus the codon/anticodon pairing. Transfer RNAs dedicated to decoding just two synonymous codons ending in a purine (R), such as leucine (UUR), are believed to have a hypermodified U in the wobble base position, depicted by an asterisk, which restricts their decoding properties as shown (right panel).

Based on our findings, we propose that structural modification of mitochondrial tRNALeu(CUN) influences its decoding properties, providing a natural, low level suppression of tRNALeu(UUR) mutations. We further propose that modification of the suppressor tRNA bearing the np 12300 mutation restricts its decoding specificity to UUR leucine codons, preventing misreading of UUY phenylalanine codons.

RESULTS

np 3243 mutant tRNALeu(UUR) is not aminoacylated in lung carcinoma cybrids

RNA was prepared under acidic conditions from lung carcinoma cybrid cells carrying different levels of np 3243 mutant mtDNA, electrophoresed on 7 M urea-6.5% polyacrylamide gels in acetate buffer at pH 5.2, electroblotted and probed with an oligonucleotide for mitochondrial tRNALeu(UUR) (Fig. 2). All preparations showed a fast migrating species, presumed to be deacylated tRNALeu(UUR), and a slower migrating species, presumed to be the charged tRNA (25). These assignments are supported by the fact that pretreatment of the RNA at pH 9.0 for 30 min at 37°C quantitatively converted the slow into the faster migrating material. Furthermore, reprobing of such blots with a mitochondrial tRNALys-specific oligonucleotide (data not shown) also showed two bands exhibiting similar behaviour, but with much greater separation, reflecting the previously reported mobility difference engendered by the two amino acids (25). Crucially, tRNALeu(UUR) from cells carrying predominantly np 3243 mutant mtDNA was mainly in the deacylated form, almost exclusively so in RNA prepared from cells 99% mutant. This is not the result of non-specific deacylation of mitochondrial tRNA during isolation or in these specific cell lines, since tRNALeu(CUN) (Fig. 3) and also tRNALys (data not shown) were almost completely acylated in all preparations. As expected, tRNALeu(UUR) remained in the deacylated form in cells 99% np 3243 mutant but also carrying the np 12300 suppressor mutation (Fig. 2c). The mutant tRNALeu(UUR) is therefore aminoacylated very poorly, if at all, in the lung carcinoma cell background.


Figure 2. tRNALeu(UUR) aminoacylation in np 3243 cybrids. (a) Northern blot of various RNA samples, run at pH 5.2 on a 7 M urea-6.5% polyacrylamide gel and probed with oligonucleotide Leu(21), specific for tRNALeu(UUR). RNA samples were from cybrid cells containing the np 3243 mutation at the levels indicated, based on last hot cycle PCR assays as described previously (21). The final lane comprises RNA from control cybrids deacylated at pH 9. (b) Longer exposure of the two left hand lanes from (a), containing RNA from cybrid cells with high levels of np 3243 mutant. (c) Equivalent blot of RNA from GT-10 cells, containing 99% np 3243 and 62% np 12300 mutant mtDNA, and from control cybrids (line B), containing 100% wild-type mtDNA at both sites.


Figure 3. tRNALeu(CUN) aminoacylation in suppressor cybrids. (a) Genotyping of lung carcinoma cybrids at np 12300. The three panels show genotyping by last hot cycle PCR of two subcloned derivatives of the original np 12300 suppressor cybrid cell line (21) plus a cybrid cell line without suppressor. The mutation creates a restriction site for AflII, cutting a 718 bp PCR product of the tRNALeu(CUN) region into two fragments of 466 and 252 bp. Both of the suppressor derivatives remained 99% mutant at np 3243, based on similar assays for the tRNALeu(UUR) region (data not shown). (b) Northern blot of various RNA samples, run at pH 5.2 on a 7 M urea-6.5% polyacrylamide gel and probed with oligonucleotide Leu(11), specific for tRNALeu(CUN). RNA samples were from cybrid cells as described above plus those containing the np 12300 suppressor mutation as depicted in (a). The final lane comprises RNA from control cybrids deacylated at pH 9. The dotted lines indicate minor species of acylated and deacylated tRNA detectable in appreciable amounts only in the control cybrids.

np 12300 suppressor mutant tRNALeu(CUN) is aminoacylated

Similar preparations of RNA obtained from two subclones of the suppressor cell line, GT-16 and GT-10, containing, respectively, 22 and 62% np 12300 mutant mtDNA (Fig. 3a), were fractionated and blotted under similar conditions. Blots were probed with an oligonucleotide for mitochondrial tRNALeu(CUN) from a region outside the anticodon and thus predicted to react equally well with the suppressor and wild-type tRNAs. The pattern of aminoacylation of tRNALeu(CUN) from the two subclones of suppressor cells was indistinguishable from that of np 3243 mutant cybrids not carrying the suppressor mutation (Fig. 3b), indicating that the suppressor tRNA is efficiently aminoacylated. Moreover, no additional band suggestive of aminoacylation by a different amino acid was evident. The suppressor tRNA is therefore charged with leucine or with an amino acid that alters its mobility in exactly the same way as leucine.

Deacylated mitochondrial tRNALeu(CUN) is present in two isoforms

In the blots shown (Fig. 3b) it is evident that an additional, minor band is present in deacylated tRNALeu(CUN) extracted from control cybrid cells (100% wild-type at both np 3243 and np 12300). This does not co-migrate with leucylated tRNALeu(CUN). Moreover, a band at similar relative abundance compared with the main species is also seen in the acylated tRNALeu(CUN) from control cybrid cells, indicating that the alternative form of the tRNA is also efficiently charged. The band representing the additional isoform was present only at very low levels in the np 3243 mutant cybrids, requiring long exposure times to be visible. This was also the case in cybrids containing, in addition, the np 12300 suppressor mutation.

Electrophoresis of RNA on denaturing 7 M urea-20% polyacrylamide gels at pH 8, followed by electroblotting and probing for tRNALeu(CUN) (Fig. 4a), also revealed two isoforms from control cybrid cells, with the slower migrating species present only at very low levels in np 3243 mutant cybrid cells. This pattern was seen reproducibly in a number of independent RNA preparations: control cybrids always showed two clear bands, one of which was greatly reduced in np 3243 mutant cybrids, whether or not the np 12300 suppressor mutation was also present. The faster migrating form appeared to resolve to a doublet on close inspection of gels showing the best fractionation (data not shown). The alkaline pH of this gel system (note also the denaturation step prior to loading) degrades aminoacylated tRNA (26), therefore the isoforms detected are not connected with aminoacylation. The `slow' and `fast' migrating isoforms were observed to differ in relative amounts between cell types (Fig. 4c), with the `slow' form clearly visible in all cells and predominant in HeLa cells. Comparable gels always showed only a single band for tRNALeu(UUR) (Fig. 4d). Re-probing stripped blots (for example Fig. 4c) for mitochondrial tRNASer(UCN), another tRNA with a U in the wobble base position that is proposed to decode four synonymous codons, revealed at least three isoforms clearly visible on a pH 8 gel, with a fourth visible at long exposure. The ratio of these isoforms also differed between cell types, with a slow migrating form predominant in HeLa cells.


Figure 4. Isoforms of mitochondrial tRNAs in human cells; 7 M urea-20% polyacrylamide gel (pH 7.8) blots were probed, as indicated, for mitochondrial tRNALeu(CUN), tRNALeu(UUR) or tRNAser(UCN). (a, b and d) RNA samples from lung carcinoma cybrids containing the np 3243 and np 12300 mutations at the levels indicated. (a) RNA isolates from two different cultures each of control cybrids and of a suppressor cybrid subclone with 22% np 12300 mutant mtDNA. (b) A shorter exposure of tracks from (a). (c) RNA samples from various human cell lines: 293-EBNA (denoted 293), HeLa, 143B, plus a 143B osteosarcoma cell cybrid (denoted 143B[prime]), containing 100% wild-type mtDNA from a patient heteroplasmic for the np 7472 mutation (39).

DISCUSSION

These results demonstrate two new molecular correlates of the np 3243 mutation in lung carcinoma cybrid cells. Firstly, 3243 mutant tRNALeu(UUR) is not aminoacylated. Secondly, the structure of the other mitochondrial tRNALeu(CUN) is altered. These findings lead us to propose a model relating mitochondrial tRNA modification, decoding specificity and np 3243 disease pathogenesis and progression.

Aminoacylation defect of np 3243 mutant tRNALeu(UUR)

The findings reported here represent the clearest demonstration to date that the np 3243 mutation provokes a translational defect by impairing the translation of leucine UUR codons. This is wholly consistent with the earlier characterization of the np 12300 suppressor mutation, which creates a novel tRNA predicted to decode the UUR codon group (21). Whilst other explanations for the pathogenic effects of the mutation cannot be entirely discounted (19), its primary molecular consequences appear to be on translation. Our findings are consistent with an earlier report (23), not yet published in full, suggesting an aminoacylation defect in osteosarcoma cybrids containing the same mutation. Nevertheless, the fact that cells containing almost no detectable leucylated-tRNALeu(UUR) are still able to make some, albeit greatly reduced amounts, of mitochondrial translation products suggests that some other tRNA is able to decode UUR codons.

Indirect effects of the np 3243 mutation on the structure of tRNALeu(CUN)

A further observation we have made is that lung carcinoma cybrids with high levels of np 3243 mutant mtDNA exhibit a consistently altered pattern of structural isoforms of another mitochondrial tRNA, previously designated as being dedicated to the translation of the CUN codon group. The two isoforms of tRNALeu(CUN) are electrophoretically separable on denaturing gels and therefore may be presumed to differ in primary structure. Since alternative processing sites and cut-and-paste type RNA editing (28) are unprecedented in mammalian mitochondrial tRNAs, differential base modification is by far the likeliest explanation, although this remains to be demonstrated directly. This hypothesis is supported by the fact that the isoforms are better resolved on the lower percentage gel run at acidic pH (Fig. 3), where mobility can be influenced by conformation (25).

The generality of this observation will now need to be tested in other mutant cells, in order to evaluate its significance. Thus far, all cybrids tested were derived from a single patient with np 3243 disease and of a common nuclear background. Although multiple, independent cybrids showed the effect, including cells also carrying the np 12300 suppressor mutation, we cannot exclude that it is influenced by mitochondrial haplotype, nuclear genotype or cell type. It will also be interesting to examine cybrids carrying other mitochondrial tRNALeu mutations.

The altered pattern of tRNALeu(CUN) isoforms in cybrids containing np 3243 mutant mtDNA is not a consequence of translational impairment per se, since cells also carrying the np 12300 suppressor mutation exhibit normal levels and patterns of mitochondrial protein synthesis, yet still show the altered pattern. Further work will be required to analyse the exact structure of the suppressor tRNA and of the isoforms of mitochondrial tRNALeu(CUN) present in np 3243 cybrids and in human cells in general. Hypermodification of the wobble base U34 is a strong candidate for involvement.

A natural suppression mechanism for the np 3243 mutation?

An alteration in tRNALeu(CUN) structure could provide an alternative mechanism for decoding UUR leucine codons; in other words a natural suppression mechanism. A scheme whereby this might be achieved is illustrated in Figure 5, which extends the generally accepted model for U34 wobble base modification already presented in Table 1. In the proposed scheme, tRNALeu(CUN) exists in all cells in two forms: one (fast form) hypermodified at the wobble base to favour decoding of CUR codons, the other (slow form) either unmodified or differently modified at U34 to favour decoding of CUY codons. Both codon groups are well represented in human mitochondrial genes (29). The hypermodified base creates strong interactions with wobble position purines, which might also enable the modified tRNA to interact weakly with UUR codons, via G-U base pairing at the first nucleotide position. First position G-U wobble is required for the decoding of GUG start codons by tRNAMet in both bacteria and mitochondria (30,31) and has been found in a plant cytosolic tRNA, also with hypermodified U in the wobble base position (32). An increase in the relative amount of the hypermodified tRNA would presumably assist the mitochondrial translational apparatus to cope with an insufficiency of functional tRNALeu(UUR). Furthermore, it could also permit segregation of the np 12300 suppressor mutation in culture, since the suppressor tRNA, if hypermodified, would be unable to mistranslate UUY phenylalanine codons.


Figure 5. A model for natural suppression of the np 3243 mutation. The figure shows the anticodon stem and loop of human mitochondrial tRNALeu(CUN), with non-Watson-Crick base pairs indicated by a (+). Wobble base U hypermodification, denoted by an asterisk, restricts specificity to codons ending in a purine (R), with the unmodified U suggested to promote interaction preferentially with the CUY codon group (Y, pyrimidine). Via first base G-U wobble, the hypermodified tRNA is proposed also to recognize UUR codons, hence compensating for the deficiency of functional tRNALeu(UUR) arising from the presence of the np 3243 mutation. Induction of the wobble base U modification system is proposed to be a response to the presence of high levels of np 3243 mutant mtDNA. The suppressor tRNA, in which the final nucleotide of the anticodon is mutated to A, recognizes exclusively UUR codons, with the wobble base U suggested to remain almost completely hypermodified in the suppressor cells, to prevent misrecognition of UUY codons.

In an extreme situation, if the hypermodification became too prevalent, it might deleteriously affect mitochondrial translation in a variety of ways. This might explain why mitochondrial translation products still appear to be synthesized in some cells with high levels of np 3243 mutant mtDNA, e.g. EBV-transformed lymphocytes (27), but are nevertheless structurally abnormal and defective for respiration. An interaction with the base modification machinery may also be relevant to the pathological features associated with several reported mutations in tRNALeu(CUN) (33,34).

At least one other human mitochondrial tRNA, for the Ser(UCN) codon group, exists in multiple isoforms (Fig. 4c) (35), hence it is tempting to speculate that this may represent a more general mechanism for expanding the decoding properties of the reduced set of tRNAs in mitochondria.

In conclusion, our findings suggest that cells can respond, either by selection or regulation, to compensate for the defects in mitochondrial translation provoked by the np 3243 and perhaps other tRNALeu(UUR) mutations. Mutations in this gene are strikingly common in mitochondrial disease and a compensation mechanism would offer some explanation for their prevalence. A variable ability to respond in this way to the mutation may also underlie some of the phenotypic variability of np 3243 disease.

MATERIALS AND METHODS

Cell lines and culture

A549 lung carcinoma cybrid cell lines containing np 3243 and np 12300 mutant mtDNAs have been described previously (21,36). All cell lines were maintained in Dulbecco's modified Eagle's medium with 10% fetal bovine serum with a 150 µg/ml uridine supplement. Subclones of line GT, which contains the np 12300 suppressor mutation, were obtained at limiting dilution and genotyped successively over several months in culture to confirm stability of their mitochondrial genotype.

Oligonucleotides

Oligonucleotides Leu(11) and Leu(21), specific to human mitochondrial tRNALeu(CUN) and tRNALeu(UUR), respectively, have been described previously (21). Oligonucleotide Ser(11), used to detect mitochondrial tRNASer(UCN) on northern blots, has the sequence AAGGAAGGAATCGAACCCCCCAAAGCTG. Oligonucleotides for PCR-based genotyping of the np 3243 and np 12300 mutations were as described previously (21).

DNA and RNA extraction

DNA was isolated from cell lines for PCR analysis as described previously (37,38). RNA was isolated from A549 lung carcinoma cybrid cells under acidic conditions, using the Trizol method, also as previously (21). RNA prepared similarly from other cell lines was kindly provided by Hans Spelbrink (293-EBNA and HeLa), Nicole Hance (143B) and Marina Toompuu [143B cybrid line containing 100% wild-type mtDNA from a patient with the np 7472 mutation (39)].

Genotyping of mtDNA by RFLP analysis of PCR products

PCR conditions, radiolabelling, restriction digestion, gel electrophoresis and phosphorimaging to analyse mtDNA genotype at np 3243 and np 12300 were as described previously (21). Briefly, the np 3243 mutation was quantified by ApaI digestion of a 690 bp last hot cycle PCR product spanning the tRNALeu(UUR) region, generating products of 230 and 460 bp from the mutant allele. The np 12300 mutation was similarly quantified using AflII digestion of a 718 bp product spanning the tRNALeu(CUN) region, generating products of 252 and 466 bp from the mutant allele.

Gel electrophoresis and northern blots

For analysis of aminoacylation, 10-20 µg aliquots of total RNA, isolated under acidic conditions, were fractionated on 1.5 mm thick 7 M urea-6.5% polyacrylamide gels run in 0.1 M sodium acetate buffer, pH 5.2, at 4°C, at 5.5 V/cm over 20 h with buffer recirculation, essentially as described by Enriquez and Attardi (26). Deacylation of specific tRNA samples was carried out in 0.3 M Tris-HCl, pH 9.0, at 37°C for 30 min. For analysis of deacylated tRNAs under denaturing conditions, RNA aliquots of 10-20 µg were heated at 65°C for 5 min in 70% formamide, 7 mM EDTA, pH 8.0, then fractionated on 7 M urea-20% polyacrylamide gels run in TBE buffer (pH 7.8), at 20-25 V/cm over 24 h. Samples were electroblotted to Zeta-Probe GT membrane (Bio-Rad) (15 min at 15 V, then 1 h at 30 V in TAE or TBE buffer at 4°C). Northern hybridization to end-labelled oligonucleotide probes was carried out as described previously (21). Prior to re-hybridization with a different probe, filters were stripped twice for 20 min in 1× SSC, 0.1% SDS at 95°C and checked by overnight autoradiography.

ACKNOWLEDGEMENTS

We thank Marina Toompuu, Nicole Hance and Hans Spelbrink for providing RNA samples from various human cell lines and Anja Rovio for technical assistance. This work was supported financially by the Academy of Finland, Juselius Foundation, Tampere University Hospital Medical Research Fund, the Muscular Dystrophy group of Great Britain, the Royal Society (University Research Fellowship to I.J.H.) and the European Union.

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*To whom correspondence should be addressed at Institute of Medical Technology and Tampere University Hospital, University of Tampere, PO Box 607, 33101 Tampere, Finland. Tel: +358 3 215 7731; Fax: +358 3 215 7731; Email: howy.jacobs@uta.fi

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