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• W2124207622 abstract "The pathogenetic mechanism of the mitochondrial tRNALeu(UUR) A3243G transition associated with the mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome has been investigated in transmitochondrial cell lines constructed by transfer of mutant mitochondrial DNA (mtDNA)-carrying mitochondria from three genetically unrelated MELAS patients or of isogenic wild-type mtDNA-carrying organelles into human mtDNA-less cells. An in vivofootprinting analysis of the mtDNA segment within the tRNALeu(UUR) gene that binds the transcription termination factor failed to reveal any difference in occupancy of sites or qualitative interaction with the protein between mutant and wild-type mtDNAs. Cell lines nearly homoplasmic for the mutation exhibited a strong (70–75%) reduction in the level of aminoacylated tRNALeu(UUR) and a decrease in mitochondrial protein synthesis rate. The latter, however, did not show any significant correlation between synthesis defect of the individual polypeptides and number or proportion of UUR codons in their mRNAs, suggesting that another step, other than elongation, may be affected. Sedimentation analysis in sucrose gradient showed a reduction in size of the mitochondrial polysomes, while the distribution of the two rRNA components and of the mRNAs revealed decreased association of mRNA with ribosomes and, in the most affected cell line, pronounced degradation of the mRNA associated with slowly sedimenting structures. Therefore, several lines of evidence indicate that the protein synthesis defect in A3243G MELAS mutation-carrying cells is mainly due to a reduced association of mRNA with ribosomes, possibly as a consequence of the tRNALeu(UUR)aminoacylation defect. The pathogenetic mechanism of the mitochondrial tRNALeu(UUR) A3243G transition associated with the mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome has been investigated in transmitochondrial cell lines constructed by transfer of mutant mitochondrial DNA (mtDNA)-carrying mitochondria from three genetically unrelated MELAS patients or of isogenic wild-type mtDNA-carrying organelles into human mtDNA-less cells. An in vivofootprinting analysis of the mtDNA segment within the tRNALeu(UUR) gene that binds the transcription termination factor failed to reveal any difference in occupancy of sites or qualitative interaction with the protein between mutant and wild-type mtDNAs. Cell lines nearly homoplasmic for the mutation exhibited a strong (70–75%) reduction in the level of aminoacylated tRNALeu(UUR) and a decrease in mitochondrial protein synthesis rate. The latter, however, did not show any significant correlation between synthesis defect of the individual polypeptides and number or proportion of UUR codons in their mRNAs, suggesting that another step, other than elongation, may be affected. Sedimentation analysis in sucrose gradient showed a reduction in size of the mitochondrial polysomes, while the distribution of the two rRNA components and of the mRNAs revealed decreased association of mRNA with ribosomes and, in the most affected cell line, pronounced degradation of the mRNA associated with slowly sedimenting structures. Therefore, several lines of evidence indicate that the protein synthesis defect in A3243G MELAS mutation-carrying cells is mainly due to a reduced association of mRNA with ribosomes, possibly as a consequence of the tRNALeu(UUR)aminoacylation defect. mitochondrial DNA mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes mitochondrial termination factor myoclonic epilepsy and ragged red fibers dimethyl sulfate light strand heavy strand Mutations in tRNA genes constitute a large proportion, ∼75% (1.Schon E.A. Bonilla E. DiMauro S. J. Bioenerg. Biomembr. 1997; 29: 131-149Crossref PubMed Scopus (387) Google Scholar), of the disease-causing mutations identified so far in human mitochondrial DNA (mtDNA).1Investigations of their pathogenetic mechanisms can provide important information on mitochondrial protein synthesis and other aspects of mitochondrial biogenesis. Among such studies are those on the A8344G mutation in tRNALys, which causes myoclonic epilepsy and ragged red fiber syndrome (MERRF) (2.Shoffner J.M. Lott M.T. Lezza A.M.S. Seibel P. Ballinger S.W. Wallace D.C. Cell. 1990; 61: 931-937Abstract Full Text PDF PubMed Scopus (1284) Google Scholar), and those on the T7445C mutation in the nucleotide immediately adjacent to the 3′-end of the tRNASer(UCN) gene, which is associated with deafness (3.Reid F.M. Vernham G.A. Jacobs H.T. Hum. Mutat. 1994; 3: 243-247Crossref PubMed Scopus (239) Google Scholar,4.Fischel-Ghodsian N. Prezant T.R. Fournier P. Stewart I.A. Maw M. Am. J. Otolaryngol. 1995; 16: 403-408Crossref PubMed Scopus (126) Google Scholar). The former tRNA mutation causes premature termination of translation, most likely due to a tRNALys aminoacylation deficiency (5.Enriquez J.A. Chomyn A. Attardi G. Nat. Genet. 1995; 10: 47-55Crossref PubMed Scopus (262) Google Scholar), while the latter mutation produces a reduction in the level of tRNASer(UCN), due to a defect of precursor processing, that also reduces the level of the co-transcribed ND6 mRNA (6.Guan M.-X. Enriquez J.A. Fischel-Ghodsian N. Puranam R.S. Lin C.P. Maw M.A. Attardi G. Mol. Cell. Biol. 1998; 18: 5868-5879Crossref PubMed Scopus (176) Google Scholar). Protein synthesis in mitochondria with the latter mutation is presumably also affected at the level of elongation, but, rather than premature termination, there appears to be a pause in elongation, followed by a resumption of synthesis. The pathogenetic mechanism of the most extensively investigated tRNA gene mutation, the A3243G transition in the tRNALeu(UUR)gene, which causes the MELAS encephalomyopathy (7.Goto Y. Nonaka I. Horai S. Nature. 1990; 348: 651-653Crossref PubMed Scopus (1766) Google Scholar), maternally inherited diabetes (8.van den Ouweland J.M.W. Lemkes H.H.P.J. Ruitenbeek W. Sandkuijl L.A. de Vijlder M.F. Struyvenberg P.A.A. van den Kamp J.J.P. Maassen J.A. Nat. Genet. 1992; 1: 368-371Crossref PubMed Scopus (1061) Google Scholar), or chronic progressive external ophthalmoplegia (9.Johns D.R. Hurko O. Lancet. 1991; 337: 927-928Abstract PubMed Scopus (26) Google Scholar), is still unresolved, despite the numerous investigations on this topic. The mutation occurs in the middle of the binding site for the mitochondrial transcription termination factor, mTERF (10.Kruse B. Narasimhan N. Attardi G. Cell. 1989; 58: 391-397Abstract Full Text PDF PubMed Scopus (225) Google Scholar) (Fig.1 a). In vitro experiments have shown that the mutation does indeed reduce the affinity of mTERF for the DNA (11.Chomyn A. Martinuzzi A. Yoneda M. Daga A. Hurko O. Johns D. Lai S.T. Nonaka I. Angelini C. Attardi G. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4221-4225Crossref PubMed Scopus (447) Google Scholar) and cause a decrease in the rate of termination of the rDNA transcription unit (12.Hess J.F. Parisi M.A. Bennett J.L. Clayton D.A. Nature. 1991; 351: 236-239Crossref PubMed Scopus (200) Google Scholar). However, an analysis of mtDNA-less human cells repopulated with mitochondria from MELAS patients (11.Chomyn A. Martinuzzi A. Yoneda M. Daga A. Hurko O. Johns D. Lai S.T. Nonaka I. Angelini C. Attardi G. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4221-4225Crossref PubMed Scopus (447) Google Scholar, 13.King M.P. Koga Y. Davidson M. Schon E.A. Mol. Cell. Biol. 1992; 12: 480-490Crossref PubMed Scopus (408) Google Scholar) did not reveal any significant difference from the controls in the relative steady-state levels of the two rRNA species, encoded upstream of the termination site, and of the mRNAs encoded downstream. On the other hand, the mitochondrial protein synthesis rate has been found to be decreased, as was the respiration rate, in cell lines carrying the mutation (11.Chomyn A. Martinuzzi A. Yoneda M. Daga A. Hurko O. Johns D. Lai S.T. Nonaka I. Angelini C. Attardi G. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4221-4225Crossref PubMed Scopus (447) Google Scholar,13.King M.P. Koga Y. Davidson M. Schon E.A. Mol. Cell. Biol. 1992; 12: 480-490Crossref PubMed Scopus (408) Google Scholar). The protein synthesis defect has been proposed to be due to stalling of translation by pseudoribosomes that have incorporated RNA 19, an incompletely processed transcript reported to accumulate in A3243 mutant cells, in place of 16 S rRNA (14.Schon E.A. Koga Y. Davidson M. Moraes C.T. King M.P. Biochim. Biophys. Acta. 1992; 1101: 206-209PubMed Google Scholar), or possibly to defective posttranscriptional modification of the tRNALeu(UUR) (15.Helm M. Florentz C. Chomyn A. Attardi G. Nucleic Acids Res. 1999; 27: 756-763Crossref PubMed Scopus (90) Google Scholar,16.Yasukawa T. Suzuki T. Suzuki T. Uedo T. Ohta S. Watanabe K. J. Biol. Chem. 2000; 275: 4251-4257Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). Yet others suggest that the mutant tRNA may cause mistranslation, leading to accelerated turnover of mitochondrial translation products (17.Flierl A. Reichmann H. Seibel P. J. Biol. Chem. 1997; 272: 27189-27196Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 18.Janssen G.M.C. Maassen J.A. van den Ouweland J.M.W. J. Biol. Chem. 1999; 274: 29744-29748Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). The results described in the present work do not fit a model in which, in MELAS mutation-carrying cells, mitochondrial translation is affected mainly at the level of elongation, either prematurely terminating at or near UUR codons or pausing at UUR codons and then resuming elongation. On the contrary, they give strong support to a model in which the A3243G mutation affects both the steady state level and the aminoacylation efficiency of the tRNALeu(UUR) and, possibly as a consequence of these changes, reduces the rate of assembly of mRNA with ribosomes, with a resulting decrease in mitochondrial translation rate. The cell lines used for these studies have been described previously (11.Chomyn A. Martinuzzi A. Yoneda M. Daga A. Hurko O. Johns D. Lai S.T. Nonaka I. Angelini C. Attardi G. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4221-4225Crossref PubMed Scopus (447) Google Scholar, 19.Chomyn A. Meola G. Bresolin N. Lai S.T. Scarlato G. Attardi G. Mol. Cell. Biol. 1991; 11: 2236-2244Crossref PubMed Scopus (274) Google Scholar, 20.Yoneda M. Chomyn A. Martinuzzi A. Hurko O. Attardi G. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11164-11168Crossref PubMed Scopus (242) Google Scholar). 143B.TK− is a human osteosarcoma-derived cell line (ATCC CRL 8303); the 94I, 43B, 2SA, 2SC, 4H1, and pT1 cell lines are mitochondrial transformants of an mtDNA-less (ρ0) derivative of 143B.TK−,i.e. ρ0206 (21.King M.P. Attardi G. Science. 1989; 246: 500-503Crossref PubMed Scopus (1465) Google Scholar). The cell line pT1 is a ρ0206 transformant that carries the A8344G MERRF mutation in the mitochondrial tRNALys gene in nearly 100% of its mtDNA (19.Chomyn A. Meola G. Bresolin N. Lai S.T. Scarlato G. Attardi G. Mol. Cell. Biol. 1991; 11: 2236-2244Crossref PubMed Scopus (274) Google Scholar). 143B.TK− cells and HeLa S3 cells in suspension were grown as described previously (21.King M.P. Attardi G. Science. 1989; 246: 500-503Crossref PubMed Scopus (1465) Google Scholar, 22.Micol V. Fernandez-Silva P. Attardi G. J. Biol. Chem. 1997; 272: 18896-18904Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). ρ0 cell transformants were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. For sequencing the tRNALeu(UUR)gene, the mtDNA fragment between positions 3206 and 3728 was amplified, using 20-nucleotide-long primers, by the polymerase chain reaction. The product was then purified from an agarose gel and sequenced with a primer corresponding to heavy (H)-strand nucleotides 3511–3491. Similarly, the fragment of mtDNA between positions 11836 and 12553 that contains the tRNALeu(CUN) gene was amplified and then sequenced with light (L)-strand primer L12080–12099 or H-strand primerH12500–12479. Total cellular DNA on slot blots was hybridized with a 498-nucleotide probe for nuclear ribosomal DNA, stripped, and then rehybridized with a 2537-nucleotide probe for mitochondrial ribosomal DNA as described (20.Yoneda M. Chomyn A. Martinuzzi A. Hurko O. Attardi G. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11164-11168Crossref PubMed Scopus (242) Google Scholar). This was done as described previously (22.Micol V. Fernandez-Silva P. Attardi G. J. Biol. Chem. 1997; 272: 18896-18904Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 23.Micol V. Fernandez-Silva P. Attardi G. Methods Enzymol. 1996; 264: 3-11Crossref PubMed Google Scholar). Briefly, intact cells were treated with dimethyl sulfate (DMS), which methylates unprotected guanines and adenines (24.Maxam A.M. Gilbert W. Methods Enzymol. 1980; 65: 499-560Crossref PubMed Scopus (9015) Google Scholar). Total nucleic acids were then extracted and incubated with piperidine, which cleaves the DNA preferentially at the methylated guanine residues (24.Maxam A.M. Gilbert W. Methods Enzymol. 1980; 65: 499-560Crossref PubMed Scopus (9015) Google Scholar). The DNA was subjected to multiple rounds of primer extension from a 32P-5′-end-labeled H strand primer, as described previously (23.Micol V. Fernandez-Silva P. Attardi G. Methods Enzymol. 1996; 264: 3-11Crossref PubMed Google Scholar), and the final products were resolved on a sequencing gel. Highly purified total mitochondrial tRNA was isolated as described (25.Enriquez J.A. Attardi G. Methods Enzymol. 1996; 264: 183-196Crossref PubMed Google Scholar). Quantification of the steady state levels of individual mitochondrial tRNA species was done by RNA gel blot analysis as described (5.Enriquez J.A. Chomyn A. Attardi G. Nat. Genet. 1995; 10: 47-55Crossref PubMed Scopus (262) Google Scholar), using 32P-5′-end-labeled oligonucleotides specific for tRNALeu(UUR), tRNALys, tRNASer(UCN), tRNAGlu, and 12 S rRNA, as detailed previously (6.Guan M.-X. Enriquez J.A. Fischel-Ghodsian N. Puranam R.S. Lin C.P. Maw M.A. Attardi G. Mol. Cell. Biol. 1998; 18: 5868-5879Crossref PubMed Scopus (176) Google Scholar). For quantification of the extent of aminoacylation of the tRNAs, a rapid procedure for isolation of the mitochondrial fraction was used. The tRNAs were extracted under acid conditions and then run on an acid-urea 6.5% polyacrylamide gel to separate the charged and uncharged tRNAs (5.Enriquez J.A. Chomyn A. Attardi G. Nat. Genet. 1995; 10: 47-55Crossref PubMed Scopus (262) Google Scholar, 26.Varshney U. Lee C.-P. RajBhandary U.L. J. Biol. Chem. 1991; 266: 24712-24718Abstract Full Text PDF PubMed Google Scholar). Quantification of band intensities was done by PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA) analysis. mtDNA-encoded proteins were labeled in 143B.TK− and 43B cells in vivo for 30 min with [35S]methionine in methionine-free medium in the presence of emetine, as described previously, and the labeled protein gel patterns have been published (11.Chomyn A. Martinuzzi A. Yoneda M. Daga A. Hurko O. Johns D. Lai S.T. Nonaka I. Angelini C. Attardi G. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4221-4225Crossref PubMed Scopus (447) Google Scholar). For the present work, the mtDNA-encoded proteins in 2SA and 2SC cells were labeled for 20 or 40 min in the same manner, using [35S]methionine (1175 Ci/mmol; Expre35S35S, NEN Life Science Products) at 150 μCi/ml in Dulbecco's modified Eagle's medium minus methionine, supplemented with 5% dialyzed fetal bovine serum. Lysates of the labeled cells were analyzed by SDS-polyacrylamide gel electrophoresis as described (27.Chomyn A. Methods Enzymol. 1996; 264: 197-211Crossref PubMed Google Scholar). The quantification of band intensities was done by NIH Image analysis of optical gel scans of appropriately exposed films. To examine the stability of proteins synthesized in mitochondria, a “pulse-chase” labeling protocol was used. 94I and 43B were exposed for 2 h to 0.2 mCi/ml of [35S]methionine in Dulbecco's modified Eagle's medium minus methionine, supplemented with 10% dialyzed fetal bovine serum and 100 μg/ml cycloheximide. The labeling medium was then replaced with complete, nonradioactive medium, and the cells were grown for 16 h at 37 °C before harvesting. For reference, other samples were labeled for 2 h with [35S]methionine in the presence of cycloheximide and then immediately harvested. For the analysis of mitochondrial polysomes, cells were labeled with [35S]methionine as described (5.Enriquez J.A. Chomyn A. Attardi G. Nat. Genet. 1995; 10: 47-55Crossref PubMed Scopus (262) Google Scholar) and mixed with 143B.TK− cells that had been labeled for 1 day with [5-3H]uridine in the absence of inhibitors (to provide size markers for the sucrose gradients, as described below). The mitochondrial fraction was then isolated and lysed with 2% Triton X-100 as described (28.Ojala D. Attardi G. J. Mol. Biol. 1972; 65: 273-289Crossref PubMed Scopus (40) Google Scholar) but in the presence of 5 mmdithiothreitol and 50 units/ml RNasin (Promega). The mitochondrial lysate was run on a 15–30% sucrose gradient (28.Ojala D. Attardi G. J. Mol. Biol. 1972; 65: 273-289Crossref PubMed Scopus (40) Google Scholar) in 100 mm Tris-HCl, pH 6.7 (25 °C), 100 mm KCl, 10 mm MgCl2 (TKM buffer) in a Beckman SW41 rotor at 32,600 rpm for 190 min at 2 °C. Approximately 0.3-ml fractions from the gradients were collected into tubes containing 10 μl of 2 mm dithiothreitol and 1 unit of RNasin (Promega), and a portion precipitated with 10% trichloroacetic acid in the presence of 50 μg of bovine serum albumin. The precipitates were collected on Millipore HAWP filters, and the radioactivity was counted. In a control experiment using puromycin, the cells were incubated with 150 μg/ml puromycin (Sigma) for 10 min immediately following the 5-min [35S]methionine labeling pulse. For the analysis of ribosomes and ribosomal subunits (28.Ojala D. Attardi G. J. Mol. Biol. 1972; 65: 273-289Crossref PubMed Scopus (40) Google Scholar), cells were treated for 20 min with 0.1 μg/ml actinomycin D to inhibit nuclear RNA synthesis and then labeled for 1 h (unless otherwise stated) with 0.05 mCi/ml [5-3H]uridine (22 Ci/mmol; NEN Life Science Products) in the continued presence of actinomycin D and pooled with ∼10 times as many unlabeled cells and with cells labeled with [2-14C]uridine in the absence of inhibitors (see below). The mitochondrial fraction was then isolated, lysed with 2% Triton X-100, and analyzed in the sucrose gradient described above by running it in the SW41 rotor at 28,000 rpm for 13.8 h at 2 °C. As mentioned above, size markers for the sucrose gradients were provided by the addition of [5-3H]uridine- or [2-14C]uridine-labeled 143B.TK− cells to the cells that had been labeled for polysome or ribosome analysis, respectively. In particular, 2.5 × 105 cells were labeled for 1 day with 0.25 μCi/ml of [5-3H]uridine or of [2-14C]uridine (60 mCi/mmol; ICN) in the absence of inhibitors. Under such labeling conditions, the stable nucleus-encoded large and small subunit rRNAs incorporate most of the radioactivity. Because the cytosolic ribosomes and ribosomal subunits, with sedimentation constants 74, 60, and 40 S, always contaminate the mitochondrial fraction, they are detectable in the sucrose gradient profiles of the mitochondrial lysates by virtue of their containing the long term 3H- or 14C-labeled nucleus-encoded rRNAs. For analysis of the RNAs in the various fractions of a sucrose gradient, pairs or triplets of fractions were pooled, and then 2 volumes of 50 mm Tris-HCl, pH 7.4, 0.3 m NaCl, 8 mm EDTA, 1% SDS, 150 μg/ml proteinase K, and 10 μg/ml tRNA were added. The mixtures were incubated at 37 °C for 30 min and then extracted twice with a 25:24:1 mixture of phenol/chloroform/isoamyl alcohol equilibrated with 10 mmTris-HCl, pH 7.4, 100 mm NaCl, 1 mm EDTA, 0.5% SDS, precipitated with ethanol, and finally dissolved in 10 mm Tris-HCl, pH 7.4, 1 mm EDTA. The RNAs from the desired fractions were pooled and divided into five equal parts. An excess of unlabeled DNA probe or mixture of probes was hybridized to each aliquot of labeled RNA in 0.4 m NaCl, 10 mm Tris-HCl, pH 7.4, 1 mm EDTA for 15 h at 60 °C. After digestion with RNases T1 (50 units/ml) and A (40 μg/ml), the RNase-resistant radioactivity was determined as described (29.King M.P. Attardi G. J. Biol. Chem. 1993; 268: 10228-10237Abstract Full Text PDF PubMed Google Scholar). The probes were as follows: for the detection of 16 S rRNA, 12 S rRNA, and ND1 RNA, plasmid clones containing 805 nucleotide pairs of the 16 S rRNA gene, 800 nucleotide pairs of the 12 S rRNA gene, and 809 nucleotide pairs of the ND1 gene, respectively, were used. To probe the RNAs downstream of theND1 gene, an equimolar mixture of clones containing sequences of the COI, COII, ATPase 8/ATPase 6,COIII, ND3, ND4L/ND4, ND5, and cytochrome b genes, spanning 7424 nucleotide pairs of mRNA coding region was used. The latter set of plasmid clones contained also the sequences of tRNAs specific for Ser (both tRNAs), Asp, Lys, Glu, Arg, His, and Leu(CUN). This was done substantially as described previously (11.Chomyn A. Martinuzzi A. Yoneda M. Daga A. Hurko O. Johns D. Lai S.T. Nonaka I. Angelini C. Attardi G. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4221-4225Crossref PubMed Scopus (447) Google Scholar). The probes for ND1 mRNA, 16 S rRNA, and 12 S rRNA were plasmids carrying the human mtDNA sequences from position 2953 to 3311, from 1768 to 2573, and from 764 to 1466, respectively. The probes for ND2 and COI mRNAs were plasmid inserts that corresponded to the human mtDNA sequence from position 4431 to 5274, and from 6203 to 6910, respectively. All were labeled with [α-32P]dCTP or [α-32P]dATP by the random primer method (30.Feinberg A.P. Vogelstein B. Anal. Biochem. 1984; 137: 266-267Crossref PubMed Scopus (5190) Google Scholar). Nonlinear curve fitting was done using the Levenberg-Marquardt algorithm (KaleidaGraph software, Synergy). The cell lines 43B and 2SC, which carried the A3243G mutation in nearly homoplasmic form (99 and 97%, respectively) had been previously shown to have decreased mitochondrial protein synthesis rates (∼20 and ∼50%, respectively, of the wild-type rates, after a 30-min labeling pulse with [35S]methionine) and markedly reduced respiration rates (<10 and ∼18%, respectively, of the wild-type rates) (11.Chomyn A. Martinuzzi A. Yoneda M. Daga A. Hurko O. Johns D. Lai S.T. Nonaka I. Angelini C. Attardi G. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4221-4225Crossref PubMed Scopus (447) Google Scholar). Another mutant cell line, 4H1, that carried 98% mutant mtDNA (20.Yoneda M. Chomyn A. Martinuzzi A. Hurko O. Attardi G. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11164-11168Crossref PubMed Scopus (242) Google Scholar), was used in some of the analyses presented here. The 143B.TK− cell line, and transmitochondrial cell lines 94I and 2SA all exhibited 100% wild-type DNA at position 3243 (11.Chomyn A. Martinuzzi A. Yoneda M. Daga A. Hurko O. Johns D. Lai S.T. Nonaka I. Angelini C. Attardi G. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4221-4225Crossref PubMed Scopus (447) Google Scholar). Cell lines 94I and 43B are isogenic except at position 3243, as are the pair 2SA and 2SC. The sequences of the tRNALeu(UUR) genes in 94I and 43B had been previously shown (15.Helm M. Florentz C. Chomyn A. Attardi G. Nucleic Acids Res. 1999; 27: 756-763Crossref PubMed Scopus (90) Google Scholar) to be identical to the Cambridge sequence (31.Anderson S. Bankier A.T. Barrell B.G. de-Bruijn M.H.L. Coulson A.R. Drouin J. Eperon I.C. Nierlich D.P. Roe B.A. Sanger F. Schreier H.P. Smith A.J.H. Stader R. Young I.G. Nature. 1981; 290: 427-465PubMed Google Scholar) outside of position 3243. Polymerase chain reaction amplification and sequencing of the tRNALeu(UUR) gene from the 2SC and 4H1 cell lines (this work) also failed to reveal any difference from the standard sequence. Furthermore, in order to investigate the possible presence in the mutant cells of a suppressor mutation in the other mitochondrial tRNALeu gene, like the one at position 12,300 that has been recently reported (32.El Meziane A. Lehtinen S.K. Hance N. Nijtmans L.G.J. Dunbar D. Holt I.J. Jacobs H.T. Nat. Genet. 1998; 18: 350-353Crossref PubMed Scopus (93) Google Scholar), the mitochondrial tRNALeu(CUN) gene of the 43B, 4H1, 2SA, and 2SC cell lines was polymerase chain reaction-amplified and sequenced. No mutation at position 12,300 or at any other position was found in this gene in any of the cell lines. The amount of mtDNA relative to nuclear DNA in the cell lines 94I, 43B, 2SA, 2SC, and 4H1 was 129, 103, 130, 100, and 83%, respectively, of that in 143B.TK− cells. Previous evidence from in vitro experiments had shown that the A3243G mutation decreased the binding of mTERF to mtDNA and the efficiency of transcription termination (11.Chomyn A. Martinuzzi A. Yoneda M. Daga A. Hurko O. Johns D. Lai S.T. Nonaka I. Angelini C. Attardi G. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4221-4225Crossref PubMed Scopus (447) Google Scholar, 12.Hess J.F. Parisi M.A. Bennett J.L. Clayton D.A. Nature. 1991; 351: 236-239Crossref PubMed Scopus (200) Google Scholar). In view of the discrepancy of these results with the observed lack of any effect of the mutation on the ratio of transcripts downstream to those upstream of the termination site (11.Chomyn A. Martinuzzi A. Yoneda M. Daga A. Hurko O. Johns D. Lai S.T. Nonaka I. Angelini C. Attardi G. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4221-4225Crossref PubMed Scopus (447) Google Scholar, 13.King M.P. Koga Y. Davidson M. Schon E.A. Mol. Cell. Biol. 1992; 12: 480-490Crossref PubMed Scopus (408) Google Scholar), in the present work in vivo footprinting experiments were carried out to determine whether the A3243G mutation reduces the occupancy of the mTERF binding sites or causes changes in the methylation interference pattern in living cells. Fig. 1 b shows the in vivo footprint of the mtDNA L strand of the rDNA transcription termination region for several cell lines that are wild-type at position 3243, namely HeLa, 143B.TK−, 94I, and 2SA, and two cell lines that carry the A3243G transition (in ≥97% of their mitochondrial genomes), 43B and 2SC. The footprints of the protein can be seen in the lanes marked E between positions 3232 and 3256, in agreement with data from in vitro DNase I protection experiments (10.Kruse B. Narasimhan N. Attardi G. Cell. 1989; 58: 391-397Abstract Full Text PDF PubMed Scopus (225) Google Scholar) and from previously reported in vivofootprinting experiments (22.Micol V. Fernandez-Silva P. Attardi G. J. Biol. Chem. 1997; 272: 18896-18904Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). The region protected from modification by DMS is the same in wild-type and mutant cells. An analysis of the patterns from mutant cells, after normalization to reference bands outside the mTERF binding site, reveals protection of the new G residue at position 3243. As the adjacent G at position 3242 is protected in both wild-type and mutant cells, it is likely that protein protects position 3243 in control cells as well as in mutant cells, but this effect is not detected because methylated A is not efficiently cleaved by piperidine (24.Maxam A.M. Gilbert W. Methods Enzymol. 1980; 65: 499-560Crossref PubMed Scopus (9015) Google Scholar). The G at position 3244 is affected in its in vivo methylation pattern, but inconsistently; it is hypermethylated in 143B.TK− and HeLa cells, in agreement with published data (22.Micol V. Fernandez-Silva P. Attardi G. J. Biol. Chem. 1997; 272: 18896-18904Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar), and also in 94I cells. By contrast, this G is protected in 43B mutant cells. However, the modification of methylation of this G is marginal in 2SA and 2SC cells. It is not clear whether any significance is to be attributed to this variable base modification. Apart from the differences at 3243 and, possibly, at 3244, there are no significant qualitative differences in the methylation interference pattern of the mitochondrial rDNA termination region between the mutants 43B and 2SC, on one hand, and their isogenic controls, 94I and 2SA, respectively, or the parental cell line 143B.TK−, on the other. Furthermore, a densitometric analysis showed that, after correction for loading differences, there is no significant consistent quantitative change between mutant and control patterns in the nucleotide protection or hypermethylation within the mTERF binding segment in the tRNALeu(UUR) gene. The transfer hybridization analysis of mitochondrial tRNAs in Fig.2 a shows that there is no obvious difference in electrophoretic mobility between the mutant tRNA and the wild-type species (compare, for example, 2SA and 2SC). Correct processing of the mutant tRNALeu(UUR) from its precursor had previously been shown by a structural analysis of the 5′- and 3′-ends of mutant and wild-type tRNALeu(UUR) (33.Koga Y. Davidson M. Schon E.A. King M.P. Nucleic Acids Res. 1993; 21: 657-662Crossref PubMed Scopus (37) Google Scholar) and confirmed recently by direct analysis of the nucleotide composition (including modified nucleotides) of the radioactively pure tRNA (15.Helm M. Florentz C. Chomyn A. Attardi G. Nucleic Acids Res. 1999; 27: 756-763Crossref PubMed Scopus (90) Google Scholar). On the other hand, the steady state amount of tRNALeu(UUR), when normalized to the values for tRNALys, tRNASer, tRNAGlu, and 12 S rRNA, appears to be reduced in 43B to 47, 35, 42, and 60%, respectively (mean 46%), of the isogenic control, 94I; in 2SC to 41, 32, 42, and 69% (mean 46%) of the isogenic control, 2SA; and in 4H1 to 63, 55, 46, and 86% o" @default.
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• W2124207622 title "The Mitochondrial Myopathy, Encephalopathy, Lactic Acidosis, and Stroke-like Episode Syndrome-associated Human Mitochondrial tRNALeu(UUR) Mutation Causes Aminoacylation Deficiency and Concomitant Reduced Association of mRNA with Ribosomes" @default.
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