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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Mitochondrion. Author manuscript; available in PMC Jul 16, 2010.
Published in final edited form as:
PMCID: PMC2905380
NIHMSID: NIHMS214112

Mutation in MTO1 involved in tRNA modification impairs mitochondrial RNA metabolism in the yeast Saccharomyces cerevisiae

Abstract

The yeast MTO1 gene encodes an evolutionarily conserved protein for the biosynthesis of the 5-carboxymethylaminomethyl group of cmnm5s2U in the wobble position of mitochondrial tRNA. However, mto1 null mutant expressed the respiratory deficient phenotype only when coupled with the C1409G mutation of mitochondrial 15S rRNA. To further understand the role of MTO1 in mitochondrial RNA metabolism, the yeast mto1 null mutants carrying either wild-type (PS) or 15S rRNA C1409G allele (PR) have been characterized by examining the steady-state levels, aminoacylation capacity of mitochondrial tRNA, mitochondrial gene expression and petite formation. The steady-state levels of tRNALys, tRNAGlu, tRNAGln, tRNALeu, tRNAGly, tRNAArg and tRNAPhe were decreased significantly while those of tRNAMet and tRNAHis were not affected in the mto1 strains carrying the PS allele. Strikingly, the combination of the mto1 and C1409G mutations gave rise to the synthetic phenotype for some of the tRNAs, especially in tRNALys, tRNAMet and tRNAPhe. Furthermore, the mto1 strains exhibited a marked reduction in the aminoacylation levels of mitochondrial tRNALys, tRNALeu, tRNAArg but almost no effect in those of tRNAHis. In addition, the steady-state levels of mitochondrial COX1, COX2, COX3, ATP6 and ATP9 mRNA were markedly decreased in mto1 strains. These data strongly indicate that unmodified tRNA caused by the deletion of MTO1 gene caused the instability of mitochondrial tRNAs and mRNAs and an impairment of aminoacylation of mitochondrial tRNAs. Consequently, the deletion of MTO1 gene acts in synergy with the 15S rRNA C1409G mutation, leading to the loss of COX1 synthesis and subsequent respiratory deficient phenotype.

Keywords: Mitochondrial tRNA, Nucleotide modification, 15S rRNA, RNA metabolism, Yeast

1. Introduction

All tRNA species contain modified nucleosides, which are derivatives of the four normal nucleotides adenosine (A), guanosine (G), uridine (U) and cytidine (C) (Björk, 1995). In particular, nucleoside at the position 34 (wobble position of anticodon) of tRNAs is more prone to modification than those at other positions of tRNA (Hopper and Phizicky, 2003). In Escherichia coli, the hyper-modified nucleoside 5-methyl-aminomethyl-2-thio-uridine (mnm5s2U34) occurs at the wobble position of tRNALys, tRNAGlu and tRNAGln (Hopper and Phizicky, 2003). The synthesis of mnm5s2U is a complicated process with the multiple steps in bacterial tRNAs (Brégeon et al., 2001). The products of trmE (Cabedo et al., 1999), gidA (homolog of MTO1) (Brégeon et al., 2001) and trmU (Kambampati and Lauhon, 2003; Yan et al., 2005) are components of the enzyme complex for the biosynthesis of mnm5s2U34 in the tRNAs (Björk, 1995). This modified nucleoside plays an important role in the structure and function of tRNAs, including stability, turnover, aminoacylation of tRNAs and codon recognition (Alexandrov et al., 2006; Hagervall and Björk, 1984; Sullivan et al., 1985).

Mto1p/GidA is involved in the biosynthesis for the C5 modification of mnm5s2U34 of tRNALys, tRNAGlu and tRNAGln in E. coli and yeast mitochondria (Yim et al., 2006; Umeda et al., 2005; Li et al., 2002, 2003). E. coli gidA mutants exhibited alterations in biosynthesis of mnm5s2U34 and translational frameshift (Brégeon et al., 2001; Yim et al., 2006). In Saccharomyces cerevisiae, mto1 null mutants conferred the complete loss of 5-carboxymethylaminomethyluridine (cmnm5s2U) of mitochondrial tRNALys (Umeda et al., 2005). Furthermore, mto1 alleles altered the processing of mitochondrial mRNA and rRNA precursors and synthesis of COXI (Colby et al., 1998). As a result, the S. cerevisiae mto1 null mutant expressed the respiratory deficient phenotype only when coexisting with a paromomycin resistance (PR) C1409G mutation of mitochondrial 15S rRNA, at the very conservative decoding site, corresponding to human deafness-associated mitochondrial 12S rRNA C1494T mutation (Yan et al., 2005; Zhao et al., 2004). These strongly indicate that the product of MTO1 functionally interacts with the decoding region of 15S rRNA, particularly at site of C1409G or A1491G mutation. To understand the role of MTO1 in mitochondrial RNA metabolism, the yeast mto1 null mutants have been characterized by examining the steady-state level and aminoacylation capacity of tRNA, the stability of mRNAs, and the frequency of petite formation.

2. Materials and methods

2.1. Yeast strains and growth analysis

Genotypes of strains were: M12 (a, ilv5, trp2, MTO1), M12-54 (a, ilv5, trp2, MTO1 [PR]) (Decoster et al., 1993), ΔMTO1 PS (α, his3-1, leu2-3, trp1, ura3-1, mto1::URA3), and ΔMTO1PR (α, leu2-3, trp1, ura3-1, mto1::URA3 [PR]) (Colby et al., 1998). The wild-type ρo strains YPH500 (a, ade2-1, his3-1, ura3-1, trp1) and 15B-6B (α, ade2-1, ura3-1, trp1, leu2-3, his3) were isolated as described elsewhere (Wang et al., 2007). These strains carried either paromomycin sensitivity (PS) or paromomycin resistance 15S rRNA C1409G allele (PR). These yeast strains carried either wild-type paromomycin sensitivity mtDNA (PS) or a paromomycin resistance C1409G allele (PR) (Wang et al., 2007). The mitochondrial genomes of the strains were identical, apart from the C1409G allele. The complete media for yeast cell growth consists of 0.5% yeast extract Difco (Y), 1% Bacto-Peptone Difco (P), and a carbon source as specified (Guan, 1997). GYP medium contains Y, P and 2% glucose. GlyYP contains Y, P and 2% glycerol.

2.2. Petite production analysis

Yeast cells were grown in liquid GYP medium overnight, then diluted and spread on GYP medium plates. After incubation at 30 °C for 2 days, cells were replica-plated onto GlyYP medium and incubated for 4 days. Petite were scored as small colonies were grown in GYP medium but were unable to grow in GlyYP medium. At least 400 colonies for each strain from three independent cultures were scored in GYP medium. Frequency of petite formation was defined as numbers of petites/total colonies in GYP medium in each strain.

To distinguish nuclear petite mutants from mitochondrial petite mutants, 100 single clones, which were unable to grow in GlyYP medium, for each strain, were crossed to the opposite mating type wild-type ρ° strain YPH500 in 5% GYP medium, plated on 2% glucose minimal medium and glycerol minimal medium. After growth at 30 °C for 5 days, the diploid progeny were checked for their growth. The diploid strains showing growth on glycerol medium revealed that the nuclear gene mutation is responsible for the petite phenotype, while the inability to grow on glycerol medium in diploid strains indicated that these petites were cytoplasmic petites (Tzagoloff and Dieckmann, 1990).

2.3. tRNA Northern-blot analysis

Extraction of total mitochondrial RNAs, electrophoresis, hybridization and quantification of tRNA were performed as detailed elsewhere (Wang et al., 2007; Li et al., 2004). Oligodeoxynucleosides used for non-radioactive digoxingenin-(DIG) labeled probes of tRNAArg2, tRNALeu, tRNALys, tRNAGly, tRNAGlu, tRNAGln, tRNAMet, and tRNAHis, were as detailed previously (Wang et al., 2007). Oligodeoxynucleoside for tRNAPhe was 5′-TTGGTGCCCTTAATGAGAATC GAACT-3′ (GenBank accession no: AJ011856) (Foury et al., 1998).

2.4. Mitochondrial tRNA aminoacylation analysis

Total mitochondrial RNAs were isolated as above but under acid condition. The total mitochondrial RNA (2 μg) was electrophoresed at 4 °C through an acid 10% polyacrylamide/7 M urea gel in a 0.1 M sodium acetate (PH 5.0) to separate the charged and uncharged tRNA, as detailed elsewhere (Enríquez and Attardi, 1996a,b). Then RNAs were electro-blotted onto a positively charged membrane (Roche) and hybridized sequentially with the specific tRNA probes as above.

2.5. Mitochondrial mRNA analysis

Total cellular RNA was obtained using a Totally RNA kit (Ambion) from midlog phase yeast cultures (2.0 × 107 cells) according to the manufacturer’s instructions. Equal amounts (20 μg) of total RNA were fractionated by electrophoresis through a 1.8% agarose-formaldehyde gel, transferred onto a positively charged membrane (Roche Applied Science), and initially hybridized with a probe specific for CYTB RNA. The probe was synthesized on the corresponding restriction enzyme-linearized plasmid using a DIG RNA labeling kit (Roche Applied Science). RNA blots were then stripped and rehybridized with DIG-labeled COX1, COX 2, COX 3, ATP6, and ATP9 probes, respectively (Yan et al., 2005; Wang et al., 2007). As an internal control, RNA blots were stripped and rehybridized with a DIG-labeled nuclear 25S rRNA probe. The plasmids used for CYTB, COX1 and 25S rRNA probes were as described previously (Yan et al., 2005). The other plasmids used for mtDNA probes were constructed by PCR-amplifying fragments of COX2 (positions 74,158–74,472), COX3 (positions 796,084–79,967), ATP6 (positions 28,829–29,227) and ATP9 (positions 46,738–46,949) (GenBank accession no: AJ011856) (Foury et al., 1998), and then cloning the fragments into the pCRII-TOPO vector carrying SP6 and T7 promoters (Invitrogen).

2.6. Southern blot analysis

Yeast geonomic DNA was extracted as detailed elsewhere (Sherman et al., 1986). Equal amounts (10 μg) of total DNA were was digested with restriction enzyme CfoI, fractionated by electrophoresis through a 1.8% agarose gel, transferred onto a positively charged membrane (Roche Applied Science), and hybridized with DIG-labeled probes specific for 15S rRNA and 21S rRNA Yan et al., 2005, respectively. As an internal control, DNA blots were stripped and rehybridized with DIG-labeled nuclear 25SrRNAprobe.

3. Results

3.1. Deletion of MTO1 lowered the steady-state levels of mitochondrial tRNAs

To examine if the mto1 mutation alters the stability of mitochondrial tRNAs, the steady-state levels of the tRNAs were determined by isolating total mitochondrial RNA from four yeast strains, separating them by a 10% polyacrylamide/7 M urea gel, electroblotting and hybridizing with non-radioactive DIG-labeled oligodeoxynucleotide probes specific for tRNALys, tRNAGlu, tRNAGln, tRNALeu, tRNAGly, tRNAMet, tRNAArg, tRNAPhe and tRNAHis, respectively. As shown in Fig. 1, the amounts of tRNALys, tRNAGlu, tRNAGln, tRNALeu, tRNAGly, tRNAArg, tRNAMet and tRNAPhe were markedly decreased but those of tRNAHis were not affected in mto1/PR mutant cells. As shown in Table 1, the average levels of tRNAs in mto1/PR strain were 64% in the tRNALys, 59% in tRNAGlu, 41% in tRNAGln, 56% in tRNALeu, 47% in tRNAGly, 55% in tRNAMet, 56% in tRNAArg, 45% in tRNAPhe and 112% in tRNAHis of control levels (M12) after normalization to 23S rRNA, respectively. Furthermore, the average levels of tRNAs in mto1/PS strain are 79% in the tRNALys, 59% in tRNAGlu, 65% in tRNAGln, 58% in tRNALeu, 61% in tRNAGly, 99% in tRNAMet, 44% in tRNAArg, 83% in tRNAPhe and 136% in tRNAHis of control levels (M12) after normalization to 21S rRNA, respectively. It is worth noting that cells carrying mto1 and C1409G double mutations exhibited lower steady-state levels of tRNALys, tRNAMet and tRNAPhe than those in cells carrying sole mto1 or C1409G mutation.

Fig. 1
Northern-blot analysis of mitochondrial tRNA. RNA blots were hybridized with DIG-labeled oligonucleoside probes for tRNALy, tRNAGlu, tRNAGln, tRNALeu, tRNAGly, tRNAMet, tRNAPhe, tRNAArg, and tRNAHis, respectively.
Table 1
Quantification of the levels of mitochondrial tRNAs.

3.2. Deletion of MTO1 impaired aminoacylation of mitochondrial tRNAs

It has been shown that the mnm5s2U34 serves as a determinant for tRNA recognition by cognate aminoacyl-tRNA synthetases in E. coli (Hagervall and Björk, 1984; Umeda et al., 2005). This led us to test whether the mto1 mutation affects the aminoacylation of mitochondrial tRNAs. For this purpose, the aminoacylation capacity of mitochondrial RNALys, tRNALeu, tRNAArg and tRNAHis in wild-type and mutant strains were carried out by the use of electrophoresis in an acid polyacrylamide/urea gel system to separate uncharged tRNA species from the corresponding charged tRNA (Enríquez and Attardi, 1996a,b). As shown in Fig. 2, the upper band represented charged tRNA, and the lower band was uncharged tRNA. Electrophoretic patterns showed that either charged or uncharged tRNALys, tRNALeu and tRNAArg in mto1 strains migrated faster than those of MTO1 strains, while there were no obvious differences in electrophoretic mobility of tRNAHis between the mto1 and MTO1 strains. The proportions of aminoacylation of tRNA in MTO1/PS strain were 41%, 38% and 44% in the tRNALys, tRNALeu and tRNAArg, respectively, while the proportions of aminoacylation of the tRNAs in strains carrying MTO1/PR, mto1/PS and mto1/PR alleles: were 32%, 33% and 25% of tRNALys, 32%, 27% and 26% of tRNALeu, 38%, 31% and 27% of tRNAArg, respectively. Aminoacylation capacity of these tRNAs were significantly decreased in mto1 cells carrying PS or PR allele, relative to MTO1 cells carrying PS allele (P = 0.01–0.05). However, there was no effect in aminoacylation efficiencies of tRNAHis among these control and mutant cells.

Fig. 2
In vivo aminoacylation assays for mitochondrial tRNA. (A) Two microgram of total mitochondrial RNA were treated with electrophoresis at 10% polyacrylamide/7 M urea gel, electro-blotted onto a membrane, and hybridized with DIG-labeled oligonucleoside probes ...

3.3. Deletion of MTO1 altered the expression of mitochondrial genome

In the previous studies, it was showed that the mto1 mutant exhibited lower levels of mature CYTB and COX1 mRNA and accumulated unprocessed or partially processed precursors in the presence of the 15S rRNA C1409G allele (Colby et al., 1998). We here tested whether the mto1 null mutation also affected the expression of other mitochondrial genes by Northern-blot analysis. RNA blots were hybridized with DIG-labeled probes for COX1, COX2, COX3, ATP6, and ATP9, respectively. As an internal control, RNA blots were stripped and rehybridized with the DIG-labeled nuclear-encoded 25S rRNA probe. As shown in Fig. 3A, the amounts of mature COX1 mRNA as well as COX2, COX3, ATP6 and ATP9 mRNA were decreased in the mto1/PR strain. The average levels of each mRNA were normalized to the average levels in the same strain for the 25S rRNA. As shown in Fig. 3B, the average steady-state levels of most mRNAs were decreased in the mto1 mutant cells, relative to the MTO1/PS strain. The steady-state levels of mRNAs in mto1/PR cells were 14% in mature COX1, 84% in COX2, 48% in COX3, 41% in ATP6 and 24% in ATP9 of control levels, while the steady-state levels of mRNAs in mto1/PS cells were 19% in mature COX1, 81% in COX2, 62% in COX3, 46% in ATP6 and 37% in ATP9 of control levels. It was noted that the levels of mature COX1 mRNA in mto1 cells with PS or PR allele were only 14% and 19% of MTO1/PS, while the levels of this mRNA in mto1/PS cells was comparable with those of controls in the previous study (Colby et al., 1998). This discrepancy is likely due to these mto1 strains derived from different nuclear background.

Fig. 3
Northern-blot analysis of mitochondrial mRNAs. (A) RNA blots were hybridized with DIG-labeled COX1, COX2, COX3, ATP6, and ATP9 probes, respectively. After re-stripping, blots were hybridized with a DIG-labeled nuclear 25S RNA probe as internal control. ...

3.4. Deletion of MTO1 increased the petite production

The previous study indicated that the mto1 mutant in the 15S rRNA PR allele exhibited an inability to grow on glycerol medium, respectively, suggesting that the mto1 mutation increased the petite formation (Colby et al., 1998). The rates of petite production in the MTO1 strain carrying PS or PR allele were 3% and 20%, respectively, while the rates of petite production in the mto1 mutant carrying PS or PR allele were 40% and 99%, respectively. To test if this phenotype was stable, mto1/PS respiratory negative and positive single colonies were grown in liquid glucose medium overnight, diluted, then spread on glucose medium plate. After incubation for 2 days, cells were replica-plated on glycerol medium. Among 400 single colonies from each strain, all progeny from respiratory negative strain remained the respiratory negative, while ~40% of progeny from respiratory positive strain became respiratory negative. To distinguish nuclear petite mutants from cytoplasmic petite mutants in mto1 strains, approximately 100 single colonies showing no growth in glycerol medium were crossed to the opposite mating type wild-type ρo strains. Diploid progeny were checked for their growth in glycerol and glucose media. Diploid strains showing growth on glycerol medium revealed that the mto1 mutation was responsible for petite phenotype, while the inability to grow on glycerol medium in diploid strains indicated that the petites were cytoplasmic petites (Tzagoloff and Dieckmann, 1990). Of these, the cytoplasmic petites account for 97%, 3% and 54% of total petite production in the MTO1 strain carrying the PR allele, the mto1 strain carrying PS or PR allele, respectively.

3.5. Southern blot analysis

To examine if the deficient RNA metabolism caused by the inactivation of MTO1 altered mitochondrial genomes, Southern blot analysis were determined by isolating total mitochondrial DNA from four yeast strains, digesting with restriction enzyme CfoI, separating them by electrophoresis, blotting and hybridizing with non-radioactive DIG-labeled 15S rRNA and 21S rRNA, respectively. As an internal control, DNA blots were stripped and rehybridized with a 32P-labeled nuclear 25S rRNA probe. Quantification of the hybridization was carried out by the Image-Quant program. For comparison of the data from different blots, the values obtained for the each strain in each blot were normalized to the values obtained for the M12 sample in the same blot. Using 15S rRNA as a probe, the average levels of mtDNA in MTO1/PR, mto1/PS, mto1/PR strains were 80%, 74% and 78% of control levels (M12) after normalization to 25S rRNA, respectively. Similarly, using 21S rRNA as a probe, the average levels of mtDNA in MTO1/PR, mto1/PS, mto1/PR strains were 82%, 57% and 65% of control levels (M12) after normalization to 25S rRNA, respectively.

4. Discussion

In this study, we investigated the role of MTO1 in mitochondrial RNA metabolism. The E. coli gidA mutants (homolog of MTO1) were defective in the biosynthesis of the hyper-modified nucleoside mnm5s2U34 (Brégeon et al., 2001). This modified nucleotide, found in the wobble position of several bacterial tRNAs specific for glutamate, lysine, and glutamine, has a pivotal role in the function of tRNAs, including both tRNA identity and the codon recognition specificity (Björk, 1995; Li et al., 2002, 2003). In fact, the inactivation of MTO1 in S. cerevisiae caused the complete loss of cmnm5s2U of tRNALys (Umeda et al., 2005). The deficiency of this nucleotide modification may alter structure and function of tRNALys, tRNAGlu and tRNAGln, including the stability, aminoacylation of tRNA and codon–anticodon interaction, thereby affecting the fidelity and efficiency of mitochondrial translation. These unmodified tRNAs caused by the deletion of MTO1 may then make tRNA more unstable, reducing levels of these tRNAs. Here, the steady-state levels of tRNALys, tRNAGlu, tRNAGln, tRNALeu, tRNAGly, tRNAArg and tRNAPhe were decreased significantly while those of tRNAMet and tRNAHis were not affected in the mto1 strains carrying PS allele. In fact, lowered levels of steady-state tRNALys, tRNAGlu and tRNAGln were consistent with the loss of C5 modification in these tRNAs (Umeda et al., 2005). Nucleosides at position 34 are uridine for tRNALys, tRNAGlu and tRNAGln, tRNALeu and tRNAGly, cytidine for tRNAMet, adenosine for tRNAArg and guanosine for tRNAPhe and tRNAHis, respectively (Björk, 1995; Foury et al., 1998). The protein encoded by MTO1 is involved in uridine modification at position 34 of tRNALys, tRNAGlu and tRNAGln, while other enzymes may be responsible for nucleoside modifications at this position of other tRNAs such as RNALeu, tRNAGly, tRNAMet, tRNAArg, tRNAPhe and tRNAHis (Björk, 1995). Deletion of MTO1 likely causes directly or indirectly transcriptional/translational defects, thereby reduces the steady-state levels of those tRNAs. Alternatively, the reduced levels of RNALeu, tRNAGly, tRNAMet, tRNAArg, tRNAPhe could be attributed to preferential deletion of the ρ cells containing particular segments of mtDNA carrying these tRNA genes. In contrast with mto2 strains (Wang et al., 2007), the combination of mto1 and C1409G mutations produced the potential synthetic phenotype for tRNALys, tRNAMet and tRNAPhe. Indeed, the C1409G mutation disrupted a C1409-1491G base-pairing at the decoding site of ribosomes where the codon–anticodon recognition occurs (Yan et al., 2005). Hyper-modified tRNAs were less efficient for the decoding of codons ending in G than C of the 15S rRNA (Yan et al., 2005; Weiss-Brummer and Huttenhofer, 1989). Therefore, the C1409G allele, acting in synergy with the mto1 mutation, may worsen deficient mitochondrial tRNA metabolisms. However, the steady-state levels of mitochondrial tRNAHis even increased in mto1 cells carrying the C1409G mutation, as compared with those in PS allele. Likely, the increased levels of tRNAHis could be attributed to preferential accumulation of the ρ cells containing particular segments of mtDNA.

The mnm5s2u34 is known to function as a recognition element for tRNA by cognate aminoacyl-tRNA synthetases (Krüger and Sørensen, 1998). Here, mitochondrial tRNALys, tRNALeu and tRNAArg were uncharged to a significantly high degree in vivo in mto1 strain carrying PS allele, compared to the MTO1/Ps strain. Unlike the mto2 strains (Wang et al., 2007), the PR allele worsened the deficient aminoacylation of these tRNAs in mto1 cells. In addition, both charged and uncharged tRNALys, tRNALeu and tRNAArg in mto1 strains migrated faster than those of MTO1 strains, while there were no obvious differences in electrophoretic mobility between wild-type and mto2 strains (Wang et al., 2007). Thus, mto1 cells exhibited more inefficient aminoacylation of tRNALys, tRNALeu and tRNAArg than those in mto2 cells. However, there was no difference in the aminocylated levels of mitochondrial tRNAHis between the mto1 mutant and wild-type strains.

Furthermore, the deletion of MTO1 altered mitochondrial mRNA metabolism. Indeed, the steady-state levels of COX1, COX2, COX3, ATP6 and ATP9 mRNAs were decreased in mto1 cells. The level of mature COX1 mRNA in mto1 cells was markedly decreased, but the levels of its precursors appeared to be unchanged. This strongly suggested that in the case of this transcript, the main effect was related to mRNA processing through expression of maturases encoded by mitochondrial genomes. The synthesis of maturases required for the removal of introns from CYTB and COX1 (Dieckmann and Staples, 1994) was impaired by translational defects (Yan et al., 2005; Colby et al., 1998; Decoster et al., 1993). Thus, introns in mitochondrial genomes were not spliced completely, thereby causing the accumulation of unprocessed or partially processed precursors of CYTB and COX1 in mto1 cells in the context of C1409G allele, as in the cases of mss1 and mto2 cells (Yan et al., 2005; Colby et al., 1998). Of other transcripts, COX2, COX3 and ATP9 transcripts were least affected. These transcripts are parts of polycistronic transcripts containing tRNA genes, while ATP6 lies in a primary polycistronic transcript downstream of the COX1 gene (Tzagoloff and Dieckmann, 1990; Dieckmann and Staples, 1994). However, COX2 transcript was not transcribed together with any tRNA gene (Dieckmann and Staples, 1994). Therefore, the lowered levels of these mitochondrial transcripts are likely due to the fact that the significant fraction of the cells, which were ρo petites, did not contain templates for the transcription of relevant genes.

Mutations in genes affecting mitochondrial translation caused the instability of mtDNA, thus producing respiratory deficient phenotypes (Myers et al., 1985). Here, failures in RNA metabolism led to the heterogeneity phenotype (40% petite formation) in mto1/PS strain, as in the case of mto2/PS strain (Wang et al., 2007). This was further supported by the fact that all progeny from respiratory negative mto1/PR strain remained the respiratory negative, while approximately 40% of progeny from respiratory positive mto1/PS strain became respiratory negative. Indeed, the 15S rRNA C1409G mutation altered aminoacylation of tRNAs (Wang et al., 2007) and mitochondrial translation (Yan et al., 2005), consequently contributing to mtDNA instability. This was supported by the Southern blot analysis that mto1 mutants exhibited lower levels of mtDNA than those in MTO1/PS cells. Thus, mto1 and C1409G double mutations led to the loss of respiration (Colby et al., 1998).

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