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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Mol Biol. Author manuscript; available in PMC 2011 February 5.
Published in final edited form as:
PMCID: PMC2818684
NIHMSID: NIHMS164514

Combination of the loss of cmnm5U34 with the lack of s2U34 modifications of tRNALys, tRNAGlu and tRNAGln altered mitochondrial biogenesis and respiration

Abstract

Yeast Saccharomyces cerevisiae MTO2, MTO1 and MSS1 genes encoded highly conserved tRNA modifying enzymes for the biosynthesis of cmnm5s2U34 in mitochondrial tRNALys, tRNAGlu and tRNAGln. In fact, Mto1p and Mss1p are involved in the biosynthesis of the cmnm5 group (cmnm5U34), while Mto2p is responsible for the 2-thiouridylation (s2U34) of these tRNAs. Previous studies showed that partial modifications at U34 in mitochondrial tRNA enabled mto1, mto2 and mss1 strains to respire. In this report, we investigated the functional interaction between MTO2, MTO1 and MSS1 genes by using the mto2, mto1 and mss1 single, double and triple mutants. Strikingly, the deletion of MTO2 was synthetically lethal with a mutation of MSS1 or deletion of MTO1 on medium containing glycerol, but not on medium containing glucose. Interestingly, there were no detectable levels of 9 tRNAs including tRNALys, tRNAGlu and tRNAGln in mto2/mss1, mto2/mto1 and mto2/mto1/mss1 strains. Furthermore, mto2/mss1, mto2/mto1 and mto2/mto1/mss1 mutants exhibited extremely low levels of COX1 and CYTB mRNA, 15S and 21S rRNA as well as the complete loss of mitochondrial protein synthesis. The synthetic enhancement combinations likely resulted from the completely abolished modification at U34 of tRNALys, tRNAGlu and tRNAGln, caused by the combination of eliminating the 2-thiouridylation by the mto2 mutation with the absence of the cmnm5U34 by the mto1 or mss1 mutation. The complete loss of modifications at U34 of tRNAs altered mitochondrial RNA metabolisms, causing a degradation of mitochondrial tRNA, mRNA and rRNAs. As a result, failures in mitochondrial RNA metabolisms were responsible for the complete loss of mitochondrial translation. Consequently, defects in mitochondrial protein synthesis caused the instability of their mitochondrial genomes, thus producing the respiratory deficient phenotypes. Therefore, our findings demonstrated a critical role of modifications at U34 of tRNALys, tRNAGlu and tRNAGln in maintenance of mitochondrial genome, mitochondrial RNA stability, translation and respiratory function.

Keywords: Mitochondrial tRNA, nucleotide modification, respiration, biogenesis, metabolism

INTRODUCTION

All tRNA species from living organisms contain modified nucleotides, which are derivatives of the four normal nucleotides adenosine (A), guanosine (G), uridine (U) and cytidine (C).1,2 To date, more than 80 different modifications have been identified in tRNA from various organisms.3 Of these, the nucleotide at the position 34 (wobble position of anticodon) of tRNA is more prone to modification than those at other positions of tRNA.4 In Escherichia coli, the uridine at position 34 of tRNA is always modified, and the modification is either xo5U-type (derivatives of 5-hydroxyuridine) or xm5(s2)U(m)-type (derivatives of 5-methyluridine, and 5-methyl-aminnomethy-2-thio-uridine).4 In particular, the nucleoside 5-methyl-aminomethy-2-thio-uridine (mnm5s2U34) occurs at the wobble position 34 of bacterial tRNAGlu, tRNALys and tRNAGln.4,5,6 The synthesis of mnm5s2U34 is a multiple step process catalyzed by an enzyme complex.7,8 Of these, MnmE (homolog of Mss1/Gtpbp3),8,9 GidA (homolog of Mto1)7,10 and MnmA/TrmU (homolog of Mto2)11,12 are components of the enzyme complex responsible for the biosynthesis of mnm5s2U34. In fact, MnmE and GidA assemble in a functional complex involved in the formation of carboxymethylaminomethyl (cmnm) group at position 5 of U34 of several tRNAs including tRNALys, tRNAGlu and tRNAGln,7,8,13,14,15 while MnmA, together with other proteins, catalyzes the thiolation at position 2 of U34 of tRNALys, tRNAGlu and tRNAGln.11,16,17 This modified nucleotide plays a pivotal role in the structure and function of tRNAs including the stabilization of anticodon structure, the ribosome binding ability to tRNA and the improvement of reading frame maintenance.1,2,3,4

In the yeast Saccharomyces cerevisiae, MSS1, MTO1 and MTO2 genes encoded highly conserved tRNA modifying enzymes for the biosynthesis of cmnm5s2U34 of mitochondrial tRNAs.12,18,19,20 Mutations in these genes altered modifications in mitochondrial tRNAs, thereby causing a failure in tRNA metabolisms.20,21,22 The deletion of MTO2 in the S. cerevisiae led to the complete loss of 2-thiouridylation in the mitochondrial tRNALys, tRNAGlu and tRNAGln, while the inactivation of MTO1 and MSS1 conferred the complete loss of 5-carboxymethylaminomethyluridine (cmnm5U) of mitochondrial tRNALys.20 The mto1, mto2 and mss1 strains were still able to grow on glycerol medium, indicating that mto1, mto2 or mss1 mutation by themselves were insufficient to produce the respiratory deficient phenotype.12,18,19,20 However, the mto1, mto2 and mss1 null mutants expressed the respiratory deficient phenotype in the context of mitochondrial 15S rRNA C1409G mutation,12,18,19 corresponding to human deafness-associated 12S rRNA C1494T mutation.23 Furthermore, mto1, mto2 and mss1 mutants altered mitochondrial protein synthesis and expression of mitochondrial genes, especially in CYTB and COX1 in the presence of 15S rRNA C1409G allele.12,18,19 These strongly indicate that products of MTO1, MTO2 and MSS1 functionally interact with the decoding region of 15S rRNA, particularly at site of C1409G mutation. Subsequent investigations indicated that the human mutated TRMU, acting as a modifier factor, modulated the phenotypic manifestation of the deafness-associated mitochondrial 12S rRNA mutations.24 To further investigate the functional interactions among MTO1, MTO2 and MSS1, mto2/mto1, mto2/mss1, mss1/mto1 double mutants and mto2/mto1/mss1 triple mutant were constructed by crossing one mto1 null strain with one mto2/mss1-18 strain. The resultant strains were characterized by examining the growth properties, the 2-thiouridine modification, the steady-state level and aminoacylation of tRNAs, the stability of mRNA and rRNAs and mitochondrial protein synthesis.

RESULTS

The combination of mto2 allele with mto1 or mss1 allele led to a respiratory-deficient phenotype

The previous investigations showed that mto1, mss1 and mto2 null mutants were able to grow in glycerol medium.12,18,19 To examine if there are functional interactions among these genes, mto2/mto1, mto2/mss1, mss1/mto1 double mutants and mto2/mto1/mss1 triple mutant were constructed by crossing one mto1 null strain with one mto2/mss1-18 ρ° strain isolated by treating mto2/mss1-18 (PR) cells12 with ethidium bromide to cause the loss of mtDNA (ρ°).25 The resulting diploids were sporulated and products of meiosis were dissected onto glucose medium. Meiotic progeny derived from the cross was examined for the genotype and then tested for the growth on glucose and glycerol media. As shown in Figure 1, mto1, mss1 and mto2 null mutant strains and mto1/mss1 double mutant were able to grow on glycerol medium, indicating that these cells were respiratory-competent. Conversely, mto2/mto1, mto2/mss1 double mutants and mto2/mto1/mss1 triple mutant were unable to grow in glycerol medium, indicating that these cells were respiratory-deficient. These data strongly suggested that the expression of respiratory deficient phenotype in the mto1 and mss1 null mutants was fully dependent on the presence of the mto2 allele.

FIG. 1
Growth properties of yeast wild-type (WT) and mutant (MT) strains

Alterations in mitochondrial genomes

To examine for the presence of mtDNA in these respiratory-deficient cells, Southern blot analysis were performed by isolating total mitochondrial DNA from yeast strains, digesting with restriction enzyme CfoI, separating them by electrophoresis, blotting and hybridizing with non-radioactive DIG-labeled CYTB, COX1, COX2, COX3, ATP6, ATP9, and 21S rRNA, respectively. As an internal control, DNA blots were stripped and rehybridized with a DIG-labeled nuclear 25S rRNA probe. As shown in Fig. 2, the patterns and levels of mtDNAs in mto2, mto1 and mss1 strains appeared to be comparable with those of wild strain M12. In mto1/mss1 strains, the levels of bands corresponding to six genes were comparable with those in control strain, while the band corresponding to COX3 exhibited very lower level. By the contrast, the deletions and very lower levels of mitochondrial genomes occurred in the mto2/mss1, mto2/mto1 and mto2/mto1/mss1 strains. In particular, the bands corresponding to COX1 and ATP6 were not detected in these mutant strains. 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. After normalization to the level of 25S rDNA, the average levels of mtDNA in mto2, mto1, mss1 and mto1/mss1 strains were 85%, 85%, 86% and 77% of control levels (M12), respectively. On the other hand, the average levels of mtDNA in mto2/mss1, mto2/mto1 and mto2/mto1/mss1 strains were 28%, 31% and 34% of control levels (M12) after normalization to 25S rRNA, respectively. These data suggested that the respiratory deficient phenotypes in the mto2/mss1, mto2/mto1 and mto2/mto1/mss1 strains resulted from the loss and decrease of their mtDNAs.

FIG. 2
Southern blot analysis of mitochondrial genomes

Defect in 2-thiouridine modification at position 34 in mitochondrial tRNALys, tRNAGlu and tRNAGln

To investigate if the mto2, mto1 and mss1 mutations impair 2-thiouridine modification at position 34 in mitochondrial tRNAs, the 2-thiouridylation levels of tRNAs were determined by isolating total mitochondrial RNA from eight strains, and qualifying the 2-thiouridine modification by the retardation of electrophoresis mobility in polyacrylamide gel containing 0.05 mg/ml [(N-acryloylamino)phenyl] mercuric chloride (APM)2628 and hybridizing specific probes for the tRNALys, tRNAGlu, tRNAGln, tRNALeu, tRNAMet and tRNAHis. As shown in Fig. 3, the upper band represented the thiouridylated tRNA, and the lower band was unthiouridylated tRNA. In this system, the mercuric compound can specifically interact with the tRNAs containing thiocarbonyl group including tRNALys, tRNAGlu and tRNAGln, thereby retarding tRNA migration.26,27 However, no 2-thiouridylation occurs in other tRNAs lacking thiocarbonyl group such as tRNALeu, tRNAMet and tRNAHis. The proportions of the 2-thiouridylation levels of the tRNA in the wild type strain were 92%, 85% and 92% in the tRNALys, tRNAGlu and tRNAGln, respectively, whereas the mto2 null strain exhibited the complete loss of the 2-thiouridylation in tRNALys, tRNAGlu and tRNAGln. In addition, the 2-thiouridylation levels of the tRNALys, tRNAGlu and tRNAGln in mss-18, mto1 and mss1/mto1 double mutants were comparable with those in wild type strain. However, there was no detectable thiouridylated and unthiouridylated tRNALys, tRNAGlu and tRNAGln as well as tRNALeu, tRNAMet and tRNAHis in the mto2/mto1, mto2/mss1 double mutants and mto2/mto1/mss1 triple mutant.

FIG. 3
APM gel electrophoresis combined with Northern blotting of yeast mitochondrial tRNAs

Decreases in the steady-state levels of mitochondrial tRNAs

To examine if the defective nucleotide modification alters the stability of mitochondrial tRNAs, the steady-state levels of nine tRNAs were determined by isolating total mitochondrial RNA from eight 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, RNAPhe and tRNAHis, respectively. For comparison, the average levels of each tRNA in the various control or mutant yeast strains were then normalized to the average levels in the same strain for the mitochondrial 21S rRNA. As shown in Fig. 4 and Table 2, the average steady-state levels of tRNAs, except tRNAHis, were significantly decreased in the mto2 mutant relative to the wild type strain. The average levels of tRNAs in the mto2 strain varied from 22% in the tRNAGln to 58% in tRNAMet of control levels. Similarly, the average levels of tRNAs in mto1 strain, except tRNAMet and tRNAHis, ranged from 43% in the tRNAArg to 74% in tRNAPhe, of control levels. Furthermore, the average levels of those tRNAs in mss1-18 strain, except tRNALys and tRNALeu, were comparable to those in the wild type strain. In particular, the average levels of tRNALys and tRNALeu in mss1-18 strain were 66% and 64% of wild type strain, respectively. Surprisingly, the average levels of those tRNAs in mss1-18/mto1 double mutant strain appeared to increase, as compared with those in the wild type strain. On the other hand, there were no detectable mitochondrial tRNAs in the mto2/mto1, mto2/mss1double mutants and mto2/mto1/mss1 triple mutant.

FIG. 4
Northern-blot analysis of mitochondrial tRNA
Table 2
Quantification of the levels of mitochondrial tRNAs.

Alteration in 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.29,30 This led us to test whether the mto1, mto2 and mss1 mutations affect the aminoacylation of mitochondrial tRNAs. For this purpose, the steady state levels of aminoacylation in RNALys, 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 un-charged tRNA species from the corresponding charged tRNA.21,22 As shown in Fig. 5, the upper band represented the charged tRNA, and the lower band was uncharged tRNA. As shown in Table 3, the proportions of aminoacylation of the tRNALys, tRNAArg and tRNAHis in the wild type strain were 46%, 40% and 44%, respectively, while the proportions of aminoacylation of the tRNAs in strains carrying mto2, mto1, mss1 and mto1/mss1 alleles: were 29%, 28%, 43% and 38% of tRNALys, 21%, 22%, 35% and 36% of tRNAArg, 43%, 41%, 47% and 44% of tRNAHis, respectively. The steady state levels of aminoacylation in these tRNAs were significantly decreased in mto2 and mto1 cells, relative to the wild type cells (P=0.01–0.05). By contrast, there was no effect in levels of aminoacylation in tRNAHis among these control and mutant cells. Furthermore, the proportions of aminoacylation of the tRNALys, tRNAArg and tRNAHis in mss1-18 and mto1/mss1-18 mutant strains were comparable with those in the wild type strain. However, there was no detectable RNALys, tRNAArg and tRNAHis in the mto2/mto1, mto2/mss1 double mutant and mto2/mto1/mss1 triple mutant strains.

FIG. 5
In vivo aminoacylation assays for mitochondrial tRNA
Table 3
Quantification of the levels of aminoacylated mitochondrial tRNA.

Deficient expression of mitochondrial genes

We examined whether the combination of mto2 null mutation with mss1 and mto1 null mutation also affects the expression of mitochondrial genes by Northern blot analysis. For this purpose, RNA blots were hybridized with DIG-labeled probes for exon regions of CYTB, COX1, 15S rRNA and 21S rRNA probes, respectively. In these mitochondrial genes, CYTB consists of six exons and five introns, COX1 contains 8 exons and 7 introns, 21S rRNA harbors two exons and one intron, but 15S rRNA lacks intron.31 As shown in Fig. 6, the amount of mature CYTB mRNA was decreased in the mto2, mto1 and mss1-18 strains, compared with the wild type strain, while the amount of mature COX1 mRNA were decreased in the mto2 and mto1 strains but not in mss1-18 strain, compared with the wild type strain. On the other hand, there were no detectable mature COX1 and CYTB mRNAs in mto2/mto1, mto2/mss1-18, mto1/mss1-18 and mto2/mto1/mss1-18 strains. Furthermore, there were the similar levels of 15S rRNA and 21S rRNA between wild-type strain and mto2, mto1, mss1-18, or mto1/mss1-18 strains. However, levels of 15S rRNA and 21S rRNA were extremely lower or undetectable in mto2/mto1, mto2/mss1-18 and mto2/mto1/mss1-18 strains, as compared with those in the wild type strain.

FIG. 6
Northern-blot analysis of mitochondrial mRNAs

Mitochondrial protein synthesis defects

To examine if a failure in RNA metabolism alters mitochondrial protein synthesis, mutant and control strains were labeled for 2.5 min with [35S] methionine in a methionine-free medium in the presence of cycloheximide to inhibit cytoplasmic protein synthesis. Fig. 7 shows SDS-PAGE electrophoretic patterns of the organelle-specific products of mutant and wild-type strains. Interestingly, the patterns of the mitochondrial translational products from mto2, mto1 and mto1/mss1-18 mutants differed from those of the wild-type strain. There was a marked reduction in the overall labeling of the mitochondrial translation products in the mto2 and mto1 mutants, when compared with wild type strain. In particular, the Cox1 and Cytb polypeptides were less labeled in mto2, mto1 and mto1/mss1-18 mutants than in wild type strain. The patterns of the mitochondrial translational products from mss1-18 mutant strain are similar to those in wild type strain. However, the mitochondrial translation was completely abolished in the mto2/mto1, mto2/mss1-18 and mto2/mto1/mss1-18 strains.

FIG. 7
Mitochondrial protein labeling analysis

DISCUSSION

In this study, we have investigated the genetic interactions among MTO1, MTO2 and MSS1 genes encoding tRNA modifying enzymes responsible for the biosynthesis of cmnm5s2U34 in the mitochondrial tRNAs. Mto1p and Mss1p are involved in the biosynthesis of the cmnm5 group (cmnm5U34), while Mto2p is responsible for the 2-thiouridylation (s2U34).20,21,22,32 In particular, Mss1p and Mto1p form a functional complex that would catalyze the production of an unknown intermediate group (X) at position 5.7,18,32 Glycine would be subsequently incorporated into tRNA by unidentified transferases to produce cmnm5U34.20,32 On the other hand, U34 undergoes thiolation at position 2 catalyzed by Mto2p, together with other proteins including Nfs1/IscS.7,11,20,32 The deletion of MTO2 abolished the 2-thiouridylation in mitochondrial tRNALys, tRNAGlu and tRNAGln, while the inactivation of MTO1 and MSS1 caused the complete loss of cmnm5U34 in tRNALys.20 These implied that the modifications at positions 2 and 5 of U34 in these tRNAs proceed independently. Despite the absence of cmnm5s2U34 of these tRNAs, the presence of cmnm5U34 or s2U34 makes mto2, mto1, mss1 or mss1/mto1 strains to respire. However, the combination of the loss of cmnm5U34 caused by the mss1 or mto1 mutation with the absence of s2U34 caused by the mto2 mutation completely abolished nucleotide modification at U34 of tRNALys, tRNAGlu and tRNAGln in mto2/mto1, mto2/mss1 or mto2/mss1/mto1 strains. As a result, the mto2 allele, acting in synergy with the mto1 or mss1 mutation, manifested the respiratory phenotype, as in the case of 15S rRNA C1409G mutation.18,19 Thus, Mto2p appeared to act as a hub of a strong set of synthetic genetic interactions.

The cmnm5s2U34 modified nucleotide plays a pivotal role in the structure and function of tRNAs including stability, turnover, aminoacylation of tRNAs and codon-recognition.4,7,29,33 Thus, the deficiency of this U34 modification may affect the structure and function of these mitochondrial tRNAs. In fact, the mto2, mto1 and mss1 mutations caused a large proportion of incompletely modified mitochondrial tRNAs.20 These unmodified tRNAs likely leave the tRNAs more exposed to degradation, thus reducing the levels of mitochondrial tRNAs.21,22,24 Here, the lowered levels of tRNALys, tRNAGlu, tRNAGln in mto2 and mto1 strains likely resulted from the deficient uridine modification at position 34 of these tRNAs. On the other hand, the reduced levels of tRNALys but not tRNAGlu and tRNAGln in mss1 strain implied that Mss1p is involved in uridine modification at position 34 of tRNALys but not tRNAGlu and tRNAGln. Of other six tRNAs, the steady-state levels of tRNALeu, tRNAGly, tRNAMet, tRNAArg, RNAPhe in mto2 mutant cells, tRNALeu, tRNAGly, tRNAMet, tRNAArg, RNAPhe in mto1 strains and only tRNALeu in mss1 mutant cells were decreased, as compared with those in the wild type strain. The lowered levels of those tRNAs likely result from directly or indirectly transcriptional/translational defects caused by mutations in MTO1, MTO2 and MSS1. 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.22 Furthermore, the deficient nucleotide modification altered the aminoacylation of tRNAs.29,30,34 Here, the tRNALys and tRNAArg were uncharged to a significantly high degree in vivo in mto2 and mto1 mutants, compared to the wild type strain. However, the proportions of aminoacylation of the tRNALys and tRNAArg in mss1 strain were comparable with those in the wild type strain. These clearly indicated that the loss of synthesis of s2U34 by the deletion of MTO2 and the loss of cmnm5U34 synthesis by mto1 mutation impaired the aminoacylation of tRNALys. Subsequently, the destabilization of tRNAs caused by the loss of cmnm5U34 resulted from mto1 or mss1 mutation was worsened when coupled with the lack of s2U34 caused by the mto2 mutation. Indeed, the rapid tRNA decay was observed in yeast cells lacking nucleotide modifications.35,36 As a result, the complete loss of modification in U34 in tRNALys, tRNAGlu and tRNAGln caused a rapid mitochondrial tRNA degradation in the mto2/mto1, mto2/mss1 and mto2/mto1/mss1 strains.

tRNA nucleotide modifications are essential for mitochondrial translation, gene expression and genome maintenance. It has been shown that the deficient tRNA nucleotide modifications affected the fidelity and efficiency of mitochondrial translation.3741 In the present investigation, the reduced levels of mitochondrial tRNAs, caused by the loss of s2U34 or cmnm5U34, most probably accounts for a marked decrease in the overall rate of mitochondrial translation in mto2 and mto1 strains. Conversely, it appears that the reduced levels of tRNALys and tRNALeu in the mss1 strain did not severely affect the mitochondrial protein synthesis, suggesting that these tRNAs are not limiting factors. However, the lack of both cmnm5U34 and s2U34 modifications of tRNALys, tRNAGlu and tRNAGln completely abolished the mitochondrial translation in mto2/mto1, mto2/mss1 and mto2/mto1/mss1 strains. Perturbations in mitochondrial translations affected the synthesis of the maturases encoded by introns of CYTB and COX1 genes.31,42,43, thereby affecting the processing of polycistronic precursors and the stability of mature transcripts. The incomplete removal of introns reduced the levels of mature CYTB and COX1 mRNAs in these mto2, mto1 or mss1 single, double or triple mutant strains. Alternatively, these transcriptional/translational defects can also lower the steady-state levels of CYTB and COX1 mRNAs and those of COX2, COX3, ATP6 and ATP9 lacking introns.31 Strikingly, the combination of mto2 with mto1 or mss1 mutation produced synthetic phenotypes for 15S rRNA and 21S rRNA. Therefore, the mto2 mutation, acting in synergy with mto1 or mss1 mutations, significantly perturbed mitochondrial mRNA and rRNA metabolism. Furthermore, it has been shown that mutations in genes affecting mitochondrial protein synthesis caused the instability of mtDNA.4446 In fact, a failure in RNA metabolism promoted the petite formation, thereby accounting for slight reductions in levels of mtDNA in the mto2, mto1 and mss1 cells.21,22 Strikingly, mtDNAs in the mto2/mto1, mto2/mss1 or mto2/mss1/mto1 mutants were by themselves very unstable. Their mtDNA levels in these mutant cells, unlike the cytoplamic petites,25 were only 30% of wild type level (comprising large deletions or loss of entire genomes). The instability of their mtDNA is likely a secondary effect by the reduced levels of transcripts or unstable transcripts caused by the impairment of translation. It appears that the instability of mtDNA is preferentially in some genes. In particular, the COX1 and ATP6 genes, whose transcripts were processed from the same polycistronic precursor,42 were not detectable in these double and triple mutants. This instability of these specific genes may be related to the loss of introns such as the intron ai5α in COX1 gene47 or a transcription-dependent DNA traction.48 However, this conclusion may not be extended to mammalian mitochondria.49

In summary, MTO2, MTO1 and MSS1 encoded enzymes responsible for the biosynthesis of cmnm5s2U34 in of mitochondrial tRNALys, tRNAGlu and tRNAGln. The unmodified mitochondrial tRNAs caused by the mutations in MTO2, MTO1 and MSS1 genes likely makes the tRNA more unstably, thus reducing the levels of mitochondrial tRNAs. The unmodified mitochondrial tRNAs are poor substrates for cognate aminoacyl-tRNA synthetases, thereby decreasing the aminoacylation levels of mitochondrial tRNAs. As shown in Table 4, the loss of cmnm5U34 by mto1 and mss1 mutations or s2U34 by mto2 mutation was insufficient to produce a respiratory deficient phenotype. On the other hand, the loss of both cmnm5U34 and s2U34 modifications lead to very significant failures in mitochondrial RNA metabolism, complete loss of mitochondrial protein synthesis, instability of mitochondrial genome and subsequent respiratory deficient phenotype. These data demonstrated the functional interaction between MTO2 and MTO1 or MSS1 and the critical role of modifications at U34 of tRNALys, tRNAGlu and tRNAGln in mitochondrial functions.

Table 4
Summary of relationship between nucleotide modifications at U34 of tRNALys and genotypes of yeast mto2, mto1 and mss1 mutants

MATERIALS AND METHODS

Yeast strains, media and genetic techniques

The genotypes and sources of strains of S. cerevisiae used in this investigation are listed in Table 1. The media used to grow yeast have been described elsewhere.50 Standard procedures were used for crossing and selecting diploids, including sporulation and dissecting tetrads.51

Table 1
Genotype and sources of yeast strains

Southern blot analysis

Yeast geonomic DNA was extracted as detailed elsewhere.51 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 digoxigenin (DIG)-labeled probes specific for CYTB, COX1, COX2, COX3, ATP6, ATP9, and 21S rRNA,12,22 respectively. As an internal control, DNA blots were stripped and rehybridized with DIG-labeled nuclear 25S rRNA probe.12 The hybridization was performed as detailed elsewhere.21,22 Quantification of density in each band was made as detailed elsewhere.21,22

APM Gel electrophoresis to quantify the 2-thiouridine modification in tRNAs

The presence of the thiouridine modification in the tRNAs was verified by the retardation of electrophoretic mobility in a polyacrylamide gel that contains 0.05 mg/ml (N-acryloylamino)phenyl)mercuric chloride (APM).2628 Total mitochondrial RNAs were obtained using TOTALLY RNA kit (Ambion) from mitochondria isolated from yeast cells, as described previously.21,22 Equal amounts (2 μg) of total mitochondrial RNA were separated by polyacrylamide gel electrophoresis and blotted onto positively charged membrane (Roche Applied Science). Each tRNA fraction was detected with a specific non-radioactive digoxingenin-(DIG) oligodeoxynucleoside probe at the 3′ termini according to the method as described elsewhere.27,28,29,50 Following oligodeoxynucleosides were used:

5′-TGGTGAGAATAGCTGGAGTTGAAC-3′ (tRNALys);

5′-TGGTAACCTTAATCGGAATCGGAATCGAAC-3′ (tRNAGlu);

5′-TGGTTGAATCGGTTTGATTCGAAC-3′ (tRNAGln);

5′-TGGTGCTATTTAAAGGACTTGAACCTT-3′ (tRNALeu);

5′-TGGTACTTGTAGAAGGAATTGAAC-3′ (tRNAMet);

5′-TGGGGTGAATACTGAGAATCGAACTCA-3′ (tRNAHis) (GenBank accession no: AJ011856).31 DIG-labeled oligodeoxynucleosides were generated by using the DIG oligonucleoside Tailing kit (Roche). APM gel electrophoresis and quantification of 2-thiouridine modification in tRNAs were conducted as detailed elsewhere.2628

Mitochondrial tRNA Northern-blot analysis

Two μg of total mitochondrial RNA were electrophoresed through a 10% polyacrylamide/7 M urea gel in Tris–borate–EDTA buffer (after heating the sample at 65°C for 10 min), and then was electroblotted onto a positively charged nylon membrane (Roche) for the hybridization analysis with specific oligodeoxynucleoside mitochondrial tRNA probes. Oligodeoxynucleosides used for (DIG) labeled probes of tRNALys, tRNAGlu, tRNAGln, tRNALeu, tRNAMet and tRNAHis were described as above. Oligodeoxynucleosides specific for tRNAArg, tRNAGly, and tRNAPhe were as follows: oligodeoxynucleosides specific for tRNAs were used:

5′-TGGTATAGATAGCGAGAATCGAAC-3′ (tRNAGly);

5′-TTGGTGCCCTTAATGAGAATCGAACT-3′ (tRNAPhe);

5′-TGGTACTCTCTCCATGATTTGAAC-3′ (tRNAArg);

(GenBank accession no: AJ011856).31 DIG-labeled oligodeoxynucleosides were generated by using the DIG oligonucleoside Tailing kit (Roche). The hybridization was performed as detailed elsewhere. 21,22 Quantification of density in each band was made as detailed elsewhere.21,22

Mitochondrial tRNA aminoacylation analysis

Total mitochondrial RNAs were isolated as above but under acid condition.52 2 μg of total mitochondrial RNA were electrophoresed at 4°C through an acid 10% polyacrylamide/7 M urea gel in a 0.1M sodium acetate (pH 5.0) to separate the charged and uncharged tRNA, as detailed elsewhere.52 Then RNAs were electro-blotted onto a positively charged membrane (Roche) and hybridized sequentially with the specific tRNA probes as above.

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 COX1 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 CYTB, 15S rRNA and 21S rRNA probes, respectively. The plasmids used for CYTB, COX1, 15S rRNA and 21S rRNA probes were as described previously.12

Analysis of mitochondrial protein synthesis

Yeast strains were pulse-labeled for 2.5 min with [35S]methionine in methionine-free medium in the presence of cycloheximide to inhibit cytoplasmic protein synthesis, as described elsewhere.53 The radiolabeled proteins were separated on SDS-exponential polyacrylamide gradient gels.12,53 The gels were treated with Me2SO/2,5-diphenyloxazole, dried, and exposed for fluorography.

Acknowledgments

This work was supported by Public Health Service grants RO1DC05230 and RO1DC07696 from the National Institute on Deafness and Other Communication Disorders. We thank Dr. Alex Tzagoloff (Columbia University) and Gèrard Faye (Institut Curie) for yeast strains. We are grateful to Li Yang, Chuck Loftice and Alisa Madden for technical and clerical support.

Footnotes

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References

1. Hopper AK, Phizicky EM. tRNA transfers to the limelight. Genes Dev. 2003;17:162–180. [PubMed]
2. Agris PF, Vendeix FA, Graham WD. tRNA’s wobble decoding of the genome: 40 years of modification. J Mol Biol. 2007;366:1–13. [PubMed]
3. Czerwoniec A, Dunin-Horkawicz S, Purta E, Kaminska KH, Kasprzak JM, Bujnicki JM, Grosjean H, Rother K. MODOMICS: a database of RNA modification pathways. 2008 update. Nucleic Acids Res. 2008;37:D118–121. [PMC free article] [PubMed]
4. Björk GR. Stable RNA modification. In: Neidhardt FC, Curtiss R III, Ingraham JL, Lin ECC, Low BK, Magasanik B, Reznikoff WS, Riley M, Schaechter M, Umbarger HE, editors. Escherichia coli and Salmonella: Cellular and Molecular Biology. American Society for Microbiology; Washington, D. C: 1996. pp. 861–886.
5. Yan Q, Guan MX. Identification and characterization of mouse TRMU gene encoding the mitochondrial 5-methylaminomethyl-2-thiouridylate-methyltransferase. Biochim Biophys Acta. 2004;1676:119–126. [PubMed]
6. Ishitani R, Yokoyama S, Nureki O. Structure, dynamics, and function of RNA modification enzymes. Curr Opin Struct Biol. 2008;18:330–339. [PubMed]
7. Brégeon D, Colot V, Miroslav M, Radman M, Taddei F. Translational misreading: a tRNA modification counteracts a +2 ribosomal framshift. Genes Dev. 2001;15:2295–2306. [PubMed]
8. Moukadiri I, Prado S, Piera J, Velazquez-Campoy A, Bjork GR, Armengod M-E. Evolutionarily conserved proteins MnmE and GidA catalyze the formation of two methyluridine derivatives at tRNA wobble positions. Nucleic Acids Res. 2009;0:gkp762v1–gkp762. [PMC free article] [PubMed]
9. Li X, Guan MX. A human mitochondrial GTP binding protein related to RNA modification may modulate the phenotypic expression of the deafness-associated mitochondrial 12S rRNA mutation. Mol Cell Biol. 2002;22:7701–7711. [PMC free article] [PubMed]
10. Li R, Li X, Yan Q, Mo JQ, Guan MX. Isolation and characterization of mouse MTO1 related to mitochondrial tRNA modification. Biochim Biophys Acta. 2003;1629:53–59. [PubMed]
11. Kambampati R, Lauhon CT. MnmA and IscS are required for in vitro 2-thiouridine biosynthesis in Escherichia coli. Biochemistry. 2003;42:1109–1117. [PubMed]
12. Yan Q, Li X, Faye G, Guan MX. Mutations in MTO2 related to tRNA modification impair mitochondrial gene expression and protein synthesis in the presence of a paromomycin resistance mutation in mitochondrial 15S rRNA. J Biol Chem. 2005;280:29151–29157. [PMC free article] [PubMed]
13. Elseviers D, Petrullo LA, Gallagher P. Novel E. coli mutants deficient in biosynthesis of 5-methylaminomethyl-2-thiouridine. Nucleic Acids Res. 1984;12:3521–3534. [PMC free article] [PubMed]
14. Meyer S, Scrima A, Versées W, Wittinghofer A. Crystal structures of the conserved tRNA-modifying enzyme GidA: implications for its interaction with MnmE and substrate. J Mol Biol. 2008;380:532–547. [PubMed]
15. Scrima A, Vetter IR, Armengod MF, Wittinghofer A. The structure of the TrmE GTP-binding protein and its implications for tRNA modification. EMBO J. 2005;24:23–33. [PubMed]
16. Ikeuchi Y, Shigi N, Kato J, Nishimura A, Suzuki T. Mechanistic insights into sulfur relay by multiple sulfur mediators involved in thiouridine biosynthesis at tRNA wobble positions. Mol Cell. 2006;21:97–108. [PubMed]
17. Numata T, Ikeuchi Y, Fukai S, Suzuki T, Nureki O. Snapshots of tRNA sulphuration via an adenylated intermediate. Nature. 2006;442:419–424. [PubMed]
18. Colby G, Wu M, Tzagoloff A. MTO1 codes for a mitochondrial protein required for respiration in paromomycin-resistant mutants of Saccharomyces cerevisiae. J Biol Chem. 1998;273:27945–27952. [PubMed]
19. Decoster E, Vassal A, Faye G. MSS1, a nuclear-encoded mitochondrial GTPase involved in the expression of COX1 subunit of cytochrome c oxidase. J Mol Biol. 1993;232:79–88. [PubMed]
20. Umeda N, Suzuki T, Yukawa M, Ohya Y, Shindo H, Watanabe K, Suzuki T. Mitochodnria-specific RNA-modifying enzymes responsible for the biosynthesis of the wobble base in mitochondrial tRNAs: implications for the molecular pathogenesis of human mitochondrial diseases. J Biol Chem. 2004;280:1613–1624. [PubMed]
21. Wang X, Yan Q, Guan MX. Deletion of the MTO2 gene related to tRNA modification causes a failure in mitochondrial RNA metabolism in the yeast Saccharomyces cerevisiae. FEBS Lett. 2007;581:4228–4234. [PubMed]
22. Wang X, Yan Q, Guan MX. Mutation in MTO1 involved in tRNA modification impairs mitochondrial RNA metabolism in the yeast Saccharomyces cerevisiae. Mitochondrion. 2009;9:180–185. [PMC free article] [PubMed]
23. Zhao H, Li R, Wang Q, Yan Q, Deng JH, Han D, Bai Y, Young WY, Guan MX. Maternally inherited aminoglycoside-induced and non-syndromic deafness is associated with the novel C1494T mutation in the mitochondrial 12S rRNA gene in a large Chinese family. Am J Hum Genet. 2004;74:139–152. [PubMed]
24. Guan MX, Yan Q, Li X, Bykhovskaya Y, Gallo-Teran J, Hajek P, Umeda N, Zhao H, Garrido G, Mengesha E, Suzuki T, del Castillo IL, Peters J, Li R, Qian Y, Wang X, Ballana E, Shohat M, Lu J, Estivill X, Watanabe K, Fischel-Ghodsian N. Mutation in TRMU related to transfer RNA modification modulates the phenotypic expression of the deafness-associated mitochondrial 12S ribosomal RNA mutations. Am J Hum Genet. 2006;79:291–302. [PubMed]
25. Clark-Walker GD, Miklos GLG. Complementation in cytoplasmic petite mutants of yeast to form respiratory competent cells. Proc Natl Acad Sci USA. 1975;72:372–375. [PubMed]
26. Moriya J, Yokogawa T, Wakita K, Ueda T, Nishikawa K, Crain PF, Hashizume T, Pomerantz SC, McCloskey JA, Kawai G, Hayashi N, Yokoyama S, Watanabe K. A novel modified nucleoside found at the first position of the anticodon of methionine tRNA from bovine liver mitochondria. Biochemistry. 1994;33:2234–2239. [PubMed]
27. Shigi N, Suzuki T, Tamakoshi M, Oshima T, Watanabe K. Conserved bases in the TΨC Loop of tRNA are determinants for thermophile-specific 2-thiouridylation at position 54. J Biol Chem. 2002;277:39128–39135. [PubMed]
28. Suzuki T, Suzuki T, Wada T, Saigo K, Watanabe K. Taurine as a constituent of mitochondrial tRNA: new insights into the functions of taurine and human mitochondrial diseases. EMBO J. 2002;21:6581–6589. [PubMed]
29. Krüger MK, Sørensen MA. Aminoacylation of hypomodified tRNAGlu in vivo. J Mol Biol. 1998;284:609–620. [PubMed]
30. Sylvers LA, Rogers KC, Shimizu M, Ohtsuka E, Soll D. A 2-thiouridine derivative in tRNAGlu is a positive determinant for aminoacylation by Escherichia coli glutamyl-tRNA synthetase. Biochemistry. 1993;32:3836–3841. [PubMed]
31. Foury F, Roganti T, Lecrenier N, Purnelle B. The complete sequence of the mitochondrial genome of Saccharomyces cerevisiae. FEBS Lett. 1998;440:325–331. [PubMed]
32. Villarroya M, Prado S, Esteve JM, Soriano MA, Aguado C, Pérez-Martínez D, Martínez-Ferrandis JI, Yim L, Victor VM, Cebolla E, Montaner A, Knecht E, Armengod ME. Characterization of human GTPBP3, a GTP-binding protein involved in mitochondrial tRNA modification. Mol Cell Biol. 2008;28:7514–7531. [PMC free article] [PubMed]
33. Sundaram M, Durant PC, Davis DR. Hypermodified nucleosides in the anticodon of tRNA(Lys) stabilize a canonical U-turn structure. Biochemistry. 2000;39:12575–12584. [PubMed]
34. Hagervall TG, Pomerantz SC, McCloskey JA. Reduced misreading of asparagine codons by Escherichia coli tRNALys with hypomodified derivatives of 5-methylaminomethyl-2-thiouridine in the wobble position. J Mol Biol. 1998;284:33–42. [PubMed]
35. Alexandrov A, Chernyakov I, Gu W, Hiley SL, Hughes TR, Grayhack EJ, Phizicky EM. Rapid tRNA decay can result from lack of nonessential modifications. Mol Cell. 2006;21:87–96. [PubMed]
36. Chernyakov I, Whipple JM, Kotelawala L, Grayhack EJ, Phizicky EM. Degradation of several hypomodified mature tRNA species in Saccharomyces cerevisiae is mediated by Met22 and the 5′-3′ exonucleases Rat1 and Xrn1. Genes Dev. 2008;22:1369–1380. [PubMed]
37. Ashraf SS, Sochacka E, Cain R, Guenther R, Malkiewicz A, Agris PF. Single atom modification (O→S) of tRNA confers ribosome binding. RNA. 1999;5:188–194. [PubMed]
38. Hagervall TG, Björk GR. Undermodification in the first position of the anticodon of supG-tRNA reduces translational efficiency. Mol Gen Genet. 1984;196:194–200. [PubMed]
39. Urbonavicius U, Qian Q, Durand JM, Hagervall TG, Björk GR. Improvement of reading frame maintenance is a common function for several tRNA modifications. EMBO J. 2001;20:4863–4873. [PubMed]
40. Yarian C, Townsend H, Czestkowski W, Sochacka E, Malkiewicz AJ, Guenther R, Miskiewicz A, Agris PF. Modified nucleoside dependent Watson–Crick and wobble codon binding by tRNALysUUU species. Biochemistry. 2000;39:13390–13395. [PubMed]
41. Yasukawa T, Suzuki T, Ishii N, Ohta S, Watanabe K. Wobble modification defect in tRNA disturbs codon–anticodon interaction in a mitochondrial disease. EMBO J. 2001;20:4794–4802. [PubMed]
42. Costanzo MC, Fox TD. Control of mitochondrial gene expression in Saccharomyces cerevisiae. Annu Rev Genet. 1990;24:91–108. [PubMed]
43. Islas-Osuna MA, Ellis TP, Marnell LL, Mittelmeier TM, Dieckmann CL. Cbp1 Is Required for Translation of the Mitochondrial Cytochrome b mRNA of Saccharomyces cerevisiae. J Biol Chem. 2002;277:37987–37990. [PubMed]
44. Myers AM, Pape LK, Tzagoloff A. Mitochondrial protein synthesis is required for maintenance of intact mitochondrial genomes in Saccharomyces cerevisiae. EMBO J. 1985;4:2087–2092. [PubMed]
45. Barrientos A, Korr D, Barwell KJ, Sjulsen C, Gajewski CD, Manfredi G, Ackerman S, Tzagoloff A. MTG1 codes for a conserved protein required for mitochondrial translation. Mol Biol Cell. 2003;14:2292–2302. [PMC free article] [PubMed]
46. Williams EH, Butler CA, Bonnefoy N, Fox TD. Translation initiation in Saccharomyces cerevisiae mitochondria: functional interactions among mitochondrial ribosomal protein Rsm28p, initiation factor 2, methionyl-tRNA-formyltransferase and novel protein Rmd9p. Genetics. 2007;175:1117–1126. [PubMed]
47. Hensgens LA, Bonen L, de Haan M, van der Horst G, Grivell LA. Two intron sequences in yeast mitochondrial COX1 gene: homology among URF-containing introns and strain-dependent variation in flanking exons. Cell. 1983;32:379–389. [PubMed]
48. Van Dyck E, Clayton DA. Transcription-dependent DNA transactions in the mitochondrial genome of a yeast hypersuppressive petite mutant. Mol Cell Biol. 1998;18:2976–2985. [PMC free article] [PubMed]
49. Burnett KG, Scheffler IE. Integrity of mitochondria in a mammalian cell mutant defective in mitochondrial protein synthesis. J Cell Biol. 1981;90:108–115. [PMC free article] [PubMed]
50. Guan MX. Cytoplasmic tyrosyl-tRNA synthetase rescues the defect in mitochondrial genome maintenance caused by the nuclear mutation mgm104-1 in the yeast Saccharomyces cerevisiae. Mol Gen Genet. 1997;255:525–532. [PubMed]
51. Sherman F, Fink GR, Hicks JB. Methods in Yeast Genetics. Cold Spring Harbor Laboratory; Cold Spring Harbor, NY: 1983.
52. Enríquez JA, Attardi G. Analysis of aminoacylation of human mitochondrial tRNAs. Methods Enzymol. 1996;264:183–196. [PubMed]
53. Barrientos A, Korr D, Tzagoloff A. Shy1p is necessary for full expression of mitochondrial COX1 in the yeast model of Leigh’s syndrome. EMBO J. 2002;21:43–52. [PubMed]