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• W2034873621 abstract "Nuclear gene(s) have been shown to modulate the phenotypic expression of mitochondrial DNA mutations. We report here the identification and characterization of the yeast nuclear gene MTO2 encoding an evolutionarily conserved protein involved in mitochondrial tRNA modification. Interestingly, mto2 null mutants expressed a respiratory-deficient phenotype when coexisting with the C1409G mutation of mitochondrial 15 S rRNA at the very conservative site for human deafness-associated 12 S rRNA A1491G and C1409T mutations. Furthermore, the overall rate of mitochondrial translation was markedly reduced in a yeast mto2 strain in the wild type mitochondrial background, whereas mitochondrial protein synthesis was almost abolished in a yeast mto2 strain carrying the C1409G allele. The other interesting feature of mto2 mutants is the defective expression of mitochondrial genes, especially CYTB and COX1, but only when coexisting with the C1409G allele. These data strongly indicate that a product of MTO2 functionally interacts with the decoding region of 15 S rRNA, particularly at the site of the C1409G or A1491G mutation. In addition, we showed that yeast and human Mto2p localize in mitochondria. The isolated human MTO2 cDNA can partially restore the respiratory-deficient phenotype of yeast mto2 cells carrying the C1409G mutation. These functional conservations imply that human MTO2 may act as a modifier gene, modulating the phenotypic expression of the deafness-associated A1491G or C1409T mutation in mitochondrial 12 S rRNA. Nuclear gene(s) have been shown to modulate the phenotypic expression of mitochondrial DNA mutations. We report here the identification and characterization of the yeast nuclear gene MTO2 encoding an evolutionarily conserved protein involved in mitochondrial tRNA modification. Interestingly, mto2 null mutants expressed a respiratory-deficient phenotype when coexisting with the C1409G mutation of mitochondrial 15 S rRNA at the very conservative site for human deafness-associated 12 S rRNA A1491G and C1409T mutations. Furthermore, the overall rate of mitochondrial translation was markedly reduced in a yeast mto2 strain in the wild type mitochondrial background, whereas mitochondrial protein synthesis was almost abolished in a yeast mto2 strain carrying the C1409G allele. The other interesting feature of mto2 mutants is the defective expression of mitochondrial genes, especially CYTB and COX1, but only when coexisting with the C1409G allele. These data strongly indicate that a product of MTO2 functionally interacts with the decoding region of 15 S rRNA, particularly at the site of the C1409G or A1491G mutation. In addition, we showed that yeast and human Mto2p localize in mitochondria. The isolated human MTO2 cDNA can partially restore the respiratory-deficient phenotype of yeast mto2 cells carrying the C1409G mutation. These functional conservations imply that human MTO2 may act as a modifier gene, modulating the phenotypic expression of the deafness-associated A1491G or C1409T mutation in mitochondrial 12 S rRNA. Interaction between the nuclear and mitochondrial genomes is necessary for controlling the phenotypic expression of mtDNA mutation(s). In humans, nuclear modifier gene(s) have been shown to modulate the phenotypic expression of the mitochondrial 12 S rRNA A1491G or C1409T mutation associated with aminoglycoside-induced and nonsyndromic deafness (1Guan M.X. Ann. N. Y. Acad. Sci. 2004; 1011: 259-271Crossref PubMed Scopus (114) Google Scholar, 2Guan M.X. Fischel-Ghodsian N. Attardi G. Hum. Mol. Genet. 2001; 10: 573-580Crossref PubMed Scopus (163) Google Scholar, 3Guan M.X. Fischel-Ghodsian N. Attardi G. Hum. Mol. Genet. 1996; 5: 963-971Crossref PubMed Scopus (195) Google Scholar, 4Zhao H. Li R. Wang Q. Yan Q. Deng J.H. Han D. Bai Y. Young W.Y. Guan M.X. Am. J. Hum. Genet. 2004; 74: 139-152Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar, 5Zhao H. Young W.Y. Yan Q. Li R. Cao J. Wang Q. Li X. Peters J.L. Han D. Guan M.X. Nucleic Acids Res. 2005; 33: 1132-1139Crossref PubMed Scopus (82) Google Scholar). As shown in Fig. 1, these mtDNA mutations are located at the A-site of the small ribosomal subunit, which is highly conserved from bacteria to mammals (6Neefs J.M. Van de Peer Y. De Rijik P. Goris A. De Wachter R. Nucleic Acids Res. 1991; 19: 1987-2018Crossref PubMed Scopus (219) Google Scholar). The homologous region of 16 S rRNA in Escherichia coli is an essential part of the decoding site of the ribosome (7Zimmermann R.A. Thomas C.L. Wower J. Hill W.E. Moore P.B. Dahlberg A. Schlessinger D. Garrett R.A. Warner J.R. The Ribosome: Structure, Function, and Evolution. American Society for Microbiology, Washington, D. C.1990: 331-347Google Scholar, 8De Stasio E.A. Dahlberg A.E. J. Mol. Biol. 1990; 212: 127-133Crossref PubMed Scopus (78) Google Scholar) and is crucial for subunit association either by an RNA-protein or RNA-RNA interaction (9Zwieb C. Jemiolo D.K. Jacob W.F. Wagner R. Dahlberg A.E. Mol. Gen. Genet. 1986; 203: 256-264Crossref PubMed Scopus (22) Google Scholar). This region is also an important locus of action for aminoglycosides (10Moazed D. Noller H.F. Nature. 1987; 327: 389-394Crossref PubMed Scopus (967) Google Scholar, 11Purohit P. Stern S. Nature. 1994; 370: 659-662Crossref PubMed Scopus (255) Google Scholar). Mutations, which disrupted the 1409-1491 base pair of E. coli 16 S rRNA or Saccharomyces cerevisiae mitochondrial 15 S rRNA, confer aminoglycoside resistance (8De Stasio E.A. Dahlberg A.E. J. Mol. Biol. 1990; 212: 127-133Crossref PubMed Scopus (78) Google Scholar, 12Gregory S.T. Dahlberg A.E. Nucleic Acids Res. 1995; 23: 4234-4238Crossref PubMed Scopus (29) Google Scholar, 13Li M. Tzagoloff A. Underbrink-Lyon K. Martin N.C. J. Biol. Chem. 1982; 257: 5921-5928Abstract Full Text PDF PubMed Google Scholar, 14Weiss-Brummer B. Huttenhofer A. Mol. Gen. Genet. 1989; 217: 362-369Crossref PubMed Scopus (28) Google Scholar). In human, the G-C or U-A pair in the mitochondrial 12 S rRNA created by the A1491G or C1409T mutation facilitates the binding of aminoglycoside (4Zhao H. Li R. Wang Q. Yan Q. Deng J.H. Han D. Bai Y. Young W.Y. Guan M.X. Am. J. Hum. Genet. 2004; 74: 139-152Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar, 5Zhao H. Young W.Y. Yan Q. Li R. Cao J. Wang Q. Li X. Peters J.L. Han D. Guan M.X. Nucleic Acids Res. 2005; 33: 1132-1139Crossref PubMed Scopus (82) Google Scholar, 15Hamasaki K. Rando R.R. Biochemistry. 1997; 36: 12323-12328Crossref PubMed Scopus (120) Google Scholar, 16Guan M.X. Fischel-Ghodsian N. Attardi G. Hum. Mol. Genet. 2000; 9: 1787-1793Crossref PubMed Scopus (165) Google Scholar). Our previous investigations revealed that the A1491G or C1409T mutation in human mtDNA is the primary contributor underlying the development of deafness but is not sufficient to produce a clinical phenotype (2Guan M.X. Fischel-Ghodsian N. Attardi G. Hum. Mol. Genet. 2001; 10: 573-580Crossref PubMed Scopus (163) Google Scholar, 3Guan M.X. Fischel-Ghodsian N. Attardi G. Hum. Mol. Genet. 1996; 5: 963-971Crossref PubMed Scopus (195) Google Scholar, 4Zhao H. Li R. Wang Q. Yan Q. Deng J.H. Han D. Bai Y. Young W.Y. Guan M.X. Am. J. Hum. Genet. 2004; 74: 139-152Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar, 5Zhao H. Young W.Y. Yan Q. Li R. Cao J. Wang Q. Li X. Peters J.L. Han D. Guan M.X. Nucleic Acids Res. 2005; 33: 1132-1139Crossref PubMed Scopus (82) Google Scholar). However, the product of nuclear modifier gene(s), which may functionally interact with the mutated 12 S rRNA, influences the phenotypic manifestation of the A1491G or C1409T mutation by enhancing or suppressing the effect of these mutations (2Guan M.X. Fischel-Ghodsian N. Attardi G. Hum. Mol. Genet. 2001; 10: 573-580Crossref PubMed Scopus (163) Google Scholar, 3Guan M.X. Fischel-Ghodsian N. Attardi G. Hum. Mol. Genet. 1996; 5: 963-971Crossref PubMed Scopus (195) Google Scholar, 4Zhao H. Li R. Wang Q. Yan Q. Deng J.H. Han D. Bai Y. Young W.Y. Guan M.X. Am. J. Hum. Genet. 2004; 74: 139-152Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar, 5Zhao H. Young W.Y. Yan Q. Li R. Cao J. Wang Q. Li X. Peters J.L. Han D. Guan M.X. Nucleic Acids Res. 2005; 33: 1132-1139Crossref PubMed Scopus (82) Google Scholar). An interesting model for nuclear-mtDNA interaction for the phenotypic expression of the A1491G or C1409T mutation has been proposed in S. cerevisiae. The mutant allele of MTO1 or MSS1, encoding mitochondrial proteins, manifests a respiratory-deficient phenotype only when coupled with the paromomycin-resistance mitochondrial 15 S rRNA C1409G mutation (PR454 or PR) (17Colby G. Wu M. Tzagoloff A. J. Biol. Chem. 1998; 273: 27945-27952Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 18Decoster E. Vassal A. Faye G. J. Mol. Biol. 1993; 232: 79-88Crossref PubMed Scopus (75) Google Scholar). These observations strongly indicate that Mss1p or Mto1p affects the phenotypic expression of the C1409G mutation by functionally interacting with the region of the C1409G mutation in mitochondrial 15 S rRNA. In E. coli, the products of mnmE (homolog of MSS1) (19Cabedo H. Macian F. Villarroya M. Escudero J.C. Martinez-Vicente M. Knecht E. Armengod M.E. EMBO J. 1999; 18: 7063-7076Crossref PubMed Scopus (95) Google Scholar), gidA (homolog of MTO1) (20Brégeon D. Colot V. Miroslav M. Radman M. Taddei F. Genes Dev. 2001; 15: 2295-2306Crossref PubMed Scopus (129) Google Scholar), and trmU (21Kambampati R. Lauhon C.T. Biochemistry. 2003; 42: 1109-1117Crossref PubMed Scopus (133) Google Scholar) have been shown to be involved in the biosynthesis of the hypermodified nucleoside 5-methyl-aminomethyl-2-thio-uridine (mnm5s2U34) (22Björk G.R. Neidhardt F.C. Curtiss III, R. Ingraham J.L. Lin E.C.C. Low B.K. Magasanik B. Reznikoff W.S. Riley M. Schaechter M. Umbarger H.E. Escherichia coli and Salmonella: Cellular and Molecular Biology. American Society for Microbiology, Washington, D. C.1996: 861-886Google Scholar). This modified nucleotide, found in the wobble position of several bacterial tRNAs specific for glutamate, lysine, arginine, and glutamine, has a pivotal role in the structure and function of tRNAs, including structural stabilization, aminoacylation, and codon recognition at the decoding site of small ribosomal RNAs (20Brégeon D. Colot V. Miroslav M. Radman M. Taddei F. Genes Dev. 2001; 15: 2295-2306Crossref PubMed Scopus (129) Google Scholar, 22Björk G.R. Neidhardt F.C. Curtiss III, R. Ingraham J.L. Lin E.C.C. Low B.K. Magasanik B. Reznikoff W.S. Riley M. Schaechter M. Umbarger H.E. Escherichia coli and Salmonella: Cellular and Molecular Biology. American Society for Microbiology, Washington, D. C.1996: 861-886Google Scholar). Recently, we demonstrated that isolated human MTO1 or GTPBP3 (homolog of MSS1) cDNA can complement the respiratory-deficient phenotype of yeast mto1 or mss1 cells carrying the 15 S rRNA C1409G mutation, suggesting that the functions of those proteins are evolutionarily conserved (23Li X. Guan M.X. Mol. Cell. Biol. 2002; 22: 7701-7711Crossref PubMed Scopus (94) Google Scholar, 24Li X. Li R. Lin X. Guan M.X. J. Biol. Chem. 2002; 277: 27256-27264Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). In E. coli, trmU has been shown to be responsible for the 2-thiolation of mnm5s2U34 in tRNALys, tRNAGlu, and tRNAGln (20Brégeon D. Colot V. Miroslav M. Radman M. Taddei F. Genes Dev. 2001; 15: 2295-2306Crossref PubMed Scopus (129) Google Scholar, 21Kambampati R. Lauhon C.T. Biochemistry. 2003; 42: 1109-1117Crossref PubMed Scopus (133) Google Scholar, 22Björk G.R. Neidhardt F.C. Curtiss III, R. Ingraham J.L. Lin E.C.C. Low B.K. Magasanik B. Reznikoff W.S. Riley M. Schaechter M. Umbarger H.E. Escherichia coli and Salmonella: Cellular and Molecular Biology. American Society for Microbiology, Washington, D. C.1996: 861-886Google Scholar). In S. cerevisiae, mto2/mtu1 (homolog of trmU) null mutants conferred defects in 2-thiouridylation in mitochondrial tRNALys, tRNAGlu, and tRNAGln but not cytoplasmic tRNALys (25Umeda N. Suzuki T. Yukawa M. Ohya Y. Shindo H. Watanabe K. Suzuki T. J. Biol. Chem. 2005; 280: 1613-1624Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). To examine whether yeast MTO2 (mitochondrial translational optimization) mediates the phenotypic expression of the 15 S rRNA C1409G mutation, this nuclear gene has been identified, and a null mto2 mutation in the wild type mitochondrial background (paromomycin-sensitive PS) or C1409G (PR) allele has been generated. These mto2 null mutants have been characterized by examining mitochondrial gene expression, mitochondrial protein synthesis, and functional complementation of human MTO2 cDNA in a yeast mto2 mutant carrying the mitochondrial C1409G (PR) allele. Furthermore, the yeast and human Mto2p were examined for subcellular localization by immunofluorescence analysis. Yeast Strains, Media, and Genetic Techniques—The genotypes and sources of strains of S. cerevisiae used in this investigation are listed in Table I. The media used to grow yeast have been described elsewhere (27Chen X.J. Guan M.X. Clark-Walker G.D. Nucleic Acids Res. 1993; 21: 3473-3477Crossref PubMed Scopus (51) Google Scholar, 28Guan M.X. Mol. Gen. Genet. 1997; 255: 525-532Crossref PubMed Scopus (12) Google Scholar). Standard procedures were used for crossing and selecting diploids, including sporulation and dissecting tetrads (29Sherman F. Fink G.R. Hicks J.B. Methods in Yeast Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1983Google Scholar).Table IGenotype and sources of yeast strainsStrainsGenotypeSourceW303-1Bα ade2-1, his3-1,15, leu2-3, trp1-1, ura3-1Ref. 17Colby G. Wu M. Tzagoloff A. J. Biol. Chem. 1998; 273: 27945-27952Abstract Full Text Full Text PDF PubMed Scopus (89) Google ScholarW1021-7Ca ade2-1, his3-1,15, leu2-3, ura3-1Ref. 26Qiu J. Guan M.X. Bailis A.M. Shen B.H. Nucleic Acids Res. 1998; 26: 3077-3083Crossref PubMed Scopus (38) Google ScholarM12a ilv5, trp2 [ρ+, PS]Ref. 18Decoster E. Vassal A. Faye G. J. Mol. Biol. 1993; 232: 79-88Crossref PubMed Scopus (75) Google ScholarM12-54a ilv5, trp2 [ρ+, PR454]Ref. 18Decoster E. Vassal A. Faye G. J. Mol. Biol. 1993; 232: 79-88Crossref PubMed Scopus (75) Google ScholarA18α, lys2, met6, mss1-18 [PR454]Ref. 18Decoster E. Vassal A. Faye G. J. Mol. Biol. 1993; 232: 79-88Crossref PubMed Scopus (75) Google ScholarW303ΔMTO1(PR)a ade2-1,his3-1, trp1-1, ura3-1,mto1::URA3[PR454]Ref. 17Colby G. Wu M. Tzagoloff A. J. Biol. Chem. 1998; 273: 27945-27952Abstract Full Text Full Text PDF PubMed Scopus (89) Google ScholarW303ΔMTO2(PS)α ade2-1, his3-1,15, leu2-3,trp1-1,ura3-1,mto2::HIS3This studyW303ΔMTO2(ρo)α ade2-1, his3-1,15, leu2-3,trp1-1,ura3-1,mto2::HIS3This studyW1021ΔMTO2(PS)a ade2-1, his3-1, leu2-3, ura3-1, mto2::HIS3This studyW1021ΔMTO2(ρo)a ade2-1, his3-1, leu2-3, ura3-1, mto2::HIS3This studyQY1a/α ade2-1/+, his3-1/+, leu2-3/+, ura3-1/+, lys2/+, met6/+, mss1-18/MSS1,mto2::HIS3/MTO2, [PR454]This studyW1021ΔMTO2(PR)a ade2-1,his3-1,lys2, ura3-1,mto2::HIS3[PR454]This studyW1021ΔMTO2/mss1(PR)a his3-1,ura3-1, mss1-18, mto2::HIS3[PR454]This study Open table in a new tab Cloning of Yeast MTO2—The peptide sequence of the E. coli trmU sequence (30Green S.M. Malik T. Giles I.G. Drabble W.T. Microbiology (N. Y.). 1996; 142: 3219-3230Crossref PubMed Scopus (20) Google Scholar) was subjected to a BLAST search of the publicly available S. cerevisiae sequence databases (NCBI/Genbank™/EMBL). This search led to the identification of one open reading frame encoding a protein (YDL033C, GenBank™ accession number Z71781) with a high degree of homology to E. coli TrmU. To obtain the full-length coding region of MTO2 DNA, PCR was performed using the high fidelity Pfu DNA polymerase (Promega) and total yeast genomic DNA isolated from W303-1B cells as a template, with primers 5′-AATTTTAAGAGCGCCGGG-3′ (nucleotides (nt) 1The abbreviations used are: nt, nucleotide(s); GFP, green fluorescent protein; DIG, digoxigenin. 143-163) and 5′-ACATGATTCAAGGGAAAAGACC-3′ (nt 1713-1734) (GenBank™ accession number AY624369). The predominant PCR product was purified by agarose gel electrophoresis and subsequently cloned into a PCR 2.1-TOPO vector (Invitrogen). Sequencing was done using a Dye Terminator cycle sequencing kit (PerkinElmer Life Sciences) and an ABI PRISM™ 3100 genetic analyzer. The resultant plasmid carrying the full-length coding region of yeast MTO2 was designed as pYMTO2. Construction of mto2 Null Mutants—mto2 null strains were generated by the one-step gene disruption technique of Rothstein (31Rothestein R.J. Methods Enzymol. 1993; 101: 202-211Crossref Scopus (2026) Google Scholar). A 1.6-kb HindIII/XbaI fragment containing the full-length coding region of the MTO2 gene was isolated by digesting pYMTO2 with HindIII and XbaI and was ligated to the vector pGEM-7Zf(+) (Promega) to produce the resultant plasmid pGEM-MTO2. A 452-base-pair EcoRV fragment (positions 614-1066) spanning the coding region of MTO2 was deleted by digesting with EcoRV. The 1.2-kb fragment containing the full-length coding region of HIS3 was obtained from plasmid pRS303 by digesting with EcoRI and filled in with Klenow PolI. The resultant fragment was ligated into the EcoRV site of pGEM-MTO2 to replace the EcoRV fragment containing the MTO2 coding region. The resultant plasmid containing the MTO2::HIS3 allele was digested with HindIII and XbaI, and the fragment was introduced into wild type yeast strains W1021-7C and W303-1B by the method of Gietz and Schestl (32Gietz R.D. Schiestl R.H. Yeast. 1991; 7: 253-263Crossref PubMed Scopus (368) Google Scholar). The integrated deletion construct was selected for the cells by growing on glucose-minimal medium in the absence of histidine. Disruption was verified by PCR amplification using primers 5′-ATGCTGGCAAGATATTTAAA-3′ (nt 305-324) and 5′-AAGGACTCATCGTCGAT-3′ (nt 1493-1510). Mitochondrial Gene Expression 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 the CYTB-specific RNA probe. 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, 15 S rRNA and 21 S rRNA probes, respectively. As an internal control, RNA blots were stripped and rehybridized with a DIG-labeled nuclear 25 S rRNA probe. The plasmids used for mtDNA probes were constructed by PCR-amplifying fragments of CYTB (positions 36595-36964), COX1 (positions 26211-26689), 15 S rRNA (positions 7532-8280), and 21 S rRNA (positions 58484-59580) (GenBank™ accession number AJ011856) (33Foury F. Roganti T. Lecrenier N. Purnelle B. FEBS Lett. 1998; 440: 325-331Crossref PubMed Scopus (323) Google Scholar), as well as a nuclear 25 S rRNA probe (position 631-1665) (GenBank™ accession number U53879) and cloning the fragments into the pCRII-TOPO vector carrying SP6 and T7 promoters (Invitrogen). 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 (34Barrientos A. Korr D. Tzagoloff A. EMBO J. 2002; 21: 43-52Crossref PubMed Scopus (141) Google Scholar). The radiolabeled proteins were separated on SDS-exponential polyacrylamide gradient gels (2Guan M.X. Fischel-Ghodsian N. Attardi G. Hum. Mol. Genet. 2001; 10: 573-580Crossref PubMed Scopus (163) Google Scholar, 35Chomyn A. Methods Enzymol. 1996; 264: 197-211Crossref PubMed Google Scholar). The gels were treated with Me2SO/2,5-diphenyloxazole, dried, and exposed for fluorography. Subcellular Localization of Yeast Mto2p—The coding sequence for MTO2 lacking its natural stop codon was obtained by PCR using pYMTO2 as a template. The primers 5′-CCGGAATTCCGGATGCTGGCAAGATATTTA-3′ (nt 305-322) and 5′-CCCAAGCTTGGGTGCATGGGTGTCATTATT-3′ (nt 1538-1555) were used for PCR amplification. PCR products were first cloned into the pCR 2.1-TOPO vector (Invitrogen). After sequence determination, the insert was subsequently subcloned into the expression vector pGFP-C-FUS under the control of the MET25 promoter (36Niedenthal R.K. Riles L. Johnston M. Hegemann J.H. Yeast. 1996; 12: 773-786Crossref PubMed Scopus (366) Google Scholar). The MTO2-GFP fusion construct or the vector pGFP-C-FUS alone was transformed into the wild type strain W303-1B. Resultant transformants were grown at 30 °C to midlog phases in 10 ml of glucose-minimal medium with 2% galactose and the appropriate auxotrophic requirements. To stain mitochondria, cells were incubated with 0.05 μm of MitoTracker™ Red CMXRos (Molecular Probes, Portland, Oregon) for 1 h at 30 °C. The cells were then examined under a Carl Zeiss confocal fluorescence microscope. Cloning and Expression of Human MTO2 cDNA—The coding region of human MTO2 cDNAs lacking its natural stop codon were obtained by reverse transcription-PCR using the high fidelity Pfu polymerase (Promega). Total RNA was extracted from 143B cells to use as a template. The primers 5′-CCGCTCGAGCGGATGCAGGCCTTGCGGCAC-3′ (nt 52-69) and 5′-CGGAATTCCGAGCAAGGGACTCAGGCC-3′ (nt 1297-1314) (GenBank™ accession number AY062123) were used for the PCR amplification. The PCR products were digested with XhoI and EcoRI and cloned into pBluescript II KS+ (Promega). After sequence determination, the inserts were subcloned into pEGFP-N1 (Clontech) to generate pEGFP-N1-MTO2. The resultant construct or vector alone was transfected into the human 143B osteosarcoma cell line using the SuperFect™ transfection reagent (Qiagen) according to the manufacturer's protocol. Immunofluorescence analysis was performed as detailed elsewhere (23Li X. Guan M.X. Mol. Cell. Biol. 2002; 22: 7701-7711Crossref PubMed Scopus (94) Google Scholar, 24Li X. Li R. Lin X. Guan M.X. J. Biol. Chem. 2002; 277: 27256-27264Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). S. cerevisiae wild type and mto2 strains used for this study were W301-1B and W303ΔMTO2(PR), respectively. A yeast expression shuttle vector pDB20 was used for the expression of human MTO2 in S. cerevisiae (37Becker D.M. Fikes J.D. Guarente L. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1968-1972Crossref PubMed Scopus (179) Google Scholar). A human MTO2 cDNA was obtained by PCR amplification using pEGFP-N1-MTO2 as the template. Primers used for PCR amplification were 5′-AAGCTTAGCTGCAGCTGGCGAAGT-3′ (nt 20-38) and 5′-AAGCTTAGCAGCAAGCTGGCCCTT-3′ (nt 1338-1355). PCR products were cloned into PCR2.1-TOPO vector (Invitrogen). After sequence determination, the insert was subsequently subcloned into pDB20 to generate pDB20-MTO2. These constructs were transformed into the yeast W303ΔMTO2(PR) strain. Ura3+ transformants were selected at 30 °C on glucose-minimal medium. Transformants were then spotted on glucose and glycerol plates and incubated at 30 °C for 3-5 days. Colonies growing on glycerol medium were subjected to further analysis. Identification of Yeast MTO2—The product of the E. coli trmU sequence (30Green S.M. Malik T. Giles I.G. Drabble W.T. Microbiology (N. Y.). 1996; 142: 3219-3230Crossref PubMed Scopus (20) Google Scholar) was subjected to a BLAST search of the publicly available S. cerevisiae sequence databases (NCBI/Genbank™/EMBL). This search led to the identification of one open reading frame encoding a protein with a high degree of homology to E. coli TrmU. The S. cerevisiae homolog MTO2 on chromosome IV (YDL033C, GenBank™ accession number Z71781) is predicted to encode a 417-amino-acid protein with a molecular mass of 47,049 Da. The predicated yeast Mto2 polypeptide revealed an extensive conservation of amino acid sequences and similarity in size by an alignment with homologs, from Homo sapiens, Mus musculus (38Yan Q. Guan M.X. Biochim. Biophys. Acta. 2004; 1676: 119-126Crossref PubMed Scopus (23) Google Scholar), Schizosaccharomyces pombe, Drosophila melanogaster, E. coli (30Green S.M. Malik T. Giles I.G. Drabble W.T. Microbiology (N. Y.). 1996; 142: 3219-3230Crossref PubMed Scopus (20) Google Scholar) to Bacillus subtilis. In particular, the overall identity of the predicted amino acid sequence of S. cerevisiae Mto2p with homologs of H. sapiens, M. musculus, D. melanogaster, S. pombe, B. subtilis, and E. coli is 35, 35, 36, 39, 41, and 39%, respectively, whereas the similarity is 47, 47, 47, 48, 51, and 49%, respectively. Mto2p is likely a mitochondrial protein, due to the presence of a typical mitochondrial target presequence (39Hartl F.U. Neupert W. Science. 1990; 247: 930-938Crossref PubMed Scopus (352) Google Scholar). The mto2 Null Mutant Expresses a Respiratory-deficient Phenotype when Combined with the 15 S rRNA C1409G Allele—To determine whether MTO2 is essential for mitochondrial function, a single copy of the gene was disrupted in a haploid strain. The resulting mto2 null strains were tested for growth on glycerol-complete medium. As can been seen in Fig. 2, the mto2 null cells in the wild type mitochondrial background (PS) were able to grow on glycerol-complete medium, indicating that these cells were respiratory-competent. To test whether the mitochondrial genetic background affects the phenotypic expression of mto2 null mutants, the W303ΔMTO2(PS) strain was treated with ethidium bromide to cause the loss of mtDNA (ρo) (28Guan M.X. Mol. Gen. Genet. 1997; 255: 525-532Crossref PubMed Scopus (12) Google Scholar). ρo derivatives were then crossed with the M12-54 strain carrying the 15 S rRNA C1409G (PR) mutation or with the M12 strain carrying the identical nuclear genetic background and the PS allele (18Decoster E. Vassal A. Faye G. J. Mol. Biol. 1993; 232: 79-88Crossref PubMed Scopus (75) Google Scholar). The resulting diploids were sporulated and products of meiosis were dissected onto glucose medium. Meiotic progeny derived from the cross between the mto2 null ρo strain and M12-54 showed 2:2 segregation of the respiratory-competent phenotype. In all cases, the respiratory-deficient phenotype coincided with histidine independence (data not shown). By contrast, all four meiotic progeny from the cross between the mto2 null ρo strain and the wild type strain M12 (PS) were respiratory-competent, even though the His3+ phenotype showed 2:2 segregation patterns. Furthermore, the W1021ΔMTO2 ρo strain was also crossed with the mss1 strain A18 carrying the 15 S rRNA C1409G allele and sporulated. The resulting mss1/mto2 double mutants were unable to grow on glycerol medium. The double mutants were then examined for the presence of mtDNA by the PCR amplification of mitochondrial 15 S rRNA. All meiotic progeny from the cross retained the 15 S rRNA (data not shown), suggesting that the strains still retained mtDNA, despite some of them showing the respiratory-deficient phenotype. These data strongly suggested that the expression of respiratory deficiency in the mto2 null mutant was fully dependent on the presence of the 15 S rRNA C1409G mutation. mto2 Mutation Affects the Expression of Mitochondrial Genome—Previous studies revealed that the mss1 or mto1 mutation had effects on the expression of mitochondrial genes when carrying the C1409G allele (17Colby G. Wu M. Tzagoloff A. J. Biol. Chem. 1998; 273: 27945-27952Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 18Decoster E. Vassal A. Faye G. J. Mol. Biol. 1993; 232: 79-88Crossref PubMed Scopus (75) Google Scholar). We therefore examined whether the mto2 null mutation also affected the expression of mitochondrial genes by Northern blot analysis. RNA blots were hybridized with DIG-labeled probes for exon regions of CYTB, COX1, 15 S rRNA, and 21 S rRNA, respectively. In these mitochondrial genes, the 15 S rRNA gene lacks any intron, whereas the 21 S rRNA gene harbors two exons and one intron (33Foury F. Roganti T. Lecrenier N. Purnelle B. FEBS Lett. 1998; 440: 325-331Crossref PubMed Scopus (323) Google Scholar). Furthermore, CYTB consists of six exons and five introns, whereas COX1 contains 8 exons and 7 introns (33Foury F. Roganti T. Lecrenier N. Purnelle B. FEBS Lett. 1998; 440: 325-331Crossref PubMed Scopus (323) Google Scholar). As an internal control, RNA blots were stripped and rehybridized with the DIG-labeled nuclear-encoded 25 S rRNA probe. As shown in Fig. 3, the 15 S rRNA and 21 S rRNA did not show obvious size changes and precursor accumulation in mto2 strains, as well as in mss1 and mto1 strains. However, there were increasing expressions of 15 S rRNA and 21 S rRNA genes in the mto2 cells in the PR background when compared with the wild type strain W303-1B. Deletion of MTO2 in the wild type mtDNA background had no obvious effect on CYTB processing, while it led to a slight accumulation of COX1 precursors. By contrast, in the presence of the 15 S rRNA PR allele, the mto2 mutant, similar to mss1 and mto1 mutants, had lower levels of mature CYTB mRNA and accumulated unprocessed or partially processed precursors. Furthermore, the COX1 probe detected an extremely low concentration of the mature mRNA and a high accumulation of unprocessed or partially processed precursors in mto2 mutants carrying the 15 S rRNA PR allele. It appeared that there were slightly different precursor accumulations of COX1 between mto2 and mss1 or mto1 mutants. Mitochondrial Protein Synthesis Defect in the mto2 Strains—To examine whether the mto2 null mutation impairs mitochondrial protein synthesis, mutant and contr" @default.
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• W2034873621 title "Mutations in MTO2 Related to tRNA Modification Impair Mitochondrial Gene Expression and Protein Synthesis in the Presence of a Paromomycin Resistance Mutation in Mitochondrial 15 S rRNA" @default.
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