mitochondrial rna splicing defects

Nucleic Acids Research, 1992, Vol. 20, No. 23 6339-6346

The yeast nuclear gene MRF1 encodes a mitochondrialpeptide chain release factor and cures severalmitochondrial RNA splicing defects

Herman J.Pel, Corien Maat, Martijn Rep and Leslie A.Grivell*Section for Molecular Biology, Department of Molecular Cell Biology, University of Amsterdam,Kruislaan 318, 1098 SM Amsterdam, The Netherlands

Received August 12, 1992; Revised and Accepted November 2, 1992

ABSTRACT

We report the molecular cloning, sequencing andgenetic characterization of the first gene encoding anorganellar polypeptide chain release factor, the MRF1gene of the yeast Saccharomyces cerevisiae. The MRF1gene was cloned by genetic complementation of arespiratory deficient mutant disturbed in the expressionof the mitochondrial genes encoding cytochrome coxidase subunit 1 and 2, COX1 and COX2. For COX1this defect has been attributed to an impairedprocessing of several introns. Sequence analysis of theMRF1 gene revealed that it encodes a protein highlysimilar to prokaryotic peptide chain release factors,especially RF-1. Disruption of the gene results in a highinstability of the mitochondrial genome, a hallmark fora strict lesion in mitochondrial protein synthesis. Therespiratory negative phenotype of mrfl mutants lackingall known mitochondrial introns and the reducedsynthesis of mitochondrial translation productsencoded by unsplit genes confirm a primary defect inmitochondrial protein synthesis. Over-expression of theMRF1 gene in a mitochondrial nonsense suppressorstrain reduces suppression in a dosage-dependentmanner, shedding new light on the role of the '530region' of 1 6S-like ribosomal RNA in translationalfidelity.

INTRODUCTION

Peptide chain release factors are required for proper terminationof polypeptide chain synthesis. The prokaryotic release factors1 and 2 (RF-1 and RF-2) participate in polypeptide chaintermination by binding to translating ribosomes that haveencountered a nonsense codon at their A site. This binding resultsin the release of the newly synthesized protein, presumably viaan alteration of the catalytic activity of the peptidyl transferasecentre from peptide-bond formation to peptidyl-tRNA hydrolysis(reviewed in 1). RF-1 and RF-2 act in a codon-specific manner:RF-1 is required for UAA and UAG-dependent termination,while RF-2 mediates UAA and UGA-dependent termination. A

EMBL accession no. X60381

third prokaryotic release factor, RF-3, stimulates the activity ofboth RF-1 and RF-2 but lacks codon-specificity (for review see2). Recently, the identification of a gene encoding a rabbitcytoplasmic release factor has been reported (3). Surprisingly,the deduced amino acid sequence shows no structural similarityto prokaryotic or mitochondrial counterparts, but appears to benear identical to human -y2 interferon-induced protein 'y2 (4, 5)and mammalian tryptophanyl-tRNA synthetases (6, 7). It has beenspeculated that tRNATrp acylating activity and peptide chainrelease are catalyzed by the same protein (4, 5), however, moreresearch is necessary to clarify this relationship (8, 9).Here we report the molecular cloning, sequencing and

characterization of a gene, designated MRFJ, encoding amitochondrial peptide chain release factor (termed mRF-laccording to nomenclature recommended in 10) of the yeastSaccharomyces cerevisiae. mRF-I exhibits a high sequencesimilarity to prokaryotic release factors, in particular toEscherichia coli and Salmonella typhimurium RF-1. The MRFJgene was cloned by genetic complementation of a mutationpresent in yeast mutant E372. This mutant is disturbed in theexpression of the mitochondrial genes encoding cytochrome coxidase subunit 1 and 2, COXJ and COX2. For subunit 1 thisdefect has been attributed to an impaired processing of at leastCOXI introns aIl and aI2 (11). Correct processing of these groupH introns depends on RNA maturases, proteins expressed fromreading frames present in these introns and specifically requiredfor removal of the intervening sequence by which they areencoded (12, 13). Therefore, the defects in RNA splicing inmutant E372 may be due to an impaired synthesis of one or moreRNA maturases. We also consider the possibility that mutationsin the MRF] gene have a more direct effect on yeastmitochondrial splicing.

MATERIALS AND METHODSStrains and mediaThe strains used in this study are listed in Table 1. Mutant E372was derived by ethylmethanesulfonate mutagenesis fromD273-lOB/Al (14) and mutant WI was derived form W303-1A.

* To whom correspondence should be addressed

.=) 1992 Oxford University Press

6340 Nucleic Acids Research, 1992, Vol. 20, No. 23

Mutant E372/L6 is a spore derived from a cross between E372and W303/1A. Spontaneous lys2 mutants were selected on

minimal plates containing a-aminoadipic acid (2% glucose,0.167% yeast nitrogen base without amino acids and ammoniumsulphate (Difco), 30 Ag/ml L-lysine, 0.2% a-aminoadipic acid,2% agar) as described by Chattoo and Sherman (15). The natureof the spontaneous mutation was established by verifying thelysine auxotrophy of diploids obtained from a cross to knownlys2 mutants of opposite mating type. Yeast strains devoid ofmtDNA (rho') were obtained by 16-24 hr growth in YPD10containing 20 Ag ethidium bromide per ml (16). The absence ofmtDNA was verified by examination of cells stained with thefluorescent dye DAPI (17).The following media were used for growth of yeast: YPD (2%

glucose, 2% peptone, 1% yeast extract); YPD1O (as YPD but10% glucose); YPEG (2% glycerol, 2% ethanol, 2% peptone,1% yeast extract); YPGal (2% galactose, 2% peptone, 1% yeastextract); WO (2% glucose, 0.67% yeast nitrogen base withoutamino acids(Difco)); WOG (as WO but 2% glycerol instead ofglucose). Where required media were supplemented withauxotrophic requirements at 20 Itg/mn. Solid media contained 2%agar.

PlasmidsYeast/E. coli shuttle vectors used in this study include YCplac 111

and YEplacl81 (18) and YEp351 (19). Low- and high-copynumber plasmids containing the MRF1 as well as the LYS2 gene(pG174/CK1 resp. pG174/SK17) were constructed by insertionof a 4.9 kb Hindll fragment containing the LYS2 gene derivedfrom plasmid pDP6 (20) into the unique HindmI site of pG174/C 1

resp. pG174/S17 (see Figure 3). Plasmid YEpLK1 consists ofthe same LYS2 fragment cloned in YEplacl81.

Northern blot analysisMitochondria were prepared from wild type and mutant yeastsgrown to stationary phase in 100 ml YPGal or selective WOGalas described by Faye et al. (21). Mitochondrial RNA (mtRNA)was isolated by lysing the mitochondria in 400 1l RNA extractionbuffer (1 mM NaAc, 5 mM NaCl, 0.1 mM MgAc, 0.5% SDS),

immediately followed by at least three extractions with an equalvolume of a 1 : 1 mixture of phenol and chloroform. Nucleicacids were precipitated with ethanol and resuspended in 250 Atlof TES (10 mM Tris.Cl pH 8.0, 1 mM EDTA, 0.1 % SDS).5 tg ofmtRNA was separated on 1% agarose gels and transferredto a nylon Hybond-N membrane (Amersham). Standardprocedures were used for Northern hybridization analysis (22).A 520 bp mtDNA fragment used to detect COX] transcriptsconsists of 515 bp of the COX] leader and 5 bp of translatedsequence (Rsal site, 23). COX2 transcripts were detected witha 761 bp fragment consisting of 52 bp of the COX2 leaderfollowed by 709 bp of the coding region (24). COB transcriptswere hybridized to a fragment containing 414 bp of the COBleader (position -440 to -26 in 25).

Inactivation of the MRF1 gene

Plasmid pG174/S2, a YEp351 derivative containing a 4.7 kbHindIll-Hindl insert harbouring the MRF] gene (see Figure 3),was digested with BclI and XAoI and blunted with Klenowenzyme. Re-ligation yielded a construct (pGl74/A1) that lacks87% of the MRF1 coding region (see Figure 3) and has an uniqueXhfoI restriction site at the ligated junction. This XhoI site was

used to insert marker genes in the disrupted MRFJ gene. A 4.9kb Sail fragment containing the LYS2 gene derived from plasmidpDP6 (20) was used to construct pG174/D3. A 2.2 kb Sallfragment containing the LEU2 gene derived from construct 6(Figure 2 in 26) was used to construct pG174/D4. StrainD273-IOB/A1/K1 was transformed with pG174/D3 cut withNarl, whereas strain W303-1A was transformed with pG174/D4cut with Hindml. Total DNA isolated from several transformantswas digested with BamHI and Bgm and probed with a 3.8 kbBamHI-HindIll containing the MRFJ gene to verify disruptionof the chromosomal copy of the MRFJ gene.

In vivo labeling of mitochondrial translation productsMitochondrial translation products were labeled in whole cellswith L-35S-methionine for 30 min in phosphate buffer containing0.3% glucose and 20 jug/ml cycloheximide as described byMcKee et al. (27). Mitochondrial protein lysates were essentially

Table 1. Genotypes and sources of Saccharomyces cerevisiae strains used

Strain Nuclear genotype Mitochondrial genotype Source

D273-IOB/A1 a; met6 Q + Tzagoloff et al., 1976D273-IOB/A1/K1 a; met6; Iys2 Q + this studyW303/1A a; ade2-1; his3-11,15; leu2-3,112; Q+ R.J.Rothsteina

ura3-1; trpl-J; can1-100KL14-4BQ0 ca; unknown auxotrophy Q Muroff and Tzagoloff, 1990CBIlQ0 a; adel Qp Muroff and Tzagoloff, 1990E372 a; met6; mrfl-l Q + Tzagoloff and Dieckmann, 1990E372Q° a; met6; mrfl-l e° this studyWI a; ade2-1; his3-11,15; leu2-3,112; e+ this study

ura3-1; trpl-J; can1-100; mrfl-2W1Q° a; ade2-1; his3-11,15; leu2-3,112; Q0 this study

ura3-1; trpl-]; canl-100; mrfl-2E372/L6 a; leu2-3,112; mrfl-] Q + this studyDD3-1l1 a; met6; lys2; MRFI::LYS2 Q- this studyWD4-1 a; ade2-1; his3-11,15; leu2-3,112; Q this study

ura3-1; trpl-1; canl-100; MRFI::LEU2MOS-5 a; met3; his4 Q +; oxil-V25; MSUI Fox and Staempfli, 1982MJV1 a; met3; his4; Ivs2 Q +; oxil-V25; MSUI this studyGF167-7B a; lys2 Q +; A introns S6raphin et al., 1987kar (167) a; trpS; karl-i e+; A introns Seraphin et al., 1987

a R.J. Rothstein, Department of Human Genetics, Columbia University, New York, USA

Nucleic Acids Research, 1992, Vol. 20, No. 23 6341

prepared as described by Douglas et al. (28), except that sampleswere not heated prior to further analysis. Mitochondrial proteinscorresponding to 5 mg (10 mg in the case of Wl) wet weightof cells were separated on a 11% SDS polyacrylamide gel. Yeaststrain D273-IOB/Al and derivatives labeled 10-20 times moreefficiently than strain W303 and derivatives. This might be relatedto the methionine auxotrophy of D273-lOB/Al.

MiscellaneousStandard methods were used for manipulation of DNA,transformation of E. coli, Northern blotting and Southern blotting(22), SDS-PAGE (29), transformation of yeast cells (30) andisolation of total DNA from yeast cells (31). DNA sequenceanalysis was performed by the method of Sanger (32).

RESULTSPhenotype of mrfl mutantsPet mutants of the yeast Saccharomyces cerevisiae grow onglucose and other fermentable carbon sources but do not growon non-fermentable substrates like ethanol and glycerol due toa mutation in a nuclear gene (33). In a previous biochemicalscreen, 18 different pet complementation groups specificallyaffected in the processing of COX] pre-mRNAs have beenselected (11). One of these groups, G174, consists of two petmutants named E372 and W1. The absorption spectrum of amitochondrial extract isolated from E372 demonstrates an absenceof cytochromes a and a3 at 605 nm and a slightly reduced levelof cytochrome b at 562 nm (data not shown). This indicates anabsence of assembled cytochrome c oxidase complex. The twomutants differ with respect to the stability of their mitochondrialDNA (mtDNA). Mutant WI accumulates up to 60% cytoplasmicpetites (cells with mutations or deletions in their mtDNA) instationary phase cultures inoculated from a freshly isolated rho+colony. Mutant E372 shows no accumulation of cytoplasmicpetites (< 1%).The mitochondrial gene encoding cytochrome c oxidase subunit

1 (COXJ) in mutant E372 contains five introns, 2 group I introns(aI3 and aI4) and 3 group II introns (aIl, aI2 and aI5; 23).

Previous Northern blot experiments demonstrated a block inexcision of intron all and aI2, correct processing of intron aI5and no clear accumulation of precursors containing intron aI3or a14 (11). Analysis of mitochondrial RNAs transcribed fromthe gene encoding apocytochrome b (COB) revealed wild typemRNA levels, indicating that the two COB introns present inE372 (the group I introns bI4 and b15) are excised normally (datanot shown). Compared to E372, mutant WI contains threeadditional COB introns, the group II intron bIl and the groupI introns bI2 and bI3 (34). Analysis of mitochondrial transcriptsof mutant WI revealed low amounts of mature COX] and COBmRNAs as well as the presence of high molecular weight pre-mRNAs (Figure 1). Although an accumulation of rho- clonesmay have led to an overall reduction in the amount of COX]and COB transcripts, the relative abundance of precursor andmature RNAs indicates that this mutant is disturbed in the excisionof several COX1 as well as COB introns.

Properties of mrfl mutants lacking mitochondrial intronsThe observation that both mutants display an impaired processingof one or more transcripts raises the question to what extent theRNA splicing defects account for the respiratory deficientphenotype of these mutants. This question can be adressed bythe construction of mrfl mutants lacking all known mitochondrialintrons. If mutant E372 and WI are exclusively disturbed inmitochondrial splicing, an intron-less mitochondrial genome isexpected to render them respiratory competent.

Derivatives of mutant E372 and WI lacking mtDNA wereobtained by ethidium bromide treatment. These rhoo mutantswere crossed to two respiratory competent yeast strains carryingmtDNA devoid of all mitochondrial introns, kar(167) andGF167-7B (35). The resulting diploids were forced to sporulateand for each diploid a number of tetrads was dissected andanalyzed for the presence of auxotrophic markers and respiratorycompetence. All tetrads yielded the expected 2: 2 segregationof trpS, leu2 or lys2 marker genes, except the met6 markerpresent in E372 (all spores are met+). The apparent loss of this

D273 E372Uu

cu

r> : Cl-:

->

W303 WI

varl

-- S ~~cox3+ a

atp6-

i.p8

_ - vgl *~I- g coi-_W.w

COX I COX2 C013

Figure 1. Complementation of splicing defects present in mrf mutant Wl bythe MRF1 gene. Mitochondrial RNA isolated from mutant Wl (WI), WItransformed with pG174/Cl (Wi/Cl), and the parental strain W303-1A (WT)was separated on a 1% agarose gel, transferred to a filter and hybridized to probesderived from the COX], COX2 and COB leader as described in Materials andMethods. Arrows point to the mature mRNAs corresponding to the probes used.

Figure 2. Mitochondrial translation products in mrfl mutants. Mitochondrialtranslation products of E372, WI and their respective wild type parents were

labeled with 35S-methionine and separated as described in Materials and Methods.The following proteins are indicated: Varl; cytochrome c oxidase subunits 1,2 and 3 (coxl, cox2 resp. cox3); cytochrome b (cob) and the ATPase subunits6, 8 and 9 (atp6, atp8 resp. atp9).


marker is probably due to phenotypic suppression by kar(167)(36). Also the growth on glycerol media segregated in a 2: 2fashion, indicating that both mutants are mutated in a single orin two or more tightly linked nuclear genes. This segregationpattern demonstrates that both mrfl mutants remain respiratorydeficient in the presence of a mitochondrial genome lackingintrons implying a defect in mitochondrial biogenesis besidessplicing.

Mitochondrial translation products present in mrfl mutantsIn order to get more insight in the mitochondrial defects of themifi mutants, mitochondrial translation products present in mrflmutants E372 and Wl were analyzed by in vivo 35S-methioninelabeling of whole cells in the presence of cycloheximide(Figure 2). Both mutants revealed a similar pattern of labeledproteins, the abnormalities being somewhat more pronounced inWI than E372. Coxl as well as cox2 appear to be present instrongly reduced amounts. Surprisingly, an increased signal wasrepeatedly found at the position of apocytochrome b. Also WI,containing low levels of mature COB mnRNA (Figure 1),displayed this phenotype. Further characterization of this bandis necessary to see whether it corresponds to increased amountsof apocytochrome b or to aberrant protein products migratingat this position.

Cloning and sequence analysis of the MRF1 geneThe wild tpe MRFI gene was cloned by transfornation of mutantE372/L6, a leu2 spore derived from E372, with a yeast genomicDNA library cloned in the plasmid vector YEpl3 (37). Restrictionanalysis of eight plasmids partially complementing the respiratorynegative phenotype revealed that two different but overlappingclones had been isolated. The same plasmids confer completerestoration of respiration of mutant WI, suggesting that E372/L6may contain a second, unknown mutation that influences growthon non-fermentable carbon sources (the 2: 2 segregation ofrespiratory growth observed during tetrad analysis may indicatethat the assumed second mutation maps close to MRFJ on thechromosome or that it is masked by the intronless mitochondrialbackground). The restriction map of the smallest plasmid,pG174/Tl, indicates a nuclear DNA insert of approximately 9.0kb. The gene responsible for complementation was isolated on

pG174JC1pG174IS17

pG174/S8

smaller DNA fragments by subcloning different regions ofpGl74/Tl in multi-copy or centromeric shutde vectors and testingthe resulting constructs for complementation of E372/L6 and WI(Figure 3). The smallest subclone capable of fullcomplementation consists of a low-copy number vector containinga 2.9 kb nuclear DNA insert (pG174/C4). The fact that one tothree copies of the cloned DNA fragment are sufficient forcomplementation provides strong evidence that this fragmentcontains the wild type allele of the gene mutated in members ofcomplementation group G174. Further evidence for bona fidecomplementation was obtained by analysis of mitochondrialtranscripts of mutant WI complemented with either low- or high-copy number constructs containing MRFJ. In both cases fullcomplementation of splicing defects is observed (Figure 1 anddata not shown).The nucleotide sequence of approximately 2.5 kb of DNA

starting at the unique Hindm site was determined on both strands(Figure 3, the sequence has been deposited at the EMBL databankwith accession no. X60381). The sequenced region contains twonon-overlapping open reading frames. One reading frame(designated MRFJ, see below) starts with an AUG codon atposition 1150 and ends with a UGA stop codon at position 2389.The fact that plasmid pG174/C4 complements WI and E372/L6while plasmid pG174/S7 does not indicates that this reading franecorresponds to the gene mutated in these strains (Figure 3). Thesecond reading frame (ORF2) probably starts upstream of thesequenced region, since no in frame stop codon marking the 5'border was found. This reading frame consists of 278 codonsand ends with an ochre codon at position 837. The amino acidsequence deduced from ORF2 shows no significant similarity toprotein sequences present in the databases (SwissProt release21.0).

Similarity of mRF-1 to prokaryotic peptide chain releasefactorsThe predicted MRFJ gene product consists of 413 amino acidsand has a calculated molecular weight of 46 741 Da. The MRFJreading frame has a codon bias index of 0.14 (38), indicatingthat it is expressed at a low level. The predicted amino acidsequence contains 57 (13.8%) acidic and 72 (17.4%) basicresidues, implying that the MRFJ product is a hydrophilic and

(+)

(-)pG174/S7

pG174OC4E H G U XG E G C

LA I I I I I I I Il

3.0

ORF2 a-4.0

MF l .

5.0

(+)

S B H E

I J6 6 kb46.0 6.8 7.8 Icb

Figure 3. Restriction map and sequenced area of yeast nuclear DNA present in plasmid pG174/T1. Only a part of the nuclear DNA present in pG174/Tl is shown.Restriction fragments derived from this DNA were subcloned in YEp351 (pG174/S-clones) or YCplacl (pG174/C-clones). B: BamHI; C: Bcll; E: EcoRI; G:BgIlI; H: HindH; S: Sall; U: Sadl; X: XoI. (+) signifies full complementation of G174 WI and partial complementation of E372/L6 by the indicated plasmid,(-) signifies lack of complementation. Solid bars represent the reading frames identified as MRFI and ORF2. The accompanying arrows depict the direction oftranscription of the putative genes. The direction and length of the arrows below the bars indicate the region sequenced for each DNA strand.

1Vv0.0 2.0


basic protein. A computer-assisted comparison of the putativeamino acid sequence to a protein database (SwissProt release 21.0searched with the FastA program, 39) revealed a high similarityto prokaryotic peptide chain release factors. The MRFJ geneproduct is 38.4% and 29.8% identical to the E.coli peptide chainrelease factor 1 (RF-1) and 2 (RF-2) respectively (40, 41, 42).Similar values were obtained for Salmonella typhimurium RF-1and RF-2, since these proteins are over 95% identical to theirE. coli counterparts (43, 44). The alignment of mRF-1 with E. colirelease factors shows that the sequence similarity is mostpronounced in the central and carboxy-terminal regions(Figure 4). The highest percentage of identity is found in a stretchof 43 amino acids between position 280 and 322 of mRF-1 (79%and 70% with RF-1 and RF-2 resp.). The mRF-l protein issomewhat larger than the bacterial release factors due to a fewinsertions and the presence of extra amino acids at the amino-terminus. The amino-terminal 47 residues include 7 basic and13 hydroxylated residues and lack acidic amino acids. Thiscomposition suggests the presence of a cleavable mitochondrialtargeting sequence (45).A screening of the EMBL database (release 30.0) with the

TFastA program (39) disclosed additional high sequencesimilarities of mRF-1 to the putative products of two as yetunassigned reading frames in Bacillus subtilis and E. coli. The

XXO 31-2

SCe aUi-1 M W L S K F 0 F P S R S I F

Bacillus reading frame is a good candidate for the prfB geneencoding RF-2 in this organism. Both gene products have beenincluded in a more detailed structural comparison of the variousmembers of the prokaryotic/mitochondrial release factor family(46). No significant similarities with reported eukaryoticcytoplasmic release factor sequences could be found.

Properties of yeast strains lacking mRF-1The characteristics of the amino acid sequence deduced from theMRFJ gene suggest that this gene encodes a mitochondrial peptidechain release factor. To confirm this assignment, the one-stepgene replacement technique (47) was used to inactivate thechromosomal copy of the gene in respiratory competent haploidyeast strains. Disrupted alleles were constructed by removal ofa 1079 bp internal XhoI-BclI fragment (Figure 3), thus deleting87% of the MRFI coding region, and insertion of marker genesinto the resulting gap (see Materials and Methods). Linear DNAfragments carrying these mrfl alleles were used to transformstrain D273-lOB/Al/K1 and W303/1A respectively.Transformants that contain the disrupted instead of the wild typeMRFJ allele were obtained for both strains (DD3-11 and WD4-1resp.) as verified by Southern blot analysis (data not shown).

Strain DD3-11 as well as WD4-1 displayed a respiratorydeficient phenotype. Moreover, neither strain was complemented

M F E I N P V 7

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sco 3-2 P T G Q RTQK K M K 0 M K[TLLI 2953CO -1 P TGI V1 E 0 H K N KAKAI L S V ILl IGA RLL 278sCx ="-1IP SII VS MI 0D ER SQ H K N KA K A I F T I R ! .AR 330

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CO 3-2 [TDQ F IEA IS L DA G L3CO3-1 ILDI M L I E I II L E H Q A D O L A A [L S E Q ESCe .- UL.D E V I E A M SLY D S T E R A K EUJL L S N

365360413

Figure 4. Comparison of the amino acid sequence of mRF-1 to the E.coli release factors 1 (RF-1) and 2 (RF-2). Positions of identity between the various releasefactors are boxed. The sequences were aligned by the FastA program of Pearson and Lipman (39). Dashes indicate gaps introduced to optimize the alignment.


Figure 5. Reduction of nonsense suppression by the MRFJ gene. (A) Outlineof the suppression assay. Yeast strain MJV1 has a nonsense (UAA) mutationin the mitochondrial COX2 gene, which normally results in a glycerol-negativephenotype. However, the strain also bears a mutation in the mitochondrial 15SrRNA (black dot) which partly suppresses the ochre mutation, thus enabling thestrain to grow slowly on glycerol. The mutant 15S rRNA allows competitionbetween a natural aminoacyl-tRNA (elongation) and a yeast mitochondrial releasefactor (termination). (B) The MRFJ gene reduces glycerol-growth of MJV1. MJV1was transformed with a 2Am-based plasmnid (High, 25 to 50 plasmids per cell)or a centromeric plasmid (Low, 1-3 plasmids per cell): YEpLK1, without MRFJgene (High -); pGl74/D3 containing disrupted MRFJ allele (High AMRFI);pG174/SK17, intact MRFJ gene (High MRFI) and pG174/CKI, intact MRFJgene (Low MRFI). Transformants were suspended in water and spotted on

selective WO (glucose) and WOG (glycerol) plates. Photographs show 2 daysof growth on WO and 7 days of growth on WOG at 28°C.

by rho' tester strains indicating that they had sustained mutations(deletions) in the mitochondrial genome. The loss of wild typemtDNA is characteristic for yeast mutants lacking mitochondrialprotein synthesis (48). Further genetic analysis revealed thatDD3-11 is complemented by wild type strain W303-1A, but notby the MATa mrf] mutants WI and E372/L6. WD4-1 iscomplemented by D273-lOB/A1, but not by MATcx mrf] mutantE372 (data not shown). The results of these crosses confirm theallelism between the disrupted reading frame and the genemutated in the various mif] strains.

Reduction of nonsense suppression by mRF-1In order to study the function of mRF-1 in mitochondrial proteinsynthesis in more detail, a homologous nonsense suppressionassay was used. Nonsense suppression represents competitionbetween a suppressor aminoacyl-tRNA and a peptide chainrelease factor at a stop codon (49). This competition enables thedetection of release factor activity, since an increase in releasefactor concentration will reduce the efficiency of nonsense

suppression. An elevated release factor concentration can beachieved by cloning the relevant gene in a high-copy number

plasmid, thus increasing the gene dosage. This kind of assay hasproven successful for the cloning of the E. coli prfA gene encodingRF-1 (50).

Yeast strain MOS-5 contains a point mutation (termed MSU])in its mitochondrial small (15S) ribosomal RNA (51). Thismutation suppresses an ochre mutation in the mitochondrial COX2gene by allowing a natural aminoacyl-tRNA to compete with amitochondrial release factor (Figure SA). The suppression resultsin readthrough at the ochre codon and slow growth on non-fermentable carbon sources. An increased concentration ofmitochondrial release factor may shift the balance of competitiontowards translational termination and hence respiratory deficiencydue to lack of cox2 protein. In order to be able to transformMOS-5 with plasmids containing the MRF] gene, a spontaneouslys2 mutant, MJV 1, was isolated. The complementing wild typeLYS2 gene was cloned in both low- and high-copy numberplasmids containing the MRF] gene (see Materials and Methods).Transformation of MJV1 with an intact MRF] gene on a low-copy number plasmid strongly reduced growth on non-fermentable media, while introduction of the gene on a high-copynumber plasmid completely abolished this growth (Figure SB).These results provide strong evidence that mRF-1 is expressedand functions as a peptide chain release factor in yeastmitochondria. Moreover, the clear effect of a slight increase inmitochondrial release factor concentration demonstrates thedelicacy of the balance between translational elongation andtermination during MSU] mediated nonsense suppression.

DISCUSSIONThe MRF1 gene encodes a mitochondrial release factorThe gene mutated in members of pet complementation groupG174 has been cloned by transformation of mutant E372/L6 witha yeast genomic DNA library. Several lines of evidence indicatethat the MRF] gene corresponds to the gene mutated incomplementation group G174. First, one to three copies of thisgene complement the glycerol negative phenotype and the highinstability of the mitochondrial genome in mutant WI, eliminatingthe possibility that a multicopy suppressor has been cloned.Second, complementation of mutant WI with the MRF] generestored the correct processing of both COX] and COBtranscripts. Third, inactivation of the MRF] gene in two wildtype strains resulted in mitochondrial defects that could not becomplemented by mi-fl mutants in either of the disruptants, thusconfirming the allelism between the MRF] gene and the genemutated in these mutants.The high similarity in primary structure between mRF-1 and

prokaryotic release factors RF-I and RF-2 suggests that mRF- 1acts as a release factor in mitochondrial protein synthesis. A rolein mitochondrial translation was confirmed by the phenotype ofyeast strains lacking an intact copy of the MRF] gene. Theobserved instability of the mitochondrial genome is characteristicfor yeast mutants lacking mitochondrial protein synthesis (48).The fact that MRF] disruptants grow normally on a fermentablecarbon source rules out the possibility that the gene is essentialfor translation on cytoplasmic ribosomes. Additional evidencethat the MRF] gene encodes a mitochondrial release factor comesfrom the observation that the gene is able to reduce nonsensesuppression in yeast mitochondria in an efficient, dosage-dependent manner. To our knowledge, this is the first report onthe cloning of an organellar release factor gene.


The strong effect of mRF-l on nonsense suppression in strainMJVl also sheds new light on the mechanism by which thesuppressor mutation (MSUI) exerts its effect. The MSUI mutationhas been characterized as a G to A substitution of nucleotide 633located at the base of the so-called '530 loop' in the mitochondrial15S ribosomal RNA (51). This loop is highly similar in virtuallyall 16S-like ribosomal RNAs and plays an essential but poorlyunderstood role in translational accuracy. Several lines ofevidence point to an interaction between the 530 region and thedecoding site (see 52 for review). O'Connor et al. (53) recentlyanalyzed the effect of deletion as well as substitution of E. coli16S rRNA nucleotide G517, the nucleotide that corresponds tothe MSUI mutation in yeast mitochondrial 15S rRNA.Interestingly, mutations of G517 resulted in significantsuppression of all three termination codons as well as a stimulationof + 1 and -1 frameshifting, establishing an important role forthis nucleotide in translational fidelity. The MSUJ mutation (Gto A substitution) turns out be lethal in E. coli.

Yeast mitochondrial ribosomes carrying the MSUI mutationsuppressed several ochre mutations, but failed to suppressmissense, frameshift, as well as an amber mutation (54, 51). Eventhough the number of tested mutations is still limited, these datasuggest that MSUI specifically suppresses translationaltermination at ochre stop codons, indicating a spectrum of defectsthat is much narrower than that observed in E. coli. In line withthe results obtained for E. coli G517 mutants it is conceivablethat MSUI permits misreading of ochre codons by one or morenatural tRNAs. In the yeast cytoplasm UAA and UAG stopcodons can be misread by CAA- respectively CAG-decodingtRNAGin by wobble at the first position of the codon (55, 56).Yeast mitochondria, however, use a restricted number of tRNAs(57) and contain only a CAA-decoding tRNAGin (58). This factmay explain why MSUI failed to suppress an amber mutation.In the present study we have shown that the effect of MSUI onstop codon recognition can be compensated by increased releasefactor concentrations (Figure 5). This implies that the suppressortRNA competes successfully with the release factor when thelatter is present in normal concentrations and that increasedmRF-l levels shift the balance back to termination.

Since current data indicate that the suppressor activity ofMSUIis restricted to (ochre) stop codons, it is also feasible that theMSUI mutation alters the release factor binding domain on theribosome, thus curtailing the recognition of these codons by therelease factor. In this respect it might be of interest that boththe release factor binding site and the '530 loop' have beenmapped to the neck region of the 30S subunit (reviewed in 59,60). In this scenario increased amounts of mRF-I apparentlycompensate for the reduced affinity of the ribosome for the releasefactor. A detailed analysis of the effect of mRF-I on thetranslational ambiguities of an MSUI mutant lacking asuppressible nonsense mutation is necessary to elucidate themechanism of MSUI suppression.

mRF-1 and the mitochondrial genetic codeIn prokaryotes two release factors are involved in the recognitionof three different stop codons. RF-1 recognizes UAA and UAGwhereas RF-2 recognizes UAA and UGA codons. In contrast,yeast mitochondria use only two different stop codons since UGAcodes for tryptophan (60). The only naturally occurringtermination codon is UAA, although amber mutations and

amber codons have been described (61, 62). This codonassignment suggests that one release factor of the RF-1 typeshould be sufficient for translational termination in yeastmitochondria. In line with this, we note that mRF-1 is moresimilar to RF-1 than to RF-2. A similar situation has beenproposed for rat mitochondria (63). This organelle uses the sameset of termination codons as yeast mitochondria. Only a singlerelease factor recognizing the codons UAA and UAG, but notUGA, could be isolated and partially purified from ratmitochondria. This indicates that the deviation of the universalgenetic code is accommodated by the presence of a single releasefactor recognizing UAA and UAG and by the absence of a factorrecognizing UGA. The well established in vitro termination assay

(64) has to be adopted in order to study the codon-specificityof mRF-1 in more detail.

Mitochondrial defects in mrfl mutants

mrfl mutant E372 revealed reduced levels of both coxl and cox2.For cox2 this defect can most easily be explained by mis-termination during its biosynthesis. For coxl this defect has beenattributed to an insufficient amount of mature mRNA due tosevere defects in the processing of at least the introns aIl anda12. At present, an additional effect on termination during matureCOX] mRNA translation cannot be excluded. Severalmitochondrial introns, including intron aIl and aI2, encode an

RNA maturase that is required for the excision of the cognateintron. The expression of the aI2 maturase requires proper

excision of the upstream intron aIl (65). Therefore, the impairedexcision of aI2 can be due to the blocked excision of aIl. In thisstudy we demonstrate that a second mrl mutant, W1, alsocontains reduced amounts of coxl and cox2. Moreover, WIrevealed reduced levels of mature COX] as well as COB mRNAs,suggesting that this mutant also contains defects in the processingof several mitochondrial introns. Taken together, these datademonstrate that both mutants are disturbed in the expressionof split as well as unsplit genes. The existence of mitochondrialdefects besides splicing was further evidenced by the respiratorynegative phenotype of spores derived from both mrfl mutantslacking all known mitochondrial introns.The observation that some mitochondrial proteins are

synthesized normally, whereas others are clearly reduced impliesa gene-specific action of the mutant release factor and is incontrast with the overall reduction of mitochondrial proteinsynthesis reported for mutants defective in other general trans-acting mitochondrial translation factors such as IF-2 or EF-G (66).The observed specificity may be interpreted as evidence thatspecific release factors are required for proper termination duringtranslation of one or more individual genes, similar to the manygene-specific factors known to be required for translationalinitiation (for review see 67). However, a more plausibleexplanation is that the mutant release factor leads to differenteffects at the various mitochondrial stop codons. Since ochrecodons are the only naturally occurring termination codons inyeast mitochondria the presumed difference in affinity can onlybe explained by the RNA context of individual stop codons.Context-effects have been described for the ribosomal selectionrates of suppressor tRNA and release factor (68, 69).

For the mrfl mutants this implicates that the observed splicingdefects are not necessarily due to a lack of intact RNA maturases.At least two alternatives cannot yet be ruled out. First, it is

frameshift mutations leading to premature chain termination by possible that the MRF] gene encodes a bi-functional protein,


similar to the mitochondrial tyrosyl-tRNA synthetase ofNeurospora crassa cyt-18 (70), the leucyl-tRNA synthetase NAM2of Saccharomyces cerevisiae (71), or the product of the yeastMRS2 gene (72). Second, stalling of ribosomes at poorlytranslatable stop codons and the consequent accumulation oftranslating ribosomes on unspliced pre-mRNAs might prevent.introns to adopt a splicing-competent folding. A more detailedanalysis of the mitochondrial splicing defects present in differentmrf] mutants is necessary to ascertain the role of themitochondrial release factor in pre-mRNA splicing.

ACKNOWLEDGEMENTS

H.J.Pel acknowledges the European Molecular BiologyOrganisation (EMBO) for a Short Term Fellowship (ASTF5809). We thank Dr. Tzagoloff for help with the screening ofhis collection ofpet mutants and for making available a numberof yeast strains. We thank Dr. T.D.Fox for strain MOS-5, ErikJan Dubbink for help with the gene disruption experiments andDr. M.O'Connor for making results available to us prior topublication. This work was in part supported by a grant toL.A.Grivell from the Netherlands Foundation for ChemicalResearch (SON), with financial aid from the NetherlandsOrganization for the Advancement of Research (NWO).

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