Proc. Nati. Acad. Sci. USAVol. 84, pp. 2185-2188, April 1987Biochemistry

In vivo aminoacylation of human and Xenopus suppressor tRNAsconstructed by site-specific mutagenesis

(simian virus 40/CV-1 cells/acid/urea/polyacrylamide gel electrophoresis)

YE-SHIH Ho* AND YUET WAI KANHoward Hughes Medical Institute Laboratory and Division of Genetics and Molecular Hematology of the Departmrnt of Medicine, University of California,San Francisco, CA 94143

Contributed by Yuet Wai Kan, December 16, 1986

ABSTRACT Amber suppressor tRNA genes were con-structed by site-specific mutagenesis of the anticodons ofhuman lysine-inserting tRNA (tRNALYS) and glutamine-insert-ing tRNA (tRNAGIn) genes, and a Xenopus laevis tyrosine-inserting tRNA (tRNA Yr) gene. As previous in vitro studies inprokaryotes have shown that substitution of nucleotides in theanticodon region can profoundly affect tRNA aminoacylation,it is important to determine whether the mutation affectsaminoacylation of these eukaryotic tRNAs. We present amethod for quantitating the tRNA aminoacylation in vivo inmammalian cells, and we have determined that the suppressortRNATyr is fully aminoacylated and suppressor tRNALys andtRNAGII are aminoacylated 40-50% and 80%, respectively.This in vivo method of estimating aminoacylation may beapplied to other mutations in the tRNA genes.

Nonsense suppressor tRNAs in bacterial and yeast cells havebeen used extensively both in genetic analysis of chain-terminating mutations and in understanding the effects ofaltered bases on the biological functions oftRNAs (1-3). Theisolation and characterization of nonsense suppressor tRNAgenes in higher eukaryotes have also been initiated. Previ-ously, a naturally occurring suppressor tRNA activity wasobserved in rabbit reticulocytes and chicken and bovinelivers (4-6). More recently, genes encoding these naturallyexisting opal suppressor tRNAs have been cloned from bothchicken and human (7, 8). Through the development oftechniques for site-specific mutagenesis (9, 10), we were ableto mutate the anticodons of two human tRNA genes to readthe amber codon (11). These two amber suppressor tRNAgenes may offer a possible means of correcting the prematuretermination of,-globin chain assembly in the type of &thalassemia due to nonsense mutations (12, 13). Laski andcoworkers have similarly generated amber and ochre sup-pressors from a Xenopus laevis tyrosine tRNA gene (14, 15).Additionally, the anticodon ofa human serine tRNA gene hasbeen mutated to read all three stop codons (16). Most oftheseartifically constructed tRNA mutants have been shown to befunctional in suppressing nonsense codons when introducedinto mammalian cells (11, 14-20). With the establishment ofpermanent cell lines carrying functional suppressor tRNAgenes, the isolation and study of various nonsense mutantsshould now also be feasible in eukaryotic cells (16, 17-20).Previous studies in prokaryotes and in yeast have shown

that mutations of the anticodon region may affect tRNAaminoacylation in vitro (21-29). In most cases, these mea-surements indicated that aminoacylation was decreased. Inthis study we describe an approach for measuring the level ofaminoacylation of three amber suppressor tRNAs in mam-malian cells. These studies were carried out in vivo and theresults may have direct relevance for understanding the

biologic function of these tRNAs. Our results indicate that inmammalian cells, as in bacteria and yeast, anticodon se-quences play a crucial role in the aminoacylation reactionbetween tRNA and its cognate amino acid-tRNA ligase, andthat mutating some of these sequences may affect theaminoacylation of the tRNAs.

MATERIALS AND METHODSThe suppressor tRNAs were constructed by mutagenesis ofthe human tRNALYs and tRNAGln and Xenopus tRNATYrgenes to produce anticodons complementary to the UAGcodon. The tRNALYS gene encodes a tRNA that is comple-mentary to the codon AAA. We altered the two base pairscorresponding to the 5' and 3' anticodon nucleotides so thatthe anticodon was complementary to the UAG instead ofAAA (11). For the other two tRNAs, only one anticodonnucleotide was altered: the 5' nucleotide of the anticodon ofthe tRNATYr gene was mutated so that the anticodon wascomplementary to UAG instead of UAC (14); the 3' nucle-otide of the anticodon of the human tRNAGin gene wasmutated so that the anticodon was complementary. to UAGrather than CAG (20). These mutated genes are designated astRNALys(Su') tRNAT(Su+), and tRNAGln(Su+), respectively.The three suppressor tRNAs and their corresponding

normal tRNA genes were inserted into the late region ofsimian virus 40 (SV40). To yield high titer virus stocks, theserecombinant viral DNAs were used to transfect CV-1 Africangreen monkey kidney cells in the presence of a helper DNA,am404, that contained an amber mutation in the large tumor(T) antigen (30). Subconfluent (80% confluent) CV-1 cells in100-mm dishes were either mock infected or infected withSV40 or different SV40 recombinant viruses carrying tRNAgenes. The cells were harvested into 2 ml of cold Dulbecco'sphosphate-buffered saline (PBS) containing 0.9 mM CaCl2and 0.49 mM MgCl2 at 50 hr after infection after incubationwith the corresponding 3H-labeled amino acid (=125 ,ACi/ml;1 Ci = 37 GBq) for 6 hr. The cells were then pelleted at 500x g for 5 min and resuspended in 1 ml of lysis buffer (50 mMsodium acetate pH 5.0/0.15 M NaCl/1.5 mM MgCl2/0.65%Nonidet P-40) and vigorously mixed. Nuclei were removedby centrifugation at 2000 x g for 5 min and the cytoplasmicsupernatant was mixed with an equal volume of urea buffer(7 M urea/50 mM sodium acetate, pH 5.0/5 mM EDTA/0.35M NaCl/1% NaDodSO4). This mixture was extracted twicewith phenol/chloroform/isoamyl alcohol (50:49:1, vol/vol,saturated with 50 mM sodium acetae, pH 5.0) and RNA wasprecipitated with 2.5 vol of ethanol. The RNA was redis-solved in a small amount of 10 mM sodium acetate, pH 5.0,and then divided into aliquots for storage at -70°C. Each

Abbreviations: SV40, simian virus 40; DPT, diazophenyl thioether;m.u., map unit.*Present address: Duke University School of Medicine, Departmentof Medicine, Box 3892, Durham, NC 27710

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aliquot was thawed only once for analysi:freezing and thawing leads to deacylationtRNAs.The RNAs were then separated on a 7.

ide/8 M urea gel buffered with 0.1 M sodiunFor RNA blot analysis, the gels were s(sodium acetate, pH 4.0, for 30 min, thediazophenyl thioether (DPT)-paper, accoscribed methods (31). RNA blot filters werefor 6 hr in 50% (vol/vol) formamide/50 Inphate, pH 7.0/3x SSC (lx SSC = 150 iisodium citrate)/5x Denhardt's solution (3S04/1% glycine containing denatured salmn200 Ag/ml. Hybridization was performed iture, except glycine was omitted and 10% dcthe respective nick-translated probes wehybridization overnight at 41'C, the filters v2x SSC/0.1% NaDodSO4 at 450C for 4 hr, t]temperature and exposed at -70°C. For autRNAs aminoacylated with 3H-labeled amiiwere fixed in 45% methanol/10%o acetic acimin, followed by washing with water for 151in Autofluor (National Diagnostics, Somemin before drying and exposure.

RESULTS

Fig. 1 shows a diagram of the tRNA genes asites in the SV40 into which they were iIkilobase (kb) DNA fragments containing theor tRNALYS(Su ) genes were inserted into tiof SV40 between the Hha I site at m.u. 0.73at m.u. 0.97 in either the same or the opposiorientation relative to late SV40 transcrijSV40 recombinants were designated as SV-WSV-Lys(Su+)-1, and SV-Lys(Su+)-2; thethat the transcription of the tRNA gene

tRNAc

= 1

HhaI 0.73

s, since repeated direction as (-1) or opposite to the direction of (-2) SV40of aminoacylated late transcription. The 0.22-kb fragment containing the

tRNAGl! or tRNAGln(Su ) gene was inserted into SV40 be-5% polyacrylam- tween the two Hha I sites at m.u. 0.73 and 0.82 andn acetate, pH 5.0. designated SV-Gln-1 and SV-Gln(Su+)-1. The 0.26-kb frag-oaked in 50 mM ments containing the tRNATYr or tRNATYr(Su ) genes wereon transferred to inserted between the Hha I (m.u. 0.73) and Hae II (m.u. 0.82)rding to the de- sites of SV40, which also contain a deletion between theincubated at41C HindIII (m.u. 0.86) and EcoRI (m.u. 1.0) sites. TheseiM sodium phos- recombinants, obtained from Laski et al. (14), were desig-iM NaCl/15 mM nated SV-Tyr-2 and SV-Tyr(Su')-2, which correspond to the12)/0.2% NaDod- designations SV-tT-2 and SV-tT-2(Su+), respectively, ofon sperm DNA at these workers.in the above mix- CV-1 cells were infected with SV40 or SV40 recombinantextran sulfate and viruses carrying these tRNA genes. The cytoplasmic RNAs,re added. After were extracted from the infected cells under acidic conditionsvere washed with (pH 5.0) to avoid hydrolysis ofthe aminoacyl-tRNA bond andhen dried at room were separated on polyacrylamide gels at acid pH. The RNAsitoradiography of were then transferred to DPT-paper and hybridized with theno acids, the gels respective nick-translated tRNA gene probes. tRNA wasid (vol/vol) for 15 produced in large amounts in the recombinant SV40 infectedmin, then soaking cells and could easily be visualized over the backgroundrville, NJ) for 15 tRNA level produced by the endogenous tRNA genes (Fig.

2). In the cells infected with the SV40 recombinant virusescontaining the wild-type tRNALYS, tRNATyr, or tRNA0lngene, a slower-migrating mature tRNA species was observed(Fig. 2). In those cells with the SV40 recombinant containing

tnd the restriction the tRNALYS(Su ) gene, an additional faster-migrating band,nserted. The 0.8- slightly more abundant than the slower-migrating species,t human tRNALYS was observed (Fig. 2a). In the cells containing tRNATYsu+)he late region (L) gene, only the slower-migrating band was observed (Fig. 2b).and the Acc I site Finally, the cells infected with the virus carrying theite transcriptional tRNAGIn(Su') gene produce a small amount (-20%) of aption (20). These faster-migrating species (Fig. 2c).Lys-1, SV-Lys-2, The experiments shown in Fig. 2 d, e, andfshow that thesuffixes indicate slower-migrating bands represent aminoacylated ("charged")is in the same tRNAs. When the cells were incubated with the three

respective 3H-labeled amino acids prior to tRNA extraction,genes only the slower-migrating bands were labeled. When tRNAs

prelabeled in vivo with 3p were deacylated by mild alkalineLys or Lys(Su ) hydrolysis, they migrated with the faster bands. The differ-3Gn or GIn(Su+) ences in mobility between the aminoacylated and unamino-ryr or Tyr(Su+) acylated tRNAs were most pronounced for the tRNALYS

because of the positive charge present on the lysine residue.By comparing the intensities of the two bands, we coulddirectly assess the degree of aminoacylation of the tRNAs invivo. All three normal tRNAs were fully aminoacylated in the

Hae II 0.82 CV-1 cells. Site-specific mutagenesis of the tRNATyr toproduce the amber tRNAT'rwSU) did not affect its aminoacyla-Hha? 0.82 tion. In contrast, the aminoacylation of the tRNALYs(Su ) was\HindIII 0.86 reduced by 50-60%, and that of the tRNA ln(su ) by approx-HifldII0.86 imiately 20%6.

DISCUSSION

'AccI0.97

EcoRI 1.0

BamHI 0.14

FIG. 1. Diagram of restriction sites in the SV40 into which thenormal or suppressor tRNALYS, tRNAG1n, and tRNATYr genes wereinserted. (The tRNA genes are drawn at a lower magnification thanthe SV40 map.) The numbers following the restriction enzymesdenote the SV40 map unit (m.u.). For details, see Results.

In previous studies using in vitro assays, it was found thatanticodon mutations often affect the aminoacylation of pro-karyotic tRNAs. Substituting the 3' nucleotide of theanticodon in missense suppressor tRNAs derived from gly-cine tRNA genes of Escherichia coli greatly reduces the rateof aminoacylation in vitro, while a substitution in the middlenucleotide has less effect, and a substitution in the 5' orwobble position has little or no effect (21-25). However, theimportance of each anticodon base on the aminoacylation ofE. coli formylmethionine tRNA appears to differ from thoseobserved on E. coli mutant glycine tRNAs. Schulman andcolleagues have measured the rate of aminoacylation of a setof E. coli tRNAfIct derivatives with anticodon nucleotidesreplaced in vitro. Their results showed that base substitutions

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Proc. NatL. Acad. Sci. USA 84 (1987) 2187

- CMl -1

l- -==E

axcucn nu u,

O XJ ~J ~Ju 't0 > > > > > >

a v&4.

6,

-mz-, EC. U:,

0>>>>C> > >C,

2 U) cn un (nb

-d LI e LI f

FIG. 2. Extent of in vivo aminoacylation of nonsor tRNAs. Cytoplasmic RNAs were extractedinfected with SV40 or the various recombinant viruand electrophoresed on acid/urea/polyacrylamide Ib, and c were transferred onto DPT-paper; thosehybridized with the 32P-labeled 0.8- and 0.22-kb insettRNALYs and tRNAOn genes, respectively. RNA in Iwith a 32P-labeled 0.53-kb Kpn I-EcoRI fragmen'0.26-kb Xenopus tRNA insert and flanking SV40markers were unlabeled and deacylated tRNAs werealkaline conditions from CV-1 cells infected with therecombinant viruses. The open and solid arrows ittions of charged and uncharged mature tRNAs, rehigher molecular weight species were probably dueprocessed tRNAs. They were more abundant in tiwith SV40 recombinants carrying the tRNATyr Sbecause these genes contain intervening sequentRNALYS and tRNAG0n genes do not. In d, e, and f,extracted under acidic conditions from cells labeledtive 3H-labeled amino acid. After electrophoresis, theand fluorographed. The markers were 32P-labeled de;harvested from Xenopus oocytes injected with eaciSV40 recombinant plasmid DNA and [a-32P]GTPmarker was too faint for photography and is markedthe extents of tRNA aminoacylation could be dinbecause equal amounts of RNA were applied. In geintensity of the SV-Gln(Su+)-l lane was due to theone-fourth the amount of tRNA.

in the 5' or wobble position drastically reduce airates by at least five orders of magnitude, substmiddle base have the least effect, and chanposition have an intermediate effect (33). Thestthat the contribution of each particular anticodito tRNA aminoacylation reaction varies from tiand that the sequence-specific contact betweenof the tRNA and the cognate amino acid-tRimportant in the aminoacylation reaction of tRIcating this issue is that mutant glycine tRNAs ca substitution in the 3' base of the anticodon ofmodification in the adjacent A residue, whizcontribute to the deficiency oftRNA aminoacylE

Thus, certain effects of mutagenesis on tRNA aminoacyla-+affi tion ofE. coli mutant glycine tRNAs may be due to abnormalE anticodon loop base modification. Similar aminoacylationCC studies have also been performed on yeast tRNAPhC and

0 3 y tRNATr with anticodon nucleotides altered in vitro (26-28).C)Xl)cl)us coThe conclusion of these studies was that substituting 5'c anticodon nucleotides has a more severe effect on the level

of aminoacylation than changing the other two anticodonnucleotides.

In this study we describe a method for measuring in vivothe aminoacylation of eukaryotic tRNA in mammalian cells.The validity ofthe method was substantiated by the completecharging of the endogenous tRNAs and of the large amountof wild-type tRNAs produced in CV-1 cells infected with therecombinant SV40 viruses. We applied this technique todetermine the effect of site-specific mutagenesis of theanticodon on tRNA aminoacylation. While the mutated

_HIO $R tRNATY1 remained completely charged, the decreased levelsof aminoacylation of suppressor tRNALYS and tRNA0G1implied that the anticodon mutation of eukaryotic tRNAscould also affect their aminoacylation in mammalian cells.The decrease could be the direct result of abnormal contactbetween the altered anticodon nucleotides and the cognateamino acid-tRNA ligase or could be mediated through alteredposttranscriptional base modification. Detailed base modifi-cation and RNA fingerprint analyses of both normal and

I ~ suppressor human tRNAs remain to be performed to distin-guish between these alternatives. The estimation of theextent of aminoacylation is derived by comparing the relativeamounts of charged and uncharged tRNAs that could be

mal and suppres- separated on the polyacrylamide gel.from CV-1 cells It is interesting to note that the in vivo measurement weises as indicated, obtained differs from the in vitro results previously reportedgels. RNAs in a, in that the in vivo effects of anticodon mutation are much lessin a and c were severe than the in vitro ones. For example, the Xenopusrts containing the tRNA was aminoacylated fully in spite ofthe alteration oftheb wcoanhybndithe 5' anticodon position. In contrast, a much larger inhibitionsequences. The occurred in vitro in a similarly mutated tRNAfmret (33).extracted under Likewise, the aminoacylation of the human tRNALYS(Su'),respective SV40 which was mutated in both the 5' and 3' anticodon nucleo-idicate the posi- tides, was reduced only 50%, whereas a decrease of several-spectively. The orders of magnitude was seen in some tRNA analyzed into incompletely vitro. The difference could be because the tRNA species wehe cells infected studied in vivo are different from the ones previously ana-genes, probably lyzed in vitro. Alternatively, the in vitro measurements mayices, while the not accurately reflect the status of aminoacylation in vivo.with the respec- Mutagenesis of the anticodon region sometimes results int gels were dried aminoacylation ofthe incorrect amino acid. For example, theacylated tRNAs amber suppressor tRNA derived from the tryptophan gene ofh corresponding E. coli containing a single nucleotide substitution in the(11). In e this middle position of the anticodon is preferentially acylated

I M. In d and c, with glutamine rather than tryptophan (37, 38). In ourectly compared experiments, the various tRNAs were apparently chargedlf, the reduced with the appropriate 3H-labeled amino acid, but we cannote application of rule out some degree of mischarging. However, in experi-

ments using these tRNAs to suppress nonsense mutations in3-globin mRNA, /3-globins of the expected charge were

minoacylation produced. Thus, when a f-globin mRNA with a codon 17:itutions in the lysine (AAG) to amber (UAG) mutation was suppressed inLges in the 3' Xenopus oocytes with this tRNALYs(Su ) gene, a normallye data suggest charged 1-,lobin was obtained. When suppressed with theon nucleotide tRNAG1n( u gene, this mRNA produced a more negativelyRNA to tRNA charged 13-globin as would be expected if the lysine werethe anticodon replaced by glutamine at codon 17 (11, 20). Thus in vivo, theseNA ligase is two suppressor tRNAs probably inserted the appropriateNAs. Compli- amino acid and were therefore correctly aminoacylated.)fE. coli with Since suppression efficiency depends on the level offten acquire a aminoacylated suppressor tRNAs (39), the development ofch may then techniques to allow the measurement of in vivo levels ofation (34-36). tRNA aminoacylation should further our understanding of

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the influence of nucleotide changes on the physiologicfunction of altered tRNAs. Similar studies could also beextended to other natural or artificially constructed suppres-sor tRNAs prior to their application to genetic analysis.

We thank Dr. P. Sharp for the SV40 recombinants containing theXenopus normal and suppressor tRNATYr genes, Dr. N. Muzyczkafor plasmid pDR404, Andree Dozy for the suppressor tRNAGOn geneand the oocyte injection, Dr. William M.-F. Lee for helpful sugges-tions, and J. Gampell for editorial comments. This research wassupported in part by National Institutes of Health Grant DK16666.Y.W.K. is an Investigator of the Howard Hughes Medical Institute,and Y.-S.H. is an Associate.

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