J. Mol. Biol. (1996) 255, 67–85

Enzymatic Formation of Modified Nucleosides intRNA: Dependence on tRNA Architecture

Henri Grosjean 1*, Johan Edqvist 2, Kerstin B. Stra˚by 2 andRichard Giege´ 3

Information is still quite limited concerning the structural requirements in1Laboratoire d’EnzymologietRNA molecules for their post-transcriptional maturation by base anddu CNRS, 1 avenue de laribose modification enzymes. To address this question, we have chosen asTerrasse, F-91198the model system yeast tRNAAsp that has a known three-dimensionalGif-sur-Yvette, Francestructure and the in vivo modifying machinery of the Xenopus laevis oocyte2Department of Microbiology able to act on microinjected tRNA precursors. We have systematically

University of Umea compared the modification pattern of wild-type tRNAAsp with that of aS-901 87 Umea, Sweden series of structural mutants (21 altogether) altered at single or multiple

positions in the D-, T- and the anticodon branch, as well as in the variable3Unite Propre de Rechercheregion. The experimental system allowed us to analyze the effects of‘‘Structure desstructural perturbations in tRNA on the enzymatic formation of modifiedMacromolecules Biologiques etnucleosides at 12 locations scattered over the tRNA cloverleaf.Mecanismes de

We found that the formation of m1G37 and C40 in the anticodon loop andReconnaissance’’, Institut destem and C13 in the D-stem, were extremely sensitive to 3D perturbations.Biologie Moleculaire etIn contrast, the formation of T54, C55 and m1A58 in the T-loop, m5C49 in theCellulaire du CNRS, 15 rueT-stem and m2G6 in the amino acid accepting stem were essentiallyRene Descartes, F-67084insensitive to change in the overall tRNA architecture; these modifiedStrasbourg Cedex, Francenucleosides were also formed in appropriate minimalist (stems and loops)tRNA domains. The formation of m2G26 at the junction between theanticodon and the D-stem, of Q34 and manQ34 in the anticodon loop weresensitive only to drastic structural perturbation of the tRNA. Altogether,these results reflect the existence of different modes of tRNA recognitionby the many different modifying enzymes. A classification of this familyof maturation enzymes into two major groups, according to theirsensitivities to structural perturbations in tRNA, is proposed.

7 1996 Academic Press Limited

Keywords: tRNA maturation; tertiary structure; yeast tRNAAsp; Xenopuslaevis; microinjection*Corresponding author

Introduction

The 3D structure of tRNAs consists of two welldefined domains assembled into an L-shapedarchitecture. The first domain, the amino acidaccepting branch of the L, is a 12 base-pair helicalstack closed by the T-loop. This domain has its5'-terminus in the first base of the acceptor stem andterminates at the 3'-side by the -CCAOH sequence

where amino acids are attached. The seconddomain, the anticodon branch of the L, is a biloopminihelix consisting of the anticodon loop and stemstacked over the D-stem and loop region. These twodomains are connected by two single-strandedregions (connectors) and they are oriented roughlyperpendicular to each other. The stability of such anarchitecture is maintained by a network of tertiaryinteractions involving most of the conserved orsemi-conserved residues of the tRNAs. These ter-tiary interactions occur in the so-called 3D core ofthe molecule at the hinge of its L-shaped fold wherethe D- and T-loops are interdigitated (reviewed byGiege et al., 1993; Dirheimer et al., 1995).

The interaction between tRNAs and the ribosomeimplies that all tRNA molecules share similarconformations. However, increasing data are pre-

H. Grosjean and J. Edqvist contributed equally.Present address: J. Edqvist, Dalhousie University,

Biochemistry Department, Halifax 3H4H7, Nova Scotia,Canada.

Abbreviations used: 2D t.l.c., two-dimensionalthin-layer chromatography; 3D structure,three-dimensional structure; WC base-pairs,Watson–Crick base-pairs; cpm, counts per minute.

0022–2836/96/010067–19 $12.00/0 7 1996 Academic Press Limited

tRNA Architecture and Formation of Modified Nucleosides68

sently accumulating which indicate that differenttRNAs are characterized by subtle conformationaldifferences linked to variabilities in the tertiaryinteraction networks and in the sequence arrange-ment and length of their D-loops and variableregions (see Giege et al., 1993; Steinberg &Cedergren, 1994; Dirheimer et al., 1995). It is likelythat this conformational variability plays a majorrole in a number of specific functions of individualtRNAs. The importance of structural parameters inthe function of tRNAs has been clearly demon-strated, e.g. for Escherichia coli tRNACys identity(Hou et al., 1993) and for optimal aminoacylationupon the transplantation of identity sets from onetRNA framework to another (Perret et al., 1992;Frugier et al., 1993, 1994a).

As far as the maturation of tRNA is concerned, ithas already been shown that the three-dimensionalstructure (3D structure) is a major recognitionelement for several enzymes involved in pre-tRNAprocessing, such as RNase P (Kirsebom & Altman,1989; Hardt et al., 1993) and the tRNA splicingendonuclease (Mattocia et al., 1988; Reyes &Abelson, 1988; Bufardeci et al., 1993; reviewed inHopper & Martin, 1992, Westaway & Abelson,1995). Concerning the tRNA-modifying enzymes,no clear picture has yet emerged, especially sincethe few examples known concern modified nu-cleosides in tRNAs of various identities and origins(reviewed in Bjork, 1995). Nevertheless, architec-tural features in tRNA have been shown to beessential parameters in the enzymatic reactionsleading to the formation of few modified nu-cleosides, as m1G37 in E. coli tRNALeu (Holmes et al.,1992, 1995)†, inosine-34 in human tRNAVal (Achsel& Gross, 1993) and m2

2G26 in yeast tRNAAsp or yeasttRNAPhe (Edqvist et al., 1992, 1994; reviewed inEdqvist et al., 1995). In contrast, the overall 3Dstructure is less crucial for the enzymatic formationof m5C49 and m1A58 in E. coli tRNAfMet catalyzed byrat liver enzymes (Wildenauer et al., 1974), of Gm18

and m1A58 in tRNA of Thermus thermophilus (Horiet al., 1989; Matsumoto et al., 1990; Yamazaki et al.,1994) or T54 and Q34 in E. coli tRNAs (Gu & Santi,1991; Curnow et al., 1993; Curnow & Garcia, 1994,1995; Guenther et al., 1994; Nakanishi et al., 1994;Kealey & Santi, 1995). Indeed, these enzymaticmodifications can occur in vitro on tRNA fragments,yet less efficiently compared to the intact tRNAmolecule.

In this work, we have specifically addressed thequestion of the role of the tRNA architecture on theenzymatic formation of modified nucleosides intRNA. As the model system, we have used yeasttRNAAsp and the enzymatic machinery of theXenopus laevis oocyte. Firstly, this choice was based

on previous observations that completely un-modified in vitro transcripts of synthetic tRNAgenes are fairly good substrates for several tRNAmodifying enzymes present in the cytosol ofX. laevis oocytes (Grosjean et al., 1990; Edqvist et al.,1993; Sturchler et al., 1994). Secondly, the 3Dstructure of yeast tRNAAsp is well known, not onlyin the crystal form (Moras et al., 1980; Westhof et al.,1985), but also in solution (Romby et al., 1987b), sothat structural mutants of this tRNA can bedesigned in a rational way. Thirdly, engineered yeasttRNAAsp variants, including minimalist tRNAAsp

variants, have already been used to demonstratethat the tRNA conformation is important to triggerefficient aminoacylation reactions (Perret et al., 1992;Frugier et al., 1993, 1994a,b) as well as in theinteraction between tRNA and the elongation factorTu (Rudinger et al., 1994).

Here, we have analyzed 21 structural variantsof yeast tRNAAsp for the enzymatic formation of12 different nucleoside modifications. Thus,a matrix of 252 elements correlating tRNA struc-ture and the presence of modified nucleosidescould be screened. Quantitative data wereobtained for 201 elements in this matrix. Our resultsdemonstrate that distortion of the tRNA architec-ture very differently affects the productiveinteraction of tRNA with various modifyingenzymes. This may reflect different modes oftRNA recognition by this family of maturationenzymes.

Results

The modification pattern of wild-typeyeast tRNA Asp microinjected intoXenopus laevis oocytes

Yeast tRNAAsp naturally contains eight modifiednucleosides (Figure 1a; Gangloff et al., 1971)while X. laevis tRNAAsp, which differs from yeasttRNAAsp in 19 positions (74% homology), containsten such residues (Haumont et al., 1984; Figure 1b).Out of these modified nucleosides, five arecommon to both tRNAs (C13, D20, m5C49, T54 andC55). In addition, yeast tRNAAsp contains D17, C32

and m1G37 (indicated by arrows in Figure 1a), whileX. laevis tRNAAsp contains m2G6, m1G9, manQ34,m5C38 and m5C48 (indicated by arrows in Figure 1b).Since the sequence and modification patterndiffer between tRNAAsp from yeast and X. laevis, itwas important to determine the modificationpattern of a wild-type yeast pre-tRNAAsp transcriptmicroinjected into X. laevis oocytes. Thisrequires that the yeast tRNAAsp transcript is stablewithin the oocytes. Figure 2 shows that this wasthe case: indeed, 60 to 80% of the initially injectedtRNAAsp transcripts, radiolabelled with 32P andvisualized by autoradiography of a polyacrylamidegel electrophoresis, were recovered from the gel asfull-length tRNAAsp after as long as 63 hours ofincubation.

† Symbols and common names of modifiednucleotides from tRNA are those of Limbach et al.(1994). Universal numbering system for tRNApositions corresponds to the one used in Steinberget al. (1993).

tRNA Architecture and Formation of Modified Nucleosides 69

Figure 1. Cloverleaf structure of aspartate specifictRNAs from yeast (a; Gangloff et al., 1971) and X. laevis(b; Haumont et al., 1984). Modified nucleosides arehighlighted and are abbreviated as in Limbach et al.(1994). Boxed nucleotides in the Xenopus tRNAAsp arethose which differ from those in yeast tRNAAsp (74%homology). Arrows in yeast tRNAAsp indicate modifiednucleosides that are not present in Xenopus tRNAAsp,while arrows in Xenopus tRNAAsp indicate those that arenot present in yeast tRNAAsp. Numbering of nucleosidesis as conventionally given in Steinberg et al. (1993).

Figure 2. Stability of tRNAAsp transcripts microinjectedin Xenopus oocytes. The figure shows autoradiography ofa 15% polyacrylamide gel with tRNA recovered fromXenopus oocytes by phenol extraction at the indicated time(in hours) after the microinjection. The main radioactiveband, as indicated by an asterisk, corresponds to full size,intact yeast tRNAAsp. The small arrow and horizontal linesbetween tracks corresponds to position of the bromophe-nol blue marker. Note that the lanes for 24 hours and16 hours of incubation have been inverted. The + (plus)and − (minus) correspond to the polarity of the electro-phoretic current. Migrations are from top to bottom.

The recovered tRNA was completely digestedinto 5'-mononucleotides and/or 3'-mononucleotideswith appropriate nucleases. The presence ofmodified nucleotides in the resulting hydrolyzateswere analyzed by two-dimensional chromatog-raphy on thin layer cellulose plates (two-dimen-sional thin-layer chromatography; 2D t.l.c.). Specificlabelling of the tRNAs during the in vitrotranscription reactions, either with [a-32P]ATP,[a-32P]CTP, [a-32P]GTP or [a-32P]UTP, alloweddetection of all the various modified nucleosideson the t.l.c. plates, as illustrated in the eightpanels of Figure 3. The time course for theformation of each individual modified nucleoside isshown in Figure 4. The extent of modification(expressed in mole/mole of tRNA after 48 hoursincubation) from three to six independent micro-injections of each the different radiolabelledwild-type tRNAAsps are given in Table 1. Only thelower and the higher values obtained are indicated.The reproducibility of the experiments was there-fore evaluated to be about (20.15) mole/moletRNA.

tRNA Architecture and Formation of Modified Nucleosides70

Figure 3. Modified nucleotides formed in in vitrotranscribed yeast wild-type tRNAAsp when microinjectedinto X. laevis oocytes. The figure shows selectedautoradiograms of thin-layer chromatography plates afterchromatography in two dimensions. The 32P-labelledtRNAs were incubated in oocytes for 48 hours. Aftermicroinjection and incubation in oocytes, the tRNA wasgel-purified and completely degraded into mononucle-otides with either nuclease P1 or RNase T2, as indicatedin the figure and in Table 2. These autoradiograms havebeen overexposed in order to reveal trace amounts ofmodified nucleotides. Identification of the modifiednucleotides was made by comparison with referencemaps, as in Keith (1995). Very small amounts of Cp fromposition 13, between Gp and Up and below m2Gp inpanel ATP/T2 (solvent system 1), could be seen only onthe original autoradiography. Dp, from position 17 inpanel GTP/T2 (solvent system 2), corresponds to thedistortion at the left of Up; it was resolved from Up onlyon 20 × 20 cm2 t.l.c (result not shown). In the last panel,UTP/T2 (solvent system 2), there are three spots withoutnames. The one at the left of Up is manQp. The one at theupper left of Gp is Cm-Up, which came from positions32-33. In panel UTP/P1 (solvent system 1), the presenceof pCm was masked by pT, but can be resolved on a20 × 20 cm2 t.l.c (results not shown). The only spot thathas not been identified was located between A and m1A(upper part in panel UTP/T2). This spot was not alwaysso intense in different experiments and correlated withopposite variations in intensity with spot m1Ap. It might

Thus, a completely unmodified yeast tRNAAsp

transcript (wild-type), lacking all the modifiednucleosides, became modified at 13 distinctpositions after its microinjection into the cytoplasmof the X. laevis oocyte. Modifications occurred at allthe eight sites that are modified in the yeast cell(shown in the cloverleaf structure in Figure 5a; seealso Table 1), at two additional sites, as in X. laevistRNAAsp (m2G6 and manQ34) and at positions26 (m2G26), 40 (C40) and 58 (m1A58; indicated byarrows in Figure 5a), yet with different efficiencies.The enzymatic formation of m2G6, m2G26, [Q34 +manQ34], m5C49, and C55 was efficient and almostcomplete (0.6 to 1.0 mole/mole of tRNA) after 10 to20 hours of incubation in the oocytes. D20, m1G37 andT54 were also efficiently formed (0.3 to 0.6 mole/mole tRNA after 48 hours of incubation). But evenafter 48 hours, other modified nucleosides (D17, C40

and m1A58) were present only in less than 20% of theavailable sites in the full length tRNAAsp (at least inthe wild-type; see below). The least efficientlyformed modified nucleosides were C13 and C32

(Figure 4 and Table 1).

Design of structural tRNA variants and theirmodification patterns in oocytes

Figure 5b shows the location of the 13 modifiednucleosides within the 3D conformation of yeasttRNAAsp. The thin dashed lines between numberednucleosides correspond to the nine tertiary inter-actions, as revealed by crystallographic and solutionstudies (Moras et al., 1980; Westhof et al., 1985;Romby et al., 1987b). Eight of the modifications,namely C13, D17, D20, m2G26, m5C49, T54, C55 andm1A58, are located in the core of the molecule, wherethe two domains of the tRNA are interdigitated andthe 3D interactions occur, whereas four are found inthe anticodon loop and stem (C32, [Q34 + manQ34],m1G37 and C40) and one in the amino acid acceptorstem (m2G6).

Several series of tRNA variants were made tosystematically test the importance of the tRNAarchitecture in the formation of these modifiednucleosides (Table 2). In the first series, mutationsaffect the 3D core of the molecule, either bydisrupting interactions between the D- and T-loopsor the D-loop and the variable region (tRNA-2 to-13). These mutants contain alterations in three outof the nine tertiary interactions naturally present inyeast tRNAAsp, namely those involving the strictlyconserved base-pairs U8-A14 and G19-C56, as well asthe semi-conserved purine-pyrimidine (R15-Y48)Levitt base-pair. This series of variants includemolecules in which the 3D interactions are restoredin the form of alternate (tRNA-3) or invertedpairings (tRNA-5 and -8). The possibility exists thatthese new 3D pairings do not restore the preciseinitial 3D conformation, but should certainly

correspond to an incomplete digestion of m1A-Up(positions 58-59), or to an isomerization of m1A into m6Ain our experimental conditions.

tRNA Architecture and Formation of Modified Nucleosides 71

Figure 4. Time courses for the formation of modified nucleosides in wild-type yeast tRNAAsp microinjected intoXenopus oocytes. Estimation of the relative radioactivities in each spot as detected in Figure 3 for different incubationtimes of the yeast tRNAAsp in the oocytes led us to determine the molar ratio of each detected modified nucleotide versusunmodified ones. The relative number of each of the four bases in tRNAAsp was known from the nucleotide sequence.

contribute to it. In order to check a progressivedisruption of the 3D architecture in the tRNA, somemutants contain alterations in two (tRNA-10 to-12) or even three different tertiary base-pairs(tRNA-13).

A second series of mutations affect the structureand the size of the T-loop (tRNA-14 to -16) or theanticodon loop (tRNA-17 and -18). Among these area mutation in a fourth tertiary interaction, namelythe reverse Hoogsteen pair between residues U54

and A58 in the T-loop (tRNA-14). The othermutations concern replacement of the T-loop by aUUCG tetraloop of high structural stability (Cheonget al., 1990; tRNA-16) or the insertion of anadditional residue into the seven nucleotide longT-loop or anticodon-loop (tRNA-15, -17 and -18).

The third and last series of tRNA variants areminimalist structures in which selected domains(stems and loops) have been deleted (tRNA-19 to-21). These were designed to test the effects ofintrinsic structural features of loops and stems aspotential recognition signals for modifying en-zymes.

Each one of these 21 tRNAAsp variants wasmicroinjected into the cytoplasm of X. laevis oocytesand analyzed as described above. Except for two ofthe minimalist variants (tRNA-20 and -21), theywere stable enough in the X. laevis oocytes to allow

estimation of their content of modified nucleosidesafter 24 and 48 hours of incubation. The twominimalist tRNA variants (tRNA-20 and -21) had tobe analyzed after shorter incubations, ten and sixhours, respectively, because they were quicklydegraded in vivo by nucleases. Table 3 and Figure 6(tRNA-1 to tRNA-21) display all the resultsobtained. The numbers in Table 3 correspond tomoles of modified nucleosides per mole of fulllength tRNAAsp recovered from the microinjectedoocytes incubated for 48 hours (tRNA-1 to -19), orfor ten and six hours in the case of tRNA-20 and -21,respectively. Except for the control experiment withthe wild-type tRNAAsp (data of entry 1 in Table 3),each value comes from only one experiment.

We relate these results to the influence of thestructural perturbations in tRNA on the modifi-cation patterns in the various tRNA domains,namely in the amino acid accepting branch (do-main 1), the anticodon branch (domain 2), and inminimalist tRNAs, below.

Some nucleoside modifications are lost whenthe tRNA structure is drastically distorted

Modifications that are especially sensitive to 3Dperturbations in tRNA are essentially located in theanticodon branch (domain 2, including the anti-

tRNA Architecture and Formation of Modified Nucleosides72

codon stem and the D-arm) and concern theformation of C13, m2G26, [Q34 + manQ34], m1G37 andC40. Only C32 deviated from this ranking. Thisconclusion is evident from the results obtained withtRNA-10 to -13, where two or three base-pairs areaffected (Table 3 and Figure 6). This trend was lessevident in variants having only one of the ninetertiary base-pairs affected, as in tRNA-2, -4, -6, -7and -9. Note that the single mutation of U8 to C8

(tRNA-9) that disrupts the tertiary interactionbetween U8 and A14 had the same drastic effect onthe formation of modified nucleosides as multiplemutations of tertiary interactions.

The change of U48 to C48 (tRNA-2), which disruptsthe Levitt pairing between A15 in the D-loop and U48

in the variable region, had a moderate effect onthe formation of most modified nucleosides. Thecompensatory mutation of A15 to G15, whichreintroduces a stronger G-C tertiary interaction(tRNA-3), restored formation of m2G26 to thewild-type level and further increased that of C13

and C40 (values underlined in Table 3). Interestingly,this tRNA was the only variant where C13 in theD-stem and C40 in the anticodon stem were formedmore efficiently than in the wild-type yeasttRNAAsp. In tRNA-4, a pyrimidine/purine transver-sion (U48 to A48) that disrupts the Levitt pair, led tothe almost total loss of m2G26, [Q34 + manQ34],m1G37, and C40 formation. These modified nu-cleosides could not be recovered by the compensa-tory mutation of A15 to U15 (tRNA-5), expected tocreate U15-A48, a putative base-pair that has noequivalent in any tRNA sequenced so far (seeSteinberg et al., 1993).

We also investigated whether the formation ofmodified nucleosides in the anticodon branch were

sensitive to changes in the sequence or the size ofthe T-loop. Clearly, there was no formation of C13,m2G26, [Q34 + manQ34], m1G37 and C40, neither intRNA-14, in which the internal reverse Hoogsteenpairing between U54 (T54) and A58 within the T-loopwas disrupted through the U54 to A54 mutation, norin tRNA-15 or -16, where the size of the loop waschanged. Thus, mutations in the T-loop within thetRNA 3D core (Figure 5b), which perturb mainten-ance of the tRNA architecture, had drastic effects onthe formation of modified nucleosides in theanticodon branch. In contrast, the formation of thesesame modified nucleosides in the T-stem and loopwere almost unaffected when the anticodon loopsize was either increased or decreased by onenucleotide, as in tRNA-17 and tRNA-18.

Formation of nucleoside modifications in theT-arm and the acceptor stem are notdependent on an intact tRNA architecture

At variance with the above results concerningmodified nucleosides in the anticodon branch(domain 2), the formation and the level of modifiednucleosides in the amino acid accepting branch(domain 1), including the T-stem-loop and theamino acid stem, namely m5C49, T54, C55, m1A58 andm2G6, were not dependent on the normallyoccurring tertiary interactions in the tRNA mol-ecule. Actually, compared to wild-type tRNAAsp

(tRNA-1), the levels of m1A58, as well as of T54,increased significantly in most of the tRNAsharbouring mutations expected to distort the tRNAstructure (tRNA-2, -6 and -10 to -13; valuesunderlined in Table 3).

Table 1. Modification pattern in wild-type yeast tRNAAsp obtained with Xenopus laevis modifying enzymes and thestrategy for identification

Modication Labelled Enzyme used t.l.c. Natural occurrence inPosition Type Extent nucleotide for analysis systema Yeast Xenopus

6 m2G 0.8–1.0 [a-32P]ATP RNase T2 1 G m2G13 C 0.05–0.2 [a-32P]ATP RNase T2 1 C C17 D 0.1–0.3 [a-32P]GTP RNase T2 2 D U20 D 0.2–0.5 [a-32P]CTP RNase T2 1 D D26 m2G 0.7–1.0 [a-32P]GTP RNase T2 1 or 2 G C32 C 0.05–0.2 [a-32P]UTP RNase T2 1 C C34 Q + manQ 0.7–0.9 [a-32P]GTP Nuclease P1 1 or 2 G manQ

[a-32P]UTP RNase T2 1 or 237 m1G 0.4–0.6 [a-32P]GTP Nuclease P1 1 or 2 m1G A

[a-32P]CTP RNase T2 1 or 240 C 0.1–0.4 [a-32P]GTP RNase T2 1 U C49 m5C 0.6–0.8 [a-32P]CTP Nuclease P1 2 m5C m5C

[a-32P]GTP RNase T2 254 T 0.4–0.5 [a-32P]UTP Nuclease P1 1 or 2 T T

[a-32P]UTP RNase T2 1 or 255 C 0.6–0.8 [a-32P]CTP RNase T2 1 C C58 m1A 0.1–0.4 [a-32P]ATP Nuclease P1 2 A A

[a-32P]UTP RNase T2 2a The figure 1 means the solvent system according to Nishimura (1979); 2 means the solvent system according to Silberklang et al.

(1979). For identification of modified nucleotides by their positions on the t.l.c., see Keith (1995). Names of modified nucleotides areabbreviated as in Limbach et al. (1994); these are in bold. Notice that the pattern of nucleoside modification is not strictly the samein yeast tRNAAsp and Xenopus tRNAAsp. When microinjected into the cytoplasm of Xenopus oocyte, all of the characteristic modifiednucleosides of the Xenopus tRNAAsp occurred in yeast tRNAAsp. However, due to sequence differences, additional modifications appearas D17, C32, m1G37, C40 and m1A58, but to various extents.

tRNA Architecture and Formation of Modified Nucleosides 73

Figure 5. Summary of the nucleoside modificationpattern in yeast wild-type tRNAAsp microinjected into thecytoplasm of Xenopus oocytes. In panel a, the results aredisplayed in the classical cloverleaf representation of the

The modified nucleosides in the T-loop were onlyaffected by mutations within the T-loop. Thus, T54

consequence of the mutation of the conserved C56

residue to G56. The formation of C55 was drasticallylowered only when its nearest neighbour, theconserved U54, was mutated to A54 (tRNA-14).Furthermore, the formation of T54 was shown to bedependent on the size of the T-loop (tRNA-15),since T54 was missing from the tRNA in whichthe T-loop was enlarged to eight nucleotides, aswell as from tRNA-16, where the T-loopcontained only four residues (A57AUU60 werereplaced by a single G residue). Residue C55 wasalso absent from tRNA-16, while neither theformation of C55, nor that of m1A58 were negativelyaffected by the insertion of an extra A betweennucleotides 56 and 57 in the seven membered T-loop(tRNA-15).

Similarly, neither the formation of m2G6 in theacceptor stem, nor of m5C49 in the T-stem, weredrastically affected in any of the tested tRNAs,although the level of m2G6 was slightly reduced inthose tRNAs where the interactions between theD-loop and the acceptor stem and between theD-loop and the extra arm were simultaneouslydisrupted (tRNA-11 and -12). We conclude thatthe enzymatic formation of m2G6, m5C49, T54,C55 and m1A58 do not depend on an intact tRNAtertiary structure. Seemingly, local structural el-ements, as the T-loop size and structure, arerequired for the optimal formation of thesemodified nucleosides.

tRNA-fragments corresponding tosub-domains are efficiently modified by sometRNA-modifying enzymes

Since the formation of modified nucleosides inthe amino acid accepting branch (domain 1) wereshown to be independent of an intact tRNAstructure, it was of interest to investigate whetherfragments of tRNAs could also be modified by thecorresponding enzymes present in the cytoplasm ofX. laevis oocytes.

We first tested a tRNA fragment lacking theD-arm (tRNA-19) for its potential to be modified

tRNA. The nucleosides naturally modified in yeast andalso modified in the oocytes are highlighted by greyboxes within the cloverleaf. Additional residues notnaturally modified in yeast, but modified by the oocyteenzymes, are indicated by grey boxes and at the end ofarrows. The boxed G-C pair at the end of theaminoaccepting branch is the one that replaces the normalU-A pairs in the wild-type yeast tRNAAsp in order toenhance the transcription yield (see Materials andMethods). In panel b, a representation of the L-shaped3D-structure of the tRNA is shown and emphasizes thetype of modified nucleosides that are located (boxed) inthe 3D core of the tRNA molecule. The dashed linesbetween numbered bases corresponds the tertiaryinteractions, while the heavy lines (arrows) correspond tothe connectors between the two domains 1 and 2.

tRNA Architecture and Formation of Modified Nucleosides74

after microinjection into X. laevis oocytes. Thisvariant resembles atypical tRNAs present in themitochondria of eukaryotic cells (e.g. de Bruijn &Klug, 1983; Garey & Wolstenholme, 1989), and itprobably folds into a 3D-structure (Steinberg &Cedergren, 1994) that obviously allows inter-actions with the protein synthesis machinery. Theresults (Table 3 and Figure 6) again revealed thatm2G6, C32, m5C49, T54, C55 and m1A58 are formed,

while [Q34 + manQ34], m1G37, C40 and m2G26 werecompletely missing from this fragment. In thefragment consisting of only the acceptor stem andthe T-arm (tRNA-20), corresponding to an amino-acylable minihelix (Frugier et al., 1992, 1994b),C55 and m1A58 were formed at almost wild-typelevels, while the level of m2G6 was very low andboth m5C49 and T54 were almost or completelyabsent after ten hours of incubation. Interestingly,

Table 2. Structural variants of yeast tRNAAsp used as substrates of nucleotide modifying enzymes from Xenopus laevistRNA variants(number in a Changes inseries) Mutations architectural featuresa Comments

tRNA-1 wild-type 75 nt long transcript The 8 modified nucleotides present in nativetRNAAsp are missing in this molecule whichhas a more flexible conformationb

Mutations affecting tertiary interactions in the core of the tRNA

a. between the D-loop and the variable region (Levitt R15-Y48 base-pair)

tRNA-2 U48 : C48 A15-U48 : A15-C48 disrupts the A-U Levitt base pairtRNA-3 A15 : G15 & U48 : C48 A15-U48 : G15-C48 restores a G-C Levitt base-pairtRNA-4 U48 : A48 A15-U48 : A15-A48 disrupts the A-U Levitt base-pairtRNA-5 A15-U15 & U48 : A48 A15-U48 : U15-A48 restores an inverted U-A Levitt base pair

b. between the D-loop and the T-loop

tRNA-6 G19 : C19 G19-C56 : C19-C56 disrupts the conserved 19-56 WC base-pairtRNA-7 C56 : G56 G19-C56 : G19-G56 disrupts the conserved 19-56 WC base-pairtRNA-8 G19 : C19 & C56 : G56 G19-C56 : C19-G56 restores an inverted 19-56 WC base-pair

c. between the D-loop and the hinge joining the acceptor and D-stems

tRNA-9 U8 : C8 U8-A14 : C8-A14 disrupts a tertiary pairing, but possibly createsan alternate reverse C-A Hoogsteen pairing(in trans)

d. multiple mutations affecting several of the above regions

tRNA-10 G19 : C19 & U48 : C48 G19-C56 : C19-C56 disrupts two tertiary pairings (the conservedA15-U48 : A15-C48 19-56 WC and the Levitt base-pairs)

tRNA-11 U8 : C8 & U48 : C48 U8-A14 : C8-A14 disrupts two tertiary pairings (the conservedA15-U48 : A15-C48 8-14 and the Levitt base-pairs)

tRNA-12 A14 : C14 & A15 : C15 U8-A14 : U8-C14 disrupts two tertiary pairings (the conservedA15-U48 : C15-U48 8-14 and the Levitt base-pairs)

tRNA-13 A14 : C14 & A15 : U8-A14 : U8-C14 disrupts three tertiary pairings (theC15 & G19 : C19 A15-U48 : C15-U48 conserved 8-14, 15-48 and 19-56

G19-C56 : C19-C56 base-pairings)

Mutations affecting loop structures

a. T-loop

tRNA-14 U54 : U54 U54-A58 : A54-A58 disruption of A-U reverse Hoogsteen pair(in trans)

tRNA-15 insertion of A57a A57A58 : A57A57aA58 8 nucleotides (instead of 7) in T-looptRNA-16 deletion & insertion A57A58UU60 : G T-loop becomes a UUCG tetraloop

b. Anticodon loop

tRNA-17 insertion of U32a U32U33 : U32U32aU33 8 nucleotides (instead of 7) in anticodon looptRNA-18 insertion of G34a G34U35 : G34G34aU35 8 nucleotides (instead of 7) in anticodon loop

Minimalist tRNAs

tRNA-19 deletion of D-arm structure similar to the eukaryoticmitochondrial tRNASer (61 nt)c

tRNA-20 deletiion of D- and anticodon arms amino acid accepting minihelix (34 nt)tRNA-21 deletion of D-, accepting, and anticodon arms structure corresponding to the T-arm (17 nt)

a Mutations are emphasized in bold and underlined characters.b Perret et al. (1990).c de Bruijn & Klug (1983).All tRNA variants from 1 to 19 have comparable stability as the wild-type yeast tRNAAsp in the cytoplasm of Xenopus

oocyte.

tRNA Architecture and Formation of Modified Nucleosides 75

Table 3. Effects of mutations in tRNAAsp transcripts on their modification levels when microinjected into Xenopus laevisoocytes

Position and extent of modificationsatRNA variants300aa Branch004300000000 Anticodon Branch000000004300000aa Branch000004300aa stem0043000 3D core 0004300 Anticodon domain 004300000 3D core 000004

serial nymber &mutation introduced

m2G6 C13 D20 m2G26 C32 Q34b m1G37 C40 m5C49 T54 C55 m1A58

1 (wild-type)c 0.9 0.1 0.3 0.8 0.1 0.7 0.5 0.3 0.7 0.4 0.7 0.2

Mutations affecting tertiary interactions in the core of the tRNA

a. between D-loop and variable region (Levitt 15-48 base-pair)

2 (U48 : C48) 0.9 0.0 0.2 0.4 0.0 0.6 0.2 0.1 0.9 0.9 0.9 0.63 (A15 : G15)

(U48 : C48) 0.9 0.4 n.d. 0.8 0.0 0.5 0.1 0.5 0.9 0.8 0.8 0.44 (U48 : A48) n.d. n.d. n.d. 0.1 0.2 0.1 0.0 0.0 0.7 0.6 0.8 0.25 (A15 : U15)

(U48 : A48) n.d. n.d. n.d. 0.0 0.1 0.0 0.0 0.0 0.6 0.8 0.8 0.2

b. between D-loop and T-loop

6 (G19 : C19) 1.0 0.0 n.d. 1.0 0.2 0.4 0.1 0.0 0.9 0.8 0.9 0.67 (C56 : G56)d 0.9 0.0 n.d. 0.4 0.1 0.4 0.1 n.dd 0.9 0.0 0.6d 0.08 (G19 : C19)

(C56 : G56)d 0.9 0.0 n.d. 0.5 0.1 0.4 0.3 n.dd 0.9 0.0 0.6d 0.0

c. between D-loop and the hinge joining the acceptor and D-stems

9 (U8 : C8) 0.8 0.0 n.d. 0.0 0.2 0.0 0.0 0.0 0.8 0.3 0.9 0.6

d. multiple mutations affecting several of the above regions

10 (G19 : C19) 0.6 0.0 n.d. 0.1 0.1 0.0 0.0 0.0 0.8 0.8 0.9 0.6(U48 : C48)

11 (U8 : C8)(U48 : C48) 0.5 0.0 n.d. 0.0 0.1 0.0 0.0 0.0 0.8 0.8 0.7 0.6

12 (A14 : C14)(A15 : C15) 0.3 n.d. n.d. 0.0 0.1 0.0 0.0 0.0 0.8 0.7 0.8 0.5

13 (A14 : C14)(A15 : C15)(G19 : C19) 0.8 n.d. n.d. 0.0 0.1 0.0 0.0 0.0 0.8 0.6 0.8 0.6

Mutations affecting loop structures

a. T-loop

14 (U54 : A54) 0.9 0.0 n.d. 0.0 0.0 0.0 0.0 0.0 0.8 N.A. 0.1 0.215 (insertion A57a) 0.6 0.0 n.d. 0.0 0.0 0.0 0.0 0.0 0.8 0.0 0.8 0.616 (deletion & insertion) 0.9 0.0 n.d. n.d. 0.0 0.0 0.0 n.d. 0.7 0.0 0.0 N.A.

b. Anticodon loop

17 (insertion U32a) n.d. n.d. n.d. 0.2 n.d. 0.02 0.1 0.0 0.4 n.d. n.d. n.d.18 (insertion G34a) 0.8 0.0 n.d. 0.4 0.1 0.0 0.2 0.0 0.4 0.6 0.8 0.2

Minimalist tRNAs

19 (deletion D-arm) 0.5 N.A. N.A. 0.0 0.1 0.0 0.0 0.0 0.7 0.7 0.9 0.420 (deletion D-, &

anticodon arms) 0.03 N.A. N.A. N.A. N.A. N.A. N.A. N.A. 0.0 [0.3]e 0.9 0.221 (deletion of D-accepting

& anticodon arms) N.A. N.A. N.A. N.A. N.A. N.A. N.A. N.A. 0.0 0.0 0.3 0.0a Modifications are expressed as moles of modified nucleosides per mole of tRNA after 48 hours incubation in the oocyte (except

for mutants 20 and 21; see text).b Corresponds to Q34 + manQ34.c Average values from Table 1. Numbers in bold correspond to values that are close to, or at least 50% of those measured for the

wild-type yeast tRNAAsp. Numbers in bold and underlined mean that they are significantly higher than the ones measured for thewild-type yeast tRNAAsp. n.d. = non-determined; N.A. = non-applicable.

d When C56 is changed to G56, C measured on t.l.c. corresponds to both C40 and C55; however, all counts were attributed to C55.e In fact, no T54 on t.l.c. plates, but C54 instead.

U54 in tRNA-20 was now modified to C54

(indicated by an arrow in Figure 6(b)). Theanalogous location of U54 in the T-loop and U32 inthe anticodon loop suggests that C54 (not C55) andC32 were formed by the same enzyme. In the

shortest T-arm fragment (tRNA-21), the onlymodification detected after hours of incubation wasC55. This modification, among all the ones we havestudied, was by far the least sensitive to tRNAarchitecture.

tRNA Architecture and Formation of Modified Nucleosides76

Discussion

Because certain modified nucleosides in tRNAare common to all tRNA species and others arerestricted to one or only few tRNAs (Bjork, 1995), itcan be anticipated that the enzymes forming themwill be sensitive either to general gross features ofthe tRNA structure or to specific architecturalcharacteristics. Furthermore, modified nucleosides

are located either in the tRNA 3D core wheretertiary interactions occur, or in the distal regions ofthe molecule (Figure 5b). From this viewpoint, itcan be deduced that the enzymes responsible formodifications in different domains will not beequally sensitive to the overall tRNA structure, but,more probably, will recognize either a domain assuch and/or specific sequence elements withinthem.

Figure 6(a) (legend opposite)

tRNA Architecture and Formation of Modified Nucleosides 77

(b)

Figure 6. Effects of mutations in tRNAAsp variants (tRNA-1 to -18) or in minimalist tRNAAsp molecules (tRNA-19 to-21) on the formation of modified nucleosides by the oocyte enzymes. A model of the cloverleaf structure is used.Nucleotides that have been mutated are indicated in boxes. Filled circles in tRNA variants at positions correspondingto modified nucleosides are those formed at a similar level (2) 0.15 moles per mole tRNA) as in wild-type tRNAAsp

(tRNA-1) after 48 hours incubation in the oocytes (see also in Table 3). Filled circles that are larger than the originalones in tRNA-1 correspond to modified nucleosides that were found at significantly higher yields than in the wild typetRNA-1. Open circles correspond to modified nucleosides that are totally absent as compared to the wild-type tRNAAsp.Open circles, partially filled, correspond to those modified nucleosides for which the level of modification is lower thanin wild-type tRNAAsp. Since the smaller fragments (tRNA-20 and -21) were degraded faster than intact tRNA, they wereincubated for shorter times than 48 hours; thus, for ten and six hours, respectively.

Two major groups of tRNA-modifying enzymes

Our data show that the tRNA structure plays amajor role in the enzymatic formation of almost allthe modified nucleosides that are present in theanticodon branch (domain 2) of yeast tRNAAsp

(Figure 5b), namely, the modified nucleosides in theanticodon loop and stem, in the D-stem and in thehinge between these two sub-domains (C13, m2G26,[Q34 + manQ34], m1G37 and C40; we have not care-fully investigated D17 and D20). Thus, it follows thatthose corresponding tRNA-modifying enzymes inthe oocyte require a quasi-intact, or at least acorrectly folded, L-shaped tRNA for a productiveinteraction. Our present results for m2G26 confirmour earlier conclusions (Edqvist et al., 1992, 1994),while our data for the enzymatic formation of m1G37

extend to a eukaryotic system similar to thoseobtained by Holmes et al. (1992, 1995) in an E. colimodification system. The case of tRNA-guaninetransglycosylase is different. This enzyme isresponsible for the incorporation of Q residue intothe anticodon wobble position in tRNA specific foraspartic acid, asparagine, histidine and tyrosine inmost prokaryotic and eukaryotic organisms (re-viewed in Kersten & Kersten, 1990). While theXenopus enzyme requires a rather intact tRNAarchitecture, as shown in our in vivo experiments,the purified E. coli tRNA-guanine transglycosylasecan efficiently modify in vitro a minimalist 17-merRNA derived from an anticodon arm (Curnow et al.,1993; Curnow & Garcia, 1995). A recent footprintingstudy confirmed that the anticodon stem-loopregion is the major target for the protein/tRNAcontacts between the E. coli tRNA-guanine transgly-cosylase and a yeast tRNAAsp (Mueller & Slany,1995). Together with the fact that the mammalianand the bacterial tRNA-guanine transglycosylase

have different molecular masses and subunitcomposition (see, e.g. Garcia et al., 1993; Morriset al., 1995, and references therein), it is probablyvalid to conclude, despite the very differentexperimental conditions used, that the bacterial andeukaryotic enzymes catalyzing the synthesis of Q34

have different structural requirements on theirtRNA substrates.

In the amino acid accepting branch (domain 1),the situation is different. The enzymatic formationof modified nucleosides m2G6 in the acceptor stemand of m5C49, T54, C55 and m1A58 in the T-arm, wereclearly not dependent on an intact tRNA 3Dstructure. Even the enzymatic formation of m5C49,C55, but mostly of T54 and m1A58, in the T-arm wereenhanced by certain structural disorders of thetRNA molecules, as in tRNA-2, tRNA-6 andtRNA-10 to tRNA-13 (Table 3 and Figure 6). Weconclude that the corresponding enzymes in theoocyte only interact with a small portion of thetRNA molecule. Alternatively, these enzymes mayunfold the tRNA conformation while interactingwith their substrates, flipping out the targetnucleoside in a way that might be similar to whathappens with several DNA-modifying enzymes(reviewed by Roberts, 1995). Thus, these tRNA-modifying enzymes may not recognize or require acomplete L-shaped tRNA, but rather local structuralelements. This became evident from our datashowing that fragments containing the T-loop(tRNA-19 and tRNA-20) were active substrates forthe modifying enzymes catalyzing the formation ofC55 and m1A58. However, the size of the fragmentseems critical, since in the smallest fragment wetested in vivo, namely the minimalist 17-mer T-arm(tRNA-21), only C55 was formed. Conclusions onhow C13 in the D-stem and of C32 in the anticodonloop are formed are less evident, since in the

tRNA Architecture and Formation of Modified Nucleosides78

wild-type tRNAAsp the levels of these modifi-cations were already very low. It seems, how-ever, as if the pseudouridylate synthase acting onU13 is dependent on the tRNA 3D structure,while the pseudouridylate synthase acting on U32 isnot.

Our results are in agreement with those of Santiand coworkers (Gu & Santi, 1991; Kealey & Santi,1995) showing that E. coli tRNA-(m5U54)-methyl-transferase efficiently modified the minimalist17-mer T-arm in vitro. They also fit with those ofWildenauer et al. (1974), showing that C48 and A58 inE. coli tRNAfMet were methylated by a partiallypurified tRNA methylase fraction from rat liver onlyin conditions where the tRNA tertiary structure wasdestabilized by cadaverine. Similarly, Yamazaki et al.(1994) demonstrated that purified tRNA (A58)methyltransferase from T. thermophilus was able tomethylate A58 in a 3'-half fragment of E. colitRNAfMet. Moreover, Nishikura et al. (1982) havealready shown that Xenopus enzymes leading to theformation of m5C48, C55 and m1A58 in yeast tRNATyr

appear rather insensitive to changes in thesecondary and tertiary structure and may recognizeonly limited regions of the 102mer intron-containingprecursor tRNATyr. As to C55 and m1A58, thisconclusion agrees with our study of the yeasttRNAAsp variants. Concerning the formation ofm5C, our results for m5C49 extend those of Nishikuraet al. (1982) for m5C48 and favor the idea thattRNA-(cytosine)-methyltransferase(s) in Xenopusoocyte recognizes a small domain where thevariable region joins the amino acid acceptingbranch of the tRNA.

According to their sensitivities to structuralperturbations in tRNA, we propose to divide thetRNA-modifying enzymes into two major groups.We use the term ‘‘groups’’ instead of ‘‘classes’’ inorder to avoid confusion with the partition of tRNAsynthetases into two major classes based onmutually exclusive sets of primary sequencemotifs (Eriani et al., 1990). Figure 7 illustrates thisidea with the data obtained in the present workwith several Xenopus enzymes. Enzymes ofgroup I only need a local structural element, suchas the T-branch, the amino acid accepting branch(domain 1) or the anticodon branch (domain 2), fortheir productive recognition with the tRNA.Disruption of tertiary base-pairs within the 3Dcore may favor these interactions. Enzymes ofgroup II include those that are sensitive toperturbations in the overall tRNA structure.Distinctions are made between those enzymes thatare hypersensitive to tRNA architecture (enzymesof group IIa) and most probably require anL-shaped tRNA fold for their productive interactionwith the tRNA, and the other enzymes (of groupIIb) that tolerate certain 3D perturbations andprobably depend only upon local architecture of the3D core. Interestingly enough, the enzymes ofgroups IIa and IIb are located on the same side ofthe tRNA, along the ‘‘inner’’ part of the L-shapedmolecule (Figure 7).

Figure 7. Classification of the enzymes catalyzing thebase and ribose modifications in tRNA into two majorgroups according to their sensitivities to 3D perturbationof the tRNA architecture. The ribose-phosphate backboneof yeast tRNAAsp is represented by a continuous dark linefrom the 5'p terminal to the 3'OH of the CCA end. Dotscorrespond to positions of each of the nucleotides in thistRNA, except where a modified nucleoside was present.All the modified nucleosides are those that are formed inthe Xenopus oocytes (D17 and D20 are not shown; see text).The modified nucleosides corresponding to the enzymesof group I are shown in filled boxes. These enzymes areessentially insensitive to global 3D perturbation of tRNA;they can even modify fragments or minimalist tRNAsubstrates (see text). The tRNA modifying enzymes ofgroup II are those that are sensitive to 3D perturbation(global or local) of tRNA. They are classified intoenzymes of group IIa when they require quasi-intact, orat least a correctly folded, L-shaped tRNA for productiveformation of modified nucleosides (those that are notboxed in the figure) and enzymes of group IIb when theyare dependent to only drastic perturbations, probablylocal perturbations, of the tRNA 3D architecture. Thesecorrespond to the modified nucleosides, which are shownin grey boxes.

All tertiary base pairs are not equallyimportant for the tRNA conformationrecognized by the tRNA-modifying enzymes

Mutagenesis of the tertiary interactions betweenthe Levitt base-pair A15-U48 (tRNA-2), or betweenG19-C56 (tRNA-6 and -7), had rather moderate effectson the formation of several modified nucleosides. Toobtain a drastic effect, both interactions had to besimultaneously disrupted (as in tRNA-10). This is in

tRNA Architecture and Formation of Modified Nucleosides 79

contrast to the situation for the interaction betweenU8-A14, which sole disruption had a pleotropic effectand caused the loss of many modified nucleosidesmostly in the anticodon branch (domain 2).Likewise, most mutations in domain 1, in particularthe disruption of the reverse Hoogsteen pairbetween U54 and A58 in the T-loop (this work), oralternatively, forming a Watson–Crick G10-C25 orA10-U25 base-pair (WC base-pair) instead of thenaturally occurring wobble pair G10-U25 (Edqvistet al., 1993), had drastic effects on the formation ofmodified nucleosides in the anticodon branch(domain 2).

These results suggest that the U8-A14 interactionand the T-loop structure within the 3D core are ofcrucial importance for the proper folding of thetRNA and corroborate other independent obser-vations. Major et al. (1993) has shown that the U8-A14

interaction is the only tertiary interaction necessary,when the 3D structure of tRNAPhe was reproducedat atomic resolution with computer modelling. Also,chemical mapping experiments, combined withfunctional studies, suggest that the U8-A14 region aswell as the D- and T-loop structures are critical forthe folding and aminoacylation properties of yeasttRNAAsp (Romby et al., 1987a; Perret et al., 1992;Puglisi et al., 1993). Additional evidence that theseregions are very important for proper tRNA foldingcomes from a circular permutation analysis of yeasttRNAPhe (Pan et al., 1991). In this analysis, only thosemolecules with termini in the U8-A14 and the G53-G57

regions were unable to fold into tRNA structuresthat could be cleaved by lead ions, indicating thatthese regions in the tRNA are most sensitive tobackbone cleavage. Moreover, when intron removalwas analyzed with yeast pre-tRNA variantsharbouring mutations in conserved or semi-con-served bases, it was found that mutagenesis of U8

strongly interfered with the cleavage by the tRNAsplicing endonucleases from yeast (Reyes &Abelson, 1988) as well as from Xenopus (Mattociaet al., 1988). Although these mutations might affectidentity elements required for enzyme–tRNArecognition, it seems likely that the effect on tRNAsplicing is also a consequence of changes in tRNAstructure.

Are the tRNA modifying enzymes ensuringcorrect folding of the tRNA?

The modified nucleosides might be formed onspecific intermediates during the processing of thepre-tRNA in eukaryotic cells (reviewed in Hopper& Martin, 1992). For example, analysis of thenucleoside content in various precursor tRNAPhe

and pre-tRNATyr, which accumulate at the nonper-missive temperature in a yeast temperature-sensi-tive mutant, reveal the presence of few modifiednucleosides that occur at early stages of the tRNAmaturation process; among them are m7G46, m5C48,T54, C55 and m1A58 (Knapp et al., 1978). Similarly,expression of the intron-containing yeast tRNATyr

gene in X. laevis showed that C35, m5C49, T54, C55 andm1A58 were already formed in the unprocessed andunspliced primary transcript, before several othermodified nucleosides like m2G26, Q34, manQ34 or C40

(Nishikura & de Robertis, 1981). Interestingly, themodified nucleosides appearing first in tRNAsmostly correspond to the tRNA modifying enzymesof group I (see above), while those appearing laterduring tRNA maturation essentially correspond toenzymes of groups IIa and IIb. This correlation isalso valid for the early formation of C34 and C36 inintron-containing yeast tRNAIle and for C35 inintron-containing yeast tRNATyr, for which it wasshown that the corresponding modifying enzymesdo not depend on the correct folding of the tRNAand even act on minimalist tRNAs (Szweykowska-Kulinska et al., 1994, and unpublished results). Itmay be that the untrimmed and unspliced primarytranscript is not fully correctly folded, or can foldinto unwanted, alternative tertiary structures. If so,an important function of the modifying enzymes ofthe group I acting in the T-loop might be to promoteand/or stabilize the folding of the precursor into acorrect tertiary structure, which can only then berecognized by the enzymes of group II. Thus, thetRNA processing pathway may be a tRNA foldingpathway, in which certain modifying enzymes(possibly of group I) play a crucial ‘‘chaperone-like’’role to ensure that only correctly and completelyfolded tRNAs are produced and hence used in thetranslational machinery. Such a role for the enzymecatalyzing the formation of m2

2G26 (classified as anenzyme of group II; see Figure 7) in eukaryotic andarchaean tRNAs has been recently proposed(Steinberg & Cedergren, 1995).

Partial modification of tRNA transcripts

Clearly, the different modified nucleosides in themicroinjected tRNA transcripts are formed withvarying efficiency. It could be due, in part, to theheterologous nature of the experimental systemused. However, the yeast tRNAAsp has 74%homology with the X. laevis tRNAAsp (Haumontet al., 1984). Even small sequence or structuralvariations between tRNAAsp from yeast andtRNAAsp from X. laevis might prevent efficientformation of some modified nucleosides. Thisseems to be the case for C13 and possibly also forC40. Indeed, the formation of both C13 and C40 isconsiderably enhanced when the A15-U48 Levittbase-pair is replaced by G15-C48, as in wild-typeXenopus tRNAAsp (tRNA-3 in Table 3 and Figure 6).Likewise, alteration of the 3D-structure of yeasttRNAAsp facilitates the formation of m5C49, T54, C55

and m1A58 in Xenopus oocytes (tRNA-2, -6 and -10to -13).

An alternative explanation might be found in thecellular localisation of the tRNA modifying en-zymes. In X. laevis, most, but not all, the tRNAmodifying reactions appear to occur in the nucleus,where the corresponding modification enzymes are

tRNA Architecture and Formation of Modified Nucleosides80

obviously present (Nishikura & de Robertis, 1981;Martin & Hopper, 1994). This does not exclude afraction of the tRNA-modifying enzymes also beingpresent, although probably in low amounts, in thecytoplasm, into which the tRNA transcripts weremicroinjected. This is certainly the case for theenzyme catalyzing the formation of i6A37 in severaltRNAs (Martin & Hopper, 1994). However, therelative amounts in the different compartments(nucleus, cytoplasm and, in some cases, mitochon-dria) may vary from one enzyme to another. Finally,certain modified nucleosides, like pseudouridine-35in yeast tRNATyr (Johnson & Abelson, 1983),pseudouridine-34 and pseudouridine-36 in yeastminor tRNAIle (Szweykowska-Kulinska et al., 1994;G. Simos, H. Tekotte, H. Grosjean, A. Segref,D. Tollervey & E. C. Hurt, unpublished results) orm5C34 in yeast tRNALeu (Strobel & Abelson, 1986)are formed only in intron-containing tRNAs. Hence,mature sized transcripts are not necessarily theoptimal substrates for some of the tRNA-modifyingenzymes.

Are some tRNA-modifying enzymes interactingalso with other RNAs?

In general, tRNA-modifying enzymes are be-lieved to specifically interact only at unique sites oftRNAs. Obviously, this is most likely to be true forthe set of enzymes that recognize and interact onlywith the intact and correctly folded L-shaped tRNA(enzymes of group IIa, as defined above). On theother hand, since modifying enzymes of group Iseem to need only very local elements in the tRNA,the possibility exists that they are also able tointeract at different sites of the tRNA molecule oreven with other types of RNAs, which also carrythe required structural identity elements. Someexperimental evidence supports this hypothesis.Firstly, while using brome mosaic virus RNA as asubstrate to isolate cap-specific methyl transferasesfrom HeLa cells, the tRNA-modifying enzymetRNA-(m5C)-methyltransferase (possibly the oneacting at position 48 and/or 49; enzymes of groupI) was unintentionally purified (Keith et al., 1980).The highest activity of the purified enzyme wasobtained with tRNA as its substrate. However,methyltransferase activity was also obtained withother types of RNAs, such as ribosomal RNA, viralRNAs from tobacco mosaic virus and brome mosaicvirus and vaccinia RNA, as well as with poly (A, C,G, U). Secondly, E. coli tRNA-(m5U54)-methyltrans-ferase, catalyzing the modification of U54 to m5U54

(also called T54), can be covalently attached to 16 SrRNA (Gustafsson & Bjork, 1993). This enzyme(also of group I) has been shown to modify E. coli16 S rRNA in vitro (Gu et al., 1994). However, aribothymidine has not been found in naturallyoccurring E. coli 16 S rRNA, while the E. coli 23 SrRNA has one such residue at position 747 (Fellner,1969; see also Wrzesinski et al., 1995). An RNAmodifying enzyme with a clear dual specificity hasbeen recently described (Wrzesinski et al., 1995;

reviewed in Ofengand et al., 1995). It is apseudouridylate synthase from E. coli that iscompletely specific for a single site (U746) in 23 SrRNA and equally specific for a single site (U32) intRNAPhe. It might not be a coincidence that therelatively inefficient formation of C32 in yeasttRNAAsp, as we report here, was also catalyzed bya similar type of dual specific pseudouridinesynthase in the Xenopus oocyte. This enzyme hasbeen classified as a tRNA modifying enzyme ofgroup I (Figure 7). To our knowledge, there is, atpresent, no information concerning the identityelements in tRNA needed for the other enzymes ofgroup I catalyzing formation of m2G and m1A inpositions 6 and 58, respectively, yet such modifiednucleosides also exist in RNAs other than tRNAs(reviewed in Limbach et al., 1994). It is therefore aninteresting possibility that these last tRNA-modify-ing enzymes are also involved in biologicallyrelevant interactions with RNA molecules otherthan tRNAs.

Analogies with other enzymes that interactwith tRNAs

We have shown that the 3D structure of the tRNAplays a major role in the formation of several, butnot all, modified nucleosides in tRNA. From thispoint of view, the modifying enzymes show thesame versatility as the aminoacyl-tRNA synthetasesor elongation factors in their tRNA recognition.While elongation factors recognize only the first tenbase-pairs of the amino acid accepting domain I(Rudinger et al., 1994; Clark et al., 1995), thesynthetases interact with more extended RNAdomains. Within their family (class I and class II),one finds members recognizing only part of thetRNA molecule (e.g. alanyl-, leucyl- or seryl-tRNAsynthetases), while other synthetases recognizesignals distributed over several domains of thetRNA architecture (e.g. aspartyl- or phenylalanyl-tRNA synthetases; see Martinis & Schimmel, 1995;McClain, 1995; Cavarelli & Moras, 1995). As faras the 3' end of tRNA is concerned, the tRNAsynthetases of class I clearly interact with the minorgroove of the acceptor stem (domain I), while thetRNA synthetases of class II approach tRNA fromthe major groove side of the acceptor stem (Moras,1992).

Thus, it becomes important to search for theidentity elements responsible for the specificities ofeach of the individual tRNA-modifying enzymesand to find out whether the same chemical rationaleis used as for the tRNA aminoacylation identities.For example, in addition to the dependence ontRNA architecture, as stressed above, the E. colitRNA (m1G37) methyltransferase recognizes two Gresidues at positions 36 and 37 (Holmes et al., 1992),while for the enzyme of the yeast or X. laevis oocyte,no such dependence on G37 was observed (thiswork). On the contrary, E. coli and XenopustRNA-guanine transglycosylase recognize the U33-G34-U35 sequence in a seven-base anticodon loop

tRNA Architecture and Formation of Modified Nucleosides 81

(Carbon et al., 1983; Nakanishi et al., 1994; Curnow& Garcia, 1995), but only the enzyme from X. laevisrequires, in addition, a correct folding of the tRNAsubstrate (this work). Despite the fact that yeastcontains methyltransferases able to catalyze theformation of m2G26 and m1A58, naturally occurringyeast tRNAAsp does not contain such base modifi-cations. Nevertheless, when the yeast tRNAAsp wasmicroinjected into the cytoplasm of X. laevis oocyte,it became a fairly good substrate for both the oocytetRNA(m2G26)- and tRNA(m1A58)-methyltransfer-ases. The same observation was made for E. colitRNAfMet, which does not naturally contain t6A37,while after microinjection into the cytoplasm of X.laevis oocytes, its A37 became fully modified intot6A37 (Grosjean et al., 1986, 1987). Clearly, the ‘‘rules’’for tRNA recognition by selected tRNA modifyingenzymes in different organisms may not be thesame, a situation which prevails also for theaminoacyl-tRNA synthetases (discussed in Giegeet al., 1993). Thus, for each tRNA modifyingenzyme, it will be important to verify whether theirspecificity is brought about by only a small numberof determinants or if other recognition strategies areused. From an evolutionary point of view, this mayhelp to understand how the different types oftRNA-modifying enzymes have emerged and to seewhether this correlates with the evolution of thetRNA aminoacylation systems, which most likelyoriginated as simplified minimalist versions andlater evolved into the more elaborate present daymodular protein/RNA systems (Schimmel et al.,1993).

Epilogue

The introduction of a yeast tRNA transcript intothe cytoplasm of living oocytes may not appear tobe an ideal method for the study of how yeast oroocyte tRNA normally matures. Nevertheless, itis clear that the various mutated yeast tRNAtranscripts provide a very useful probe for therecognition properties of the oocyte enzymes, astudy that would otherwise have been difficult toperform in vitro where the optimal conditions forthe many different modification enzymes aredifficult to reproduce. By using our in vivo system,we were able to draw general conclusions on therelative sensitivity of various modifying enzymestowards structural disorders in a tRNA frameworkthat is conserved among all biological species. Ourresults and conclusions now provide a basis formore selected in vitro tests, using truly homologoussystems with either homologous Xenopus tRNAs,for which the precise 3D structures first have to beestablished, or with yeast modifying enzymes.

Materials and Methods

tRNA transcripts

All tRNA molecules studied in this work were derivedfrom yeast tRNAAsp of known nucleotide sequence

(Gangloff et al., 1971). They were produced by in vitrotranscription of synthetic genes by bacteriophage T7 RNApolymerase as described in Perret et al. (1990). To improvethe yield of the transcription reactions, the original U1-A72

base-pair in the amino acceptor stem was changed toG1-C72 in all tRNAAsp variants. In this way, thetranscription yield was increased about fivefold (about500 tRNA molecules were produced per tRNA gene).

The construction of tRNAAsp variants was done bysite-directed mutagenesis on single-stranded DNA asdescribed by Sayers et al. (1988). Mutations wereconfirmed with dideoxy sequencing of plasmid DNAwith T7 DNA polymerase. DNA-sequencing and alsolarge-scale and small-scale plasmid preparations weredone according to standard procedures (Sambrook et al.,1989). Before transcription, plasmids carrying tRNAgenes were linearized with restriction enzyme Mva1 orBstN1, according to the recommendation of the enzymesuppliers. Linearization was confirmed by gel electro-phoresis in 1% (w/v) agarose. Transcription of minimalisttRNAs was done on synthetic DNA templates with adouble-stranded promoter region of 17 base-pairs and asingle-stranded overhang corresponding to the tran-scribed region (Milligan & Uhlenbeck, 1989). Whenlabelling transcripts with [a-32P]GTP, 20 ml of [a-32P]GTP(10 mCi/ml, 400 Ci/mmol) was lyophilized with 15 mlof 100 mM unlabelled GTP before the start of thetranscription reaction. In the case of labelling with[a-32P]ATP, [a-32P]UTP or [a-32P]CTP, 10 ml of theradiolabelled nucleotide (10 mCi/ml, 400 Ci/mmol) waslyophilized together with 5 ml of 100 mM of thecorresponding unlabelled nucleotide. Transcription reac-tions and gel purification of full length transcripts weredone as previously described (Perret et al., 1990; Edqvistet al., 1992). Purified transcripts were dissolved in 5 ml ofwater and kept refrigerated at minus 20°C until used.

Oocyte microinjections

The fully matured oocytes (stages five and six) wereobtained from Xenopus ovaries. They were separatedmanually with watchmaker’s forceps (not with collagen-ase) and kept at 20°C in salt solution as described in Smithet al. (1991). The 32P-labelled tRNA transcripts (40 to50 nl/oocyte, usually at 1000 Cerenkov counts per million(cpm)/nl) were then injected into the cytoplasm ofX. laevis oocytes, as described earlier (Grosjean & Kubli,1986). The amount of injected tRNA transcript rangedbetween 0.2 and 1.0 fmol of foreign tRNA/oocyte.Incubation was at 19°C in salt solution, as described inOpresko (1991). After 3 to 48 hours of incubation, samplesof 10 to 15 ‘‘good looking’’ oocytes (with no white spoton the dark pole) were withdrawn and frozen in liquidnitrogen. The samples were stored at minus 80°C until thenucleic acids were extracted and purified by polyacryl-amide gel electrophoresis as described in Grosjean et al.(1990). Only full-length tRNAAsp recovered from suchfractionation (see Figure 2) was further processed asdescribed below.

Analysis of modified nucleotides

The tRNAs from the X. laevis oocytes, obtained afterelectrophoresis, were completely hydrolyzed either into5'-mononucleotides with nuclease P1, or into 3'-mononu-cleotides with RNase T2, as described previously (Edqvistet al., 1992). The hydrolyzates (usually 3000 to 5000 cpm,as counted with a Geiger-Muller counter) were loaded

tRNA Architecture and Formation of Modified Nucleosides82

onto t.l.c. cellulose plates (0.1 mm cellulose, 10 × 10 cm2)together with nucleotide markers. The chromatographswere done in two dimensions, the first one being inisobutyric acid:NH4OH:water (66:1:33, by vol.) and thesecond one either in isopropanol:HCl:water (68:17.6:14.4,by vol.; system 1) or in 0.1 M Na2HPO4-NaH2PO4 buffer(pH 6.8):(NH4)2SO4:n-propanol (100:60:2, v:w:v; system2) (Nishimura, 1979; Silberklang et al., 1979). The pos-itions of the nucleotide markers were examined under UVlight. The radioactivity associated with modified nu-cleosides, as determined after autoradiography of the t.l.cplates (usually overnight at minus 80°C, using anintensifier screen), was determined by liquid scintillationtechniques or by the use of a phosphor imager (MolecularDynamics, USA) supplied with the Image Quantsoftware. These conditions were adequate for effectiveseparation and identification of nucleoside monophos-phates (both the canonical and modified ones) asindividual spots on the t.l.c. in at least one of the twochromatographic solvent systems, except for dihy-drouridine residues which require separation on20 × 20 cm2 cellulose plates to properly resolve them fromUMP. Such time consuming analyses were done only forthe wild-type yeast tRNAAsp; therefore, we do not havecomplete data about the influence of the tRNA tertiarystructure on the formation of D17 and D20. Identificationof the modified nucleosides were done by comparisonwith reference maps (Keith, 1995).

Enzymes, chemicals, and biological material

[a-32P]Radiolabelled nucleoside triphosphates and theOligo-directed in vitro mutagenesis system, version 2.1were from Amersham, UK. Nuclease P1 and RNase T2

were from Sigma (St Louis, USA). Restriction enzymeswere from Boehringer-Mannheim (Germany) or NewEngland Biolabs (Beverly, MA, USA). Bacteriophage T7

RNA polymerase was from New England Biolabs orPromega (USA). Thin layer cellulose plates were fromSchleicher & Schuell (Dassel, Germany). All otherchemicals were from Merck Biochemicals (Germany).Adult female frogs were from South Africa andpurchased in France through STAACEL in MontpellierCedex (BP 5051).

AcknowledgementsWe thank C. Florentz and J. Puglisi in Strasbourg,

D. Foiret in Gif-sur-Yvette and S. Holma in Umea forplasmid preparations. We acknowledge K. Blomqvist andM. Holmes for critical reading of the manuscript. J.E. wassupported by EMBO short term fellowships and by atravel fellowship from the Swedish Natural ScienceResearch Council (NFR) for several visits to Strasbourgand Gif-sur-Yvette. This work was supported by CNRS(R. G. & H. G.), Actions de Recherche Concertees(H. G., 1993 to 1996), Ministere de la Recherche et del’Enseignement Superieur (R. G.), Universite Louis Pas-teur, Strasbourg (R.G., and a Visiting Professor fellowshipto H. G.) and grant 2407 from NFR to K.B.S. in Sweden.

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Edited by A. R. Fersht

(Received 19 June 1995; accepted 6 October 1995)