ABSTRACT
The protein products of two yeast Saccharomyces cerevisiae genes (YNL292w and CBF5) display a remarkable sequence homology with Escherichia coli tRNA:pseudouridine-55 synthase (encoded by gene truB). The gene YNL292w coding for one of these proteins was cloned in an E.coli expression vector downstream of a His6-tag. The resulting recombinant protein (Pus4) was expressed at high level and purified to homogeneity by metal affinity chromatography on Ni2+-NTA-agarose, followed by ion-exchange chromatography on MonoQ. The purified Pus4p catalyzes the formation of pseudouridine-55 in T7 in vitro transcripts of several yeast tRNA genes. In contrast to the known yeast pseudouridine synthase (Pus1) of broad specificity, no other uridines in tRNA molecules are modified by the cloned recombinant tRNA:[Psi]55 synthase. The disruption of the corresponding gene YNL292w in yeast, which has no significant effect on the growth of yeast cells, leads to the complete disappearance of the [Psi]55 formation activity in a cell-free extract. These results allow the formal identification of the protein encoded by the yeast ORF YNL292w as the only enzyme responsible for the formation of [Psi]55 which is almost universally conserved in tRNAs. The substrate specificity of the purified YNL292w-encoded recombinant protein was shown to be similar to that of the native protein present in yeast cell extract. Chemical mapping of pseudouridine residues in both cytoplasmic and mitochondrial tRNAs from the yeast strain carrying the disrupted gene reveals that the same gene product is responsible for [Psi]55 formation in tRNAs of both cellular compartments.
Due to the success of large-scale genome sequencing projects (1 -4 ), researchers are faced by the daunting task of having to assign biological functions to an ever-increasing number of newly identified (putative) proteins. One logical approach to this task is the systematic analysis of sequence homology between a novel polypeptide and protein(s) whose function has already been established experimentally. In many cases a novel polypeptide could be identified as a potential member of a functionally distinct protein family (5 ,6 ), thus providing guidance to experimental testing. Such a homology-based approach was recently employed to analyze the family of RNA:pseudouridine synthases with different substrate specificity (7 ) and for an iterative search for new RNA-modifying enzymes (RNA/DNA:methyltransferases and RNA:pseudouridine synthases) (8 ,9 ). As a result, 13 new genes encoding putative RNA-modification enzymes were identified in Escherichia coli. The activity and substrate specificity of some of the enzymes encoded by these genes were subsequently confirmed experimentally (8 ,10 ).
Four distinct families of homologous RNA:pseudouridine synthases were found based on homology with the four known E.coli enzymes designated truA (also known as HisT), truB, rluA and rsuA. The members of the first two groups specifically introduce [Psi] in tRNA at positions 38, 39 and 40 (truA, `region-specificity') (11 ) and position 55 (truB, `site-specificity') (12 ), respectively. The enzyme encoded by the rluA gene catalyzes the formation of [Psi] at position 32 of tRNA and at position 746 of E.coli 23S rRNA (`dual-specificity') (13 ). The last group (rsuA) appears to be specific to a unique [Psi]516 in E.coli 16S rRNA complexed with 30S ribosomal proteins (14 ,15 ).
While the truB gene encoding tRNA:[Psi]55 synthase is unique in the E.coli genome, the Saccharomyces cerevisiae genome contains two genes, CBF5 (16 ) and YNL292w (17 ) [SwissProt protein database entry ORF YNL292w (locus YN32_YEAST) accession number P48567, or Z71568 (EMBL protein database) was previously refered to as Ynl0480 (GeneBank accession number U23084) by Koonin (7 )], that display significant sequence homology with truB. A similar situation exists for the tRNA:[Psi]38/39/40 synthase family, represented by a unique truA gene in prokaryotes (E.coli, Bacillus subtilis....), of which at least three homologs have been detected in yeast [PUS1, PUS2 (18 ) and DEG1 (7 ,18 ,19 )]. However, the degree of homology between the prokaryotic and yeast genes in this case is lower than in the case of the truB family.
Despite the sequence homology between the yeast PUS/DEG genes and E.coli truA, the substrate specificity of the various enzymes differs. Only the Deg1p (Pus3p) protein, which has the highest degree of homology with its prokaryotic counterpart, catalyzes pseudouridine formation at the same positions in tRNA (38/39) as the E.coli enzyme (20 ). Pus1p causes [Psi] to be formed at positions 27 and 34/36 in intron-containing yeast tRNAs (18 ), while the specificity of the Pus2p enzyme remains to be established. These findings demonstrate that sequence homology can provide useful guidance in functional characterization of novel proteins but experimental verification remains necessary.
In this paper we present experimental evidence showing that the protein encoded by the yeast YNL292w gene is tRNA:[Psi]55 synthase (Pus4). We have, therefore, designated this gene PUS4.
[alpha]-32P-radiolabelled nucleotide triphosphates (400 Ci/mmol) were from Amersham (UK). Tris, DTT, nucleoside triphosphates, Penicillium citricum nuclease P1, Aspergillus oryzae RNAse T2, phenylmethylsulfonylfluoride (PMSF) and spermidine were from Sigma (USA), diisopropylfluorophosphate (DFP) from Boehringer-Mannheim (Germany), 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide metho-p-toluenesulfonate (CMCT) from Aldrich (USA), bacteriophage T7 RNA polymerase, restriction enzymes and isopropyl-[beta]-d-thiogalactoside (IPTG) from MBI Fermentas (Vilnius, Lithuania), RNasin and AMV reverse transcriptase from Promega (USA). Thin layer cellulose plates and nitrocellulose membranes BA85 were from Schleicher & Schuell (Germany), all other chemicals from Merck Biochemicals (Germany). Chemically synthesized deoxyoligonucleotides were purchased from MWG-Biotech (Germany). They were used without further purification.
The plasmids bearing various tRNA genes under the T7-promoter of wild-type yeast tRNAPhe (GAA) (p67YF0) and its mutant PheY67 (p67YF92), bearing the mutations G30C, C40G, A31C and U39G in the anticodon stem (21 ,22 ), mutant yeast tRNAAsp (mutations U1G and A72C; 23 ), tRNAAsp with deleted D-stem-loop (24 ) and T[Psi]-stem-loop minihelices (25 ,26 ), yeast pre-tRNAIle (27 ) and yeast tRNAVal(CAU) (28 ) were described elsewhere. Yeast tRNATrp(CUA) was recloned by PCR amplification using the corresponding mutant genes (29 ,30 ) and inserted in pUC118 vector. The plasmids with the cloned domains of yeast ribosomal 25S RNA [domain 2, nucleotides 650-1453 (5'-nt = 1)] and domain 4, nucleotides 1869-2389, were provided by Prof. H.Raué (Vrije Universiteit, Amsterdam, The Netherlands).
T7-polymerase transcription of tRNA genes using [alpha]-32P-radiolabelled nucleotide triphosphates and purification of the resulting tRNA transcripts by urea gels were performed as described previously (31 ).
The gene YNL292w (initially designated YNL0480; 17 ) was disrupted in strain FY1679 (Mata/[alpha]; ura3-52/ura3-52; leu2[Delta]1/+; trp1[Delta]63/+; his3[Delta]200/+) by replacing the coding region with kanMX4 by PCR-targeting of the kanMX marker cassette flanked by short regions homologous to the target locus (32 ). The marker cassette (in plasmid pFA6a-kanMX4; 32 ) is a hybrid of the coding sequence of the kanr gene and transcriptional and translational control sequences from the TEF gene of the filamentous fungus Ashbya gossypii, and confers geneticin (G418) resistance on yeast.
The haploid strain FY73 (Mat[alpha]) with the deleted YNL292w gene was obtained by tetrad analysis of the heterozygous deletant. The strains were grown in YPD containing 200 µg/ml of geneticin.
The gene YNL292w (17 ) was amplified from the pBluescript plasmid carrying this gene on a 4 kb EcoRI-generated fragment of yeast genomic DNA using two oligonucleotides, corresponding to the 5'-end (AAAACATATGaatggaatatttgctattg) and complementary to the 3'-end (AAAAAAGCTTttacacctgttcgatttttct) of the gene, respectively. The sequence of the part of the oligonucleotides corresponding to the gene YNL292w is shown in lower-case. The 5'-oligonucleotide contained the restriction site for NdeI (underlined), while the 3' sequence contained a site for HindIII (underlined). The amplified PCR product was cleaved by the two restriction enzymes and inserted into the NdeI/HindIII sites of pET28b-plasmid downstream to the T7-promoter (Stratagene, USA). The sequence of the insert was verified by DNA sequencing using the set of synthetic primers complementary to the gene YNL292w.
Expression of YNL292w-encoded recombinant protein in transformed E.coli BL21(DE3), bearing the T7 RNA-polymerase gene under control of the lac-UV5 promoter (33 ), was performed by IPTG induction in MM9 medium. Escherichia coli cells were grown at 37oC to an OD600nm of ~0.7 and expression was induced by IPTG (1 mM) at 37oC for 2 h. All subsequent operations were performed at 4oC. Cells were disrupted by sonication (20 times 10 s each) in twice their weight of buffer A (50 mM potassium phosphate, pH 8.0, 200 mM NaCl, 10 mM [beta]-mercaptoethanol, 0.1% Triton) plus 1 mM of PMSF and DFP. The homogenate was centrifuged at 10 000 g for 10 min. The resulting S10 was further fractionated into ribosomal pellet and supernatant S100 by centrifugation at 80 000 r.p.m. in a TL-100 ultracentrifuge (Beckman) (rotor TLA100.3) for 2 h. The supernatant was then directly applied onto a Ni2+-NTA-agarose column (Qiagen, Germany) and the column was washed with buffer A without Triton, then with buffer A containing 20 mM imidazol. The recombinant protein was eluted with the same buffer A but containing 150 mM imidazol. Further purification to homogeneity was performed at room temperature by ion-exchange chromatography on MonoQ HR5/5 (Pharmacia, Sweden) in 25 mM Tris-HCl buffer, pH 8.8, 2 mM [beta]-mercaptoethanol, 10% glycerol, 1 mM MgCl2, 2 mM EDTA (buffer B). Elution was performed by a linear gradient of NaCl from 0 to 0.3 M in buffer B in a total volume of 30 ml.
The activity of yeast extracts (0.6 mg/ml) and of purified enzymatic fractions (0.1 µg/ml) was tested at 30oC (37oC for E.coli extract) in 100 mM Tris-HCl, pH 8.0, 100 mM ammonium acetate, 5 mM MgCl2, 2 mM DTT, 0.1 mM EDTA and 1-2 fmol of 32P-radiolabelled T7-runoff transcripts as substrate. The reaction was terminated by phenol-chloroform extraction and RNA samples were treated as described previously (31 ). Digestion by nuclease P1 and RNAse T2, two-dimensional thin-layer chromatography on cellulose plates and identification of modified nucleotides were performed according to previously published procedures (34 ). Radioactivity in the spots of modified 3'- and 5'-mononucleotides was quantified by means of a PhosphorImager (Molecular Dynamics, USA) using ImageQuant software.
Electrophoresis under denaturing conditions was performed according to Laemmli (35 ). The separated proteins were electrophoretically transferred onto nitrocellulose membranes BA85. Blotting was performed in 25 mM Tris-192 mM glycine buffer for 1 h at 100 V. For immunochemical detection the membrane was saturated by blocking agent (low fat milk) and incubated with specific anti-His6-tag rabbit antibodies (His-probe G18, Santa Cruz Biotechnology, USA) followed by incubation with the anti-rabbit IgG-alkaline phosphatase conjugate (Sigma, USA). Activity of alkaline phosphatase was revealed using color substrates 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitro blue tetrazolium (Sigma, USA).
Localization of pseudouridine residues in tRNA was performed as described (36 ,37 ) with the following modifications: 10 µg of total tRNA extracted from wild-type or mutant yeast strain was treated by 0.17 M 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide metho-p-toluenesulfonate (CMCT) in bicine-urea buffer pH 7.5 for 5 min at 42oC. Reverse transcription was done at 42oC for 30 min using 5'-labelled synthetic primer and CMCT-modified tRNA (1 µg when analyzing cytoplasmic tRNA or 3 µg for mitochondrial tRNA). Reverse transcription products were separated on 15% denaturing PAGE and radioautographed overnight using X-OMAT Kodak film.
Analysis of protein sequences for the presence of cellular targeting signals was performed using PSORT software (38 ) (available at http://psort.nibb.ac.jp/).
Sequence analysis of a 30 kb DNA segment from yeast chromosome XIV (17 ) revealed the presence of an open reading frame (ORF N0480, later designed YNL292w) having a high degree of sequence homology with the E.coli truB gene encoding tRNA:[Psi]55 synthase (12 ). The yeast gene was amplified by means of PCR and cloned into an E.coli expression vector under control of the phage T7 promoter as described in Materials and Methods. The resulting construct encodes the YNL292w protein preceded by 20 amino acids (MGSSHHHHHH SSGLVPRGSH) including a His6-tag and a thrombin site (underlined) sequences.
Expression of the tagged, recombinant protein was achieved by induction of the T7 RNA polymerase gene in E.coli BL21(DE3) transformants using IPTG. Figure 1 A shows a separation of total cellular proteins from non-induced and IPTG-induced E.coli cells on SDS-PAGE. A major protein band with apparent molecular mass of 55 kDa is visible in the extract from the induced cells. This value is in good agreement with the calculated molecular mass of the protein encoded by YNL292w (47.4 kDa, 423 amino acids including the His6-tag and cleavage signal). The recombinant polypeptide remained soluble and could be efficiently purified from an S100 extract by metal-affinity chromatography on Ni2+-NTA-agarose. This simple, one-step procedure led to a protein that was ~95% pure (Fig. 1 B, lane 5). The yield was ~3-4 mg/l of induced E.coli culture.
On the basis of its sequence homology with E.coli truB, the product of the yeast YNL292w gene is expected to catalyze the formation of [Psi]55 in yeast tRNA. We measured pseudouridine synthase activity using a synthetic wild-type yeast tRNAAsp and a mutant form of yeast tRNAPhe (PheY67) as the substrates. This tRNAPhe variant carries mutations at positions 31 and 39 and hence can only be pseudouridylated at position 55. Incubation of these tRNA transcripts with extracts from either yeast or untransformed E.coli cells in both cases led to the efficient formation of a single [Psi] residue at position 55 as detected by thin layer chromatography of an RNase T2 or nuclease P1 digests (Fig. 2 A and B).
In vitro studies using S100 yeast extract have shown that yeast tRNA:[Psi]55 synthase catalyzes the isomerization of U55 in a large variety of tRNA substrates, including several truncated tRNA molecules (26 ). We have tested the substrate specificity of the recombinant Pus4p protein using four different yeast tRNAAsp variants (Fig. 4 A): tRNAAsp carrying a deletion of the D-stem-loop, T[Psi]-stem-loop minihelices bearing the amino acid acceptor branch or with shortened T[Psi]-stem-loop minihelices having 3 or 2 bp in the stem, respectively. As shown in Figure 4 B, recombinant Pus4p is able to modify three of these substrates at 30oC but fails to recognize the substrate with the 2 bp long T[Psi]-stem-loop. However the reaction rate for the minisubstrates correlates with the length of the stem-loop. These results fully agree with those obtained for the tRNA:[Psi]55 synthase activity present in yeast S100 extracts where we tested 43 yeast tRNAAsp variants as substrates (26 ).
We also tested recombinant Pus4p for a possible`dual-specificity' function by incubation with synthetic transcripts corresponding to domains II and IV of yeast 25S rRNA. In natural 25S rRNA each of these domains contains 11 [Psi] residues (39 ). Neither transcript was found to be modified, indicating that Pus4p is not involved in pseudouridylation of these domains of 25S rRNA.
Previous experiments have shown that in several instances a single nuclear gene is responsible for modification of both cytoplasmic and mitochondrial tRNA (40 -42 ). In order to determine whether a similar situation applies to the case of yeast tRNA:[Psi]55 synthase, we studied the effects of disrupting the PUS4 gene. Haploid yeast cells (FY73) carrying this disruption proved to be viable and to grow normally and, thus, can be assayed for the presence of tRNA:[Psi] synthase activity.
S100 extracts prepared from this disrupted strain were tested using an array of synthetic tRNA transcripts as described previously (18 ). These transcripts correspond to variants of yeast tRNAAsp, tRNATrp, tRNAVal and tRNAPhe (mutant PheY67). Altogether, nine potential sites in tRNA molecules were checked (positions 13, 26/27/28, 32, 34/36, 39 and 55) among 15 possible ones in yeast tRNAs. The results indicate that only the activity for pseudouridine-55 formation in tRNA is lacking in the extract from the disrupted strain, while the activities of other pseudouridine synthases remain unaffected. Time courses of [Psi]55 formation in yeast tRNAPhe mutant (PheY67) in presence of S100 yeast extract containing or not Pus4p are shown in Figure 5 . These results indicate that the disruption of the PUS4 gene abolishes only the isomerization of U55 and leaves the enzymes responsible for modification at the remaining positions intact. Therefore, we conclude that the PUS4 gene indeed encodes the only cytoplasmic tRNA-[Psi]55 synthase present in S.cerevisiae.
Our results clearly identify the product of the S.cerevisiae YNL292w (PUS4) gene as the enzyme that catalyzes the formation of [Psi]55 in tRNA molecules (tRNA:[Psi]55 synthase). The recombinant enzyme expressed in E.coli is active in vitro and displays the same substrate specificity towards minisubstrates as previously observed for the natural enzyme present in yeast extract (26 ). The results obtained with disrupted yeast strain allow to conclude that the gene YNL292w is one only gene for tRNA:[Psi]55 synthase in yeast.
Pus4p has a high degree of sequence homology with its functional counterpart from E.coli encoded by the truB gene, which is in fact able to modify yeast tRNA (Fig. 3 ). A second yeast protein, CBF5, showing also a high homology to the E.coli truB product has been identified (7 ,16 ). This protein was studied extensively by genetic approach (43 ) and shown to bind in vitro to yeast centromeres and microtubules (16 ). Genetic depletion of CBF5 causes arrests of cell division in G1/S phase (16 ). In contrast, cells carrying a disruption of the PUS4 gene remain viable, appear to grow normally and have no specific phenotype under standard conditions, although they do not contain any detectable tRNA:[Psi]55 synthase activity (Fig. 5 ). Our data also suggest that only one yeast homologue of truB (YNL292w) is implicated in the synthesis of pseudouridine residue at position 55 in all cellular tRNA molecules. Thus, CBF5, while expected to show pseudouridylation activity, must have a substrate specificity different from that of Pus4p. Experiments performed in the laboratory of Dr D.Tollervey (University of Edinburgh) have provided evidence that CBF5 may be involved in snoRNA-guided formation of most (if not all) [Psi] residues present in yeast ribosomal RNA (44 ), a process taking place in the nucleolus (45 ,46 ; reviewed in 47 ).
Our results obtained in vitro and in vivo with the strain carrying a disruption of the PUS4 gene allow the firm conclusion that the product of the PUS4 gene is the only tRNA:[Psi]55 synthase present in yeast and catalyzes [Psi]55 formation in both cytoplasmic and mitochondrial tRNA. As the enzyme is the fourth one identified that catalyzes [Psi] formation in yeast tRNA we have named it Pus4p.
This work was supported by research grants from CNRS and ARC to H.G. Salary to Y.M. was provided by an operating grant of the Medical Research Council of Canada (Grant MRC-MT-1226) awarded to Dr B.Lane, University of Toronto, Canada. The plasmids containing various tRNA genes were kindly provided by Drs O.Uhlenbeck (Boulder, CO, USA), R.Giegé and F.Fasiolo (IBMC, Strasbourg, France), J.Bell (University of Alberta, Canada) and Z.Sweykowska-Kulinska (University of Poznan, Poland). The plasmids containing 25S rRNA domains were kindly provided by Dr H.A.Raué (Vrije Universiteit, Amsterdam, The Netherlands). We thank Miss C.P.A.M.Kolen (VU, Amsterdam) for skillful technical assistance, and Drs H.A.Raué and J-P.Waller (CNRS, Gif-sur-Yvette) for critical reading of the manuscript and useful comments.
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