Pseudouridylation at Position 32 of Mitochondrial and Cytoplasmic tRNAs Requires Two Distinct Enzymes in Saccharomyces cerevisiae*

Cytoplasmic and mitochondrial tRNAs contain several pseudouridylation sites, and the tRNA:Ψ-synthase acting at position 32 had not been identified in Saccharomyces cerevisiae. By combining genetic and biochemical analyses, we demonstrate that two enzymes, Rib2/Pus8p and Pus9p, are required for Ψ32 formation in cytoplasmic and mitochondrial tRNAs, respectively. Pus9p acts mostly in mitochondria, and Rib2/Pus8p is strictly cytoplasmic. This is the first case reported so far of two distinct tRNA modification enzymes acting at the same position but present in two different compartments. This peculiarity may be the consequence of a gene fusion that occurred during yeast evolution. Indeed, Rib2/Pus8p displays two distinct catalytic activities involved in completely unrelated metabolism: its C-terminal domain has a DRAP-deaminase activity required for riboflavin biogenesis in the cytoplasm, whereas its N-terminal domain carries the tRNA:Ψ32-synthase activity. Pus9p has only a tRNA:Ψ32-synthase activity and contains a characteristic mitochondrial targeting sequence at its N terminus. These results are discussed in terms of RNA:Ψ-synthase evolution.

Pseudouridine (⌿) 1 and 2Ј-O-methylated nucleotides are the most abundant and universally found modified residues in RNAs of all types of organisms. Pseudouridine residues are formed post-transcriptionally by a group of enzymes called RNA:⌿-synthases (1,2). For a long time, the Escherichia coli RNA:⌿-synthases were the only ones to be identified (3)(4)(5).
Search for homologs of E. coli RNA:⌿-synthases in genomic data banks of Bacteria, Archaea, and Eukarya led to the identification of four families of RNA:⌿-synthases (families TruA, TruB, RluA, and RsuA) (2). Using this sequence homology approach, nine genes encoding putative RNA:⌿-synthases were identified in the Saccharomyces cerevisiae genome. Six of them correspond to the already characterized RNA:⌿-syn-thases (pseudouridine synthases Pus1p, Pus3p, Pus4p, Pus5p, Pus6p, and Cbf5p). Pus1p and Pus3p are TruA-related proteins. Pus1p is a multisite-specific RNA:⌿-synthase acting at seven different positions in various cytoplasmic tRNAs (positions 26,27,28,34,36,65, and 67), as well as at position 44 in U2 snRNA (6 -8). Pus3p catalyzes ⌿ formation at positions 38 and 39 in the anticodon stem-loop of several cytoplasmic and mitochondrial tRNAs (9). Like the E. coli TruB enzyme, the related yeast Pus4p is responsible for the formation of the highly conserved ⌿ residue at position 55 in both cytoplasmic and mitochondrial tRNAs (10). Probably as much as 40 ⌿ residues in yeast cytoplasmic rRNAs are formed by a unique enzyme of the TruB family, Cbf5p, that is guided by H/ACA small nucleolar RNAs (11,12). A unique ⌿ residue is present in yeast mitochondrial rRNA, and its formation is catalyzed by Pus5p, a member of the RluA family (13). Pus6p, another member of the RluA family, is responsible for ⌿ formation at position 31 in both cytoplasmic and mitochondrial tRNAs (14). Pus7p, which was not listed initially among the putative RNA: ⌿-synthases because of its divergent amino acid sequence, was identified as an U2 snRNA:⌿-synthase (15). We recently demonstrated that it acts at position 13 in cytoplasmic tRNAs and position 35 in the pre-tRNA Tyr (16).
Presently, only three of the putative RNA:⌿-synthases identified by Koonin (2) have no assigned RNA substrate: Pus2p, a TruA-related protein, and two proteins of the RluA family. They are encoded by the YDL036 and RIB2 (YOL066) ORFs, respectively. In addition to its potential RNA:⌿-synthase encoding capacity, the S. cerevisiae RIB2 gene was described a long time ago as encoding a DRAP-deaminase. This enzyme catalyzes the deamination of the 2,5-diamino-6-ribitylamino-4(3H)-pyrimidinone 5Ј-phosphate (DRAP) into 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione 5Ј-phosphate in the riboflavin biosynthesis pathway (17). The implication of the RIB2 gene in riboflavin biosynthesis was demonstrated by genetic approaches (18,19). However, the deaminase activity of Rib2p was never demonstrated directly. On the other hand, the activity and function of the putative RNA:⌿-synthase domain present in Rib2p remained obscure.
In yeast, only rRNAs, tRNAs, and UsnRNAs are known to be the substrates of RNA:⌿-synthases. The S. cerevisiae cytoplasmic tRNAs contain altogether 15 pseudouridylation sites, and 8 additional ones were detected in mitochondrial tRNAs (20) (Fig. 1). Several of these modification sites are common to cytoplasmic and mitochondrial tRNAs, and the RNA:⌿-synthases responsible for their formation have been identified (Fig. 1). Only the enzymes responsible for the formation of residue ⌿1 found in two cytoplasmic tRNAs (tRNA Arg (ACG) and tRNA Lys (UUU)), ⌿72 found in the mitochondrial tRNA Met i-(CAU), and ⌿32, a frequent modification, present in the anticodon loop of 14 cytoplasmic tRNAs and 7 mitochondrial tRNAs, have not been characterized yet. The presence of ⌿32 was shown to stabilize the three-dimensional structure of the anticodon loop of tRNA Gln by its implication in a water bridge interaction (21,22). It was thus important to look for the enzyme(s) responsible for ⌿32 formation in tRNAs. As far as the UsnRNAs are concerned, only the enzymes responsible for the formation of 2 of the 6 pseudouridines identified in these RNAs are known (8,15). The enzymes that catalyze the U to ⌿ conversion at positions 5 and 6 in U1 snRNA, 42 in U2 snRNA, and 99 in U5 snRNA remain to be characterized.
In this work, by use of strains with disrupted RIB2 and/or YDL036 ORF(s), and by production of the recombinant proteins, encoded by these two ORFs, we demonstrated that these two proteins, now renamed Rib2/Pus8p and Pus9p, respectively, are tRNA:⌿32-synthases. Rib2/Pus8p acts on cytoplasmic tRNAs, whereas Pus9p modifies the same position in mitochondrial tRNAs. Accordingly, the corresponding ORFs are renamed as RIB2/PUS8 and PUS9.
Complementation of the Disrupted Strains-To generate S. cerevisiae plasmids expressing proteins encoded by PUS9 and RIB2/PUS8 ORFs in a functional form, these ORFs were amplified by PCR from the genomic DNA of the S. cerevisiae BMA64 strain using oligonucleotides that generated NheI and HindIII restriction sites at the N and C termini, respectively. The amplified DNA fragments were subcloned at the SmaI site of plasmid pUC18. After sequencing, the resulting constructs (pUC18-PUS9 and pUC18-RIB2/PUS8) were cleaved by the NheI and HindIII restriction endonucleases, and the fragments containing the complete ORFs were inserted, downstream from the GalS promoter, between the XbaI and HindIII sites of plasmid p416GalS (24). Several mutants of these constructions were prepared: point mutations in the regions coding the putative RNA:⌿-synthase active site of both ORFs were introduced by PCR-mediated site-directed mutagenesis (D238A, GAT 3 GCT for PUS9 and D211A, GAC 3 GCC for RIB2/ PUS8). By application of the megaprimer approach to plasmid pUC18-RIB2/PUS8, we also produced genes encoding N-terminally (amino acids 1-448) or C-terminally (amino acids 450 -591) truncated Rib2/ Pus8p. The truncated genes were PCR-amplified, cut by the NheI and BamHI restriction endonucleases, and inserted into plasmid p416GalS to generate the p416GalS-RIB2/PUS8⌬PUS8 and the p416GalS-RIB2/ PUS8⌬DRAP plasmids. For PCR amplification of the C-terminally truncated RIB2/PUS8 gene, we used an oligonucleotide that created a UAA STOP codon. Deletion of the mitochondrial targeting sequence in the PUS9 ORF (amino acids 8 -30) was done by PCR amplification. Yeast transformations were performed by the standard lithium acetate procedure (25).
Recombinant Proteins-Plasmids pET28-PUS9 and pET28-RIB2/ PUS8 were built for overexpression of the recombinant His 6 -Pus9p and His 6 -Rib2/Pus8p proteins in E. coli. To this end, the NheI-HindIII fragment of plasmid pUC18-PUS9 or the NheI-BamHI fragment of pUC18-RIB2/PUS8 was inserted into plasmid pET28b (Novagen), that was cut by the same enzyme pairs. Point mutations in the region coding the putative RNA:⌿-synthase active site of both ORFs were introduced as described above (D238A, GAT to GCT for PUS9 and D211A, GAC to GCC for RIB2/PUS8). The resulting constructs were used to transform E. coli BL21CodonPlus(DE3)RIL cells (Stratagene). Recombinant His 6 -Pus9p and His 6 -Rib2/Pus8p were overproduced in cells grown at 18°C in LB medium containing 50 g/ml kanamycin by using an isopropyl 1-thio-␤-D-galactopyranoside concentration of 50 M for the induction. The recombinant proteins were purified from E. coli cell extracts by adsorption chromatography on nickel-nitrilotriacetic acid-agarose (Qiagen, France), as described earlier (7,26). The protein fractions eluted by 250 mM imidazole were diluted twice with 87% of glycerol and stored at Ϫ20°C.
In Vitro Tests-Cell-free S10 extracts from yeast were prepared as described previously (27). The activities of RNA:⌿-synthases acting at positions 1, 13, 32, and 72 in tRNAs were tested using T7 transcripts of the yeast tRNA Arg (anticodon ACG), tRNA Asp (anticodon GUC), and tRNA Met i(anticodon CAU), respectively (16). In vitro tests of the tRNA: ⌿32-synthase activities of the recombinant His 6 -Pus9p and His 6 -Rib2/ Pus8p were performed on yeast tRNA His (anticodon GUG) and tRNA Asp (anticodon GUC) T7 transcripts, or on total RNA extracted from the mutated strains. The plasmids, pTFM-Asp and pTFM-His, used for in vitro transcription of tRNA Asp and tRNA His were kindly provided by C. Florentz (Institut de Biologie Moléculaire et Cellulaire, Strasbourg, France). For in vitro enzymatic assays, total yeast RNA (10 g) or tRNA transcript (4 pmol) was incubated for 2 h with about 0.5 g of recombinant protein in 10 l of reaction mixture containing 100 mM

FIG. 1. Pseudouridylation sites detected in the cytoplasmic (A) and the mitochondrial (B) tRNAs of S. cerevisiae.
The tRNAs are schematically represented with their cloverleaf structures, and each of the positions, where a ⌿ residue was detected in one or more of the cytoplasmic or mitochondrial tRNAs, is indicated by "⌿." When identified, the enzymes acting at the indicated positions are given. For the other sites, the number of tRNAs containing a ⌿ residue or an U residue is given. These data were compiled from the tRNA data base (20).
CMCT/RT Mapping of ⌿ Residues in RNA-Preparation of total RNA from yeast strains and CMCT/RT mapping of pseudouridine residues were performed as described previously (8,28). This method is based on the production of alkaline-resistant modification of ⌿ residues by a water-soluble carbodiimide CMCT, followed by detection of the modified residues by primer extension. The oligonucleotides, used for analysis of the yeast snRNAs are described in Ref. 8. CMCT/RT mapping of ⌿ residue at position 31 in cytoplasmic tRNA Met (anticodon CAU) and mitochondrial tRNA Met (anticodon CAU) was done with oligonucleotides complementary to residues 40 -57 and 46 -67, respectively. CMCT/RT mapping of ⌿ residue at position 32 in cytoplasmic tRNA Gly -(anticodon GCC) and tRNA Val (anticodon UAC) was performed with oligonucleotides complementary to residues 59 -76 and 39 -57, respectively. Mitochondrial tRNA Ser (anticodon GCU) and tRNA Trp (anticodon UCA) were also tested for ⌿32 formation, using oligonucleotides complementary to residues 44 -57 and to residues 58 -76, respectively.
Sequence Analysis-Subcellular localization of Pus9p and Rib2/ Pus8p was predicted by the PSORTII (Ref. 29 and psort.nibb.ac.jp) and NNPSL (Ref. 30 and predict.sanger.ac.uk/nnpsl/) prediction software. Screening of the non-redundant protein data base at the National Center for Biotechnology Information was performed with the BLASTP2 software (www.ncbi.nlm.nih.gov/BLAST/). Analysis of protein sequence was done by construction of multiple sequence alignments using the ClustalW software.

RESULTS
The RIB2/PUS8 and PUS9 ORFs Encode Proteins with Highly Homologous RNA:⌿-synthase Domains-Schematic representations of the organization and sequence homologies of several proteins of the RluA RNA:⌿-synthases family, including Pus5p, Pus6p, and the proteins encoded by the PUS9 and RIB2/PUS8 ORFs are presented in Fig. 2. The RNA:⌿-synthase domains of Rib2/Pus8p and Pus9p are more similar to the one of Pus6p, than to that of Pus5p (Fig. 2). Because Pus5p is a mitochondrial rRNA:⌿-synthase (13), whereas Pus6p is a tRNA:⌿-synthase (14), this was a hint for possible activity of Rib2/Pus8p and Pus9p on tRNAs. In agreement with the finding that Rib2/Pus8p is implicated in riboflavin biosynthesis (19), it contains a long C-terminal extension (160 amino acids), which displays sequence similarity with members of the bacterial RibD family of deaminases. These enzymes are involved in riboflavin biosynthesis in bacteria (19) (Fig. 2). Altogether, the sequence alignments suggested the presence in Rib2/Pus8p of an N-terminal domain with a U to ⌿ conversion activity and a C-terminal domain with a DRAP-deaminase activity. Analysis with the PSORTII (29) and NNPSL (30) software suggested that Rib2/Pus8p is a cytoplasmic enzyme, which was experimentally confirmed (see ygac.med.yale.edu/ygac-cgi/). In contrast, Pus9p was predicted to be targeted in mitochondria by its N-terminal sequence.
The PUS9 and RIB2/PUS8 ORFs Are Not Essential in S. cerevisiae-To study the functions of the Pus9p and Rib2/ Pus8p proteins, each of the two corresponding ORFs was disrupted in the S. cerevisiae BMA64A strain by the one-step PCR-based approach (23). The PUS9 ORF was replaced by the S. cerevisiae TRP1 auxotrophic marker in the BMA64A strain. The resulting pus9⌬::TRP1 strain showed no marked growth phenotype, both in rich (YPD yeast extract, peptone, dextrose) and in minimal (YNB yeast nitrogen base) media, and this, at any of the tested temperatures (data not shown). The doubling times, measured at 30°C in YPD medium for the WT and pus9⌬::TRP1 strains were identical (about 90 Ϯ 5 min). The RIB2/PUS8 ORF was substituted either by the TRP1 marker (rib2/pus8⌬::TRP1 strain) or by a bacterial Kan r resistance gene (rib2/pus8⌬::Kan r strain). After verification by PCR amplification that the expected DNA recombination had occurred, growth phenotypes were analyzed. Unexpectedly, the growth phenotype generated by the RIB2/PUS8 ORF substitution depended upon the kind of substitution. The rib2/pus8⌬::TRP1 substitution had no detectable effect on growth in YPD medium (doubling time of 95 min, which is identical to the value obtained for the WT strain), whereas the rib2/pus8⌬::Kan r replacement led to a marked decrease of growth rate (Fig. 3). The rib2/pus8⌬::Kan r substitution even had a greater effect in the genetic background of the BY474 strain, because no growth was detected on YPD medium (see also www-deletion. stanford.edu/cgi-bin/deletion/search3.pl). We confirmed these data with a haploid strain (BY4742 rib2/pus8⌬::Kan r ) generated by A. P. Gerber (Fig. 3). Disruption of the RIB2 DRAPdeaminase gene in the yeast Pichia guilliermondii was also found to be lethal, but growth could be restored by addition of riboflavin in the culture medium (31). We also found that a normal growth of the BMA64A rib2/pus8⌬::Kan r strain on both YPD and YPG media is restored by addition of 15 mg/liter riboflavin (Fig. 3). To test whether expression of the DRAPdeaminase domain can also restore growth, the two Rib2/Pus8p domains were delineated with the Predict Protein program, and their coding regions were inserted in the p416GalS yeast expression vector. As shown in Fig. 3B, expression of the deaminase (p416GalS-RIB2/PUS8⌬PUS8), but not of the RNA: ⌿-synthase domain (p416GalS-RIB2/PUS8⌬DRAP), restored normal growth on YPG medium in the absence of riboflavin. To verify that the growth defect was not related to the absence of Rib2/Pus8p RNA:⌿-synthase activity, we produced a RIB2/ PUS8 ORF mutated in the codon corresponding to the postulated essential Asp residue of the RNA:⌿-synthase active site (D211, Fig. 2). Expression of the variant D211A protein in the rib2/pus8⌬::Kan r strain restored normal growth on YPG medium in the absence of riboflavin (Fig. 3B). Altogether, the data demonstrated that, as previously found for the Pus1p, Pus4p, Pus5p, and Pus6p RNA:⌿-synthases, the Pus9p and Rib2/ Pus8p RNA:⌿-synthase activities can be abolished without marked effect on cell growth. The different behaviors of rib2/pus8⌬::TRP1 and rib2/pus8⌬::Kan r strains suggest that the Kan r gene product may interfere with some metabolic pathways in S. cerevisiae, leading to the requirement of an increased riboflavin concentration.
Only ⌿ 32 Formation in tRNAs Is Affected by Individual Disruption of the PUS9 and RIB2/PUS8 ORFs-Because several UsnRNA:⌿-synthases remained to be identified, we first tested the effect of individual PUS9 and RIB2/PUS8 ORF disruptions on UsnRNA pseudouridylation. Total RNA was extracted from the BMA64A strain and its derivatives (pus9⌬::TRP1 and rib2/pus8⌬::Kan r ), which will now be designated as ⌬pus9 and ⌬rib2/pus8 strains, for simplification. The presence of ⌿ residues was analyzed by the CMCT/RT approach (8). Neither of the two ORF substitutions was found to alter the pattern of UsnRNA pseudouridylation (data not shown). Thus, Rib2/Pus8p and Pus9p are probably not involved in UsnRNA modification.
We then tested the possible activity of each enzyme at the tRNA positions, which had no assigned RNA:⌿-synthases: namely, positions 1 and 32 in cytoplasmic tRNAs, and positions 32 and 72 in mitochondrial tRNAs (Fig. 1). The tRNA:⌿32synthase activity of yeast extracts from the ⌬rib2/pus8 and ⌬pus9 strains was tested using as substrate an in vitro transcribed S. cerevisiae cytoplasmic tRNA Asp labeled by incorporation of [␣-32 P]UTP (7). In this tRNA, only ⌿32 is located 5Ј to a U residue and thus can be labeled and detected after RNase T2 digestion. As a control we measured the tRNA:⌿13-synthase activity of the extracts, as described previously (16). Whereas the disruption of the PUS9 ORF had no detectable effect on the tRNA:⌿32-synthase activity, the replacement of RIB2/PUS8 ORF decreased this activity by a factor of about 2 (Table I and Fig. 4). Hence, Rib2/Pus8p might have a tRNA: ⌿32-synthase activity that is partially redundant with another RNA:⌿-synthase activity present in total yeast extract. The PUS9-encoded protein was a possible candidate. It might indeed be a tRNA:⌿32-synthase specific for the mitochondrial compartment. To test for this possibility, ⌿32 formation in cytoplasmic and mitochondrial tRNAs of the ⌬rib2/pus8 and ⌬pus9 strains was tested by the CMCT/RT approach. Because of natural post-transcriptional modifications that impair primer extension analysis, the cytoplasmic tRNA Asp , used in the above experiments, could not be analyzed by this method. Hence, the mitochondrial tRNA Ser and the cytoplasmic tRNA Gly , which both contain a ⌿ residue at position 32, were selected for this experiment. As illustrated in Fig. 5A, ⌿32 formation in the mitochondrial tRNA Ser was completely abolished in the ⌬pus9 strain, but remained unaffected in the ⌬rib2/pus8 strain. The   FIG. 3. Growth properties of the WT and ⌬pus9 and ⌬rib2/pus8 S. cerevisiae strains. A, the growth properties of the WT BMA64A and BY4742 S. cerevisiae strains and their rib2/pus8⌬::TRP1 and rib2/pus8⌬::Kan r derivatives were tested on YPD medium with or without addition of 15 mg/liter riboflavin. Starting from a dilution of 1.5 ϫ 10 7 cells/ml, 5-fold serial dilutions (5 l each) were spotted on YPD agarose plates and incubated for 48 h at 30°C. In B, the BMA64A rib2/pus8⌬::Kan r strain was transformed either with an empty p416GalS plasmid (Ϫ) or with p416GalS-derived plasmid encoding WT Rib2/Pus8p (RIB2WT), the Rib2/Pus8p variant (RIB2D211A), the N-terminally truncated Rib2/Pus8p (RIB2⌬PUS8) or the C-terminally truncated Rib2/Pus8p (RIB2⌬DRAP). Serial dilutions and incubations were done as described in the legend to A, except that YPG agarose plates with or without 15 mg/liter riboflavin were used. same result was obtained for the mitochondrial tRNA Trp (not shown). In contrast, the PUS9 deletion had no significant effect on ⌿32 formation in the cytoplasmic tRNA Gly (Fig. 5B, lanes 7 and 8) and tRNA Val (not shown). These data strongly suggested that Pus9p catalyzes ⌿32 formation in mitochondrial tRNAs. A confirmation was obtained by complementation of the ⌬pus9 strain by a plasmid bearing the WT or a mutated PUS9 ORF (Fig. 6). Indeed, the p416GalS-PUS9WT plasmid expressing the active protein was able to restore ⌿32 formation in the mitochondrial tRNA Ser (Fig. 6, lanes 7 and 8), whereas its derivative p416GalS-PUS9D238A, encoding a Pus9p with a D238A amino acid substitution in the GRLD motif of the RNA: ⌿-synthase domain (Fig. 2), did not restore the activity (Fig. 6,  lanes 9 and 10). Hence, we concluded that the S. cerevisiae PUS9 ORF encodes the tRNA:⌿32-synthase responsible for modification of mitochondrial tRNAs.
The effects of the RIB2/PUS8 and PUS9 ORF deletions on the tRNA:⌿1-and tRNA:⌿72-synthase activities of cellular extracts were analyzed by the nearest neighbor approach using transcripts corresponding to the S. cerevisiae cytoplasmic tRNA Arg and mitochondrial tRNA Met i, respectively. The tRNA Arg transcript was labeled with [␣-32 P]UTP, because among the ⌿ residues present in this tRNA, only residue ⌿1 is followed by an U residue. The tRNA Met i transcript was labeled with [␣-32 P]ATP, because in this tRNA residue ⌿72 is followed by an A residue. Comparison of the activities of the WT, ⌬pus9 and ⌬rib2/pus8 yeast extracts showed that neither Rib2/Pus8p, nor Pus9p is implicated in ⌿1 formation in cytoplasmic tRNAs or ⌿72 formation in mitochondrial tRNAs (Table I).
The Recombinant Pus9p and Rib2/Pus8p Both Possess a tRNA:⌿32-synthase Activity in Vitro-The above experiments suggested that both Pus9p and Rib2/Pus8p have a tRNA:⌿32-synthase activity. To complete this demonstration, the E. coli pET28b expression vector was used to produce recombinant His-tagged Rib2/Pus8p and Pus9p proteins, which were affinity-purified on nickel-nitrilotriacetic acid-agarose beads. We also produced Rib2/Pus8p and Pus9p proteins with an aspartic  4. Deletion of the RIB2/PUS8 but not the PUS9 ORF alters the tRNA:⌿32-synthase activity of yeast extract. The figure represents time courses for formation of residue ⌿32 in the in vitro transcript of yeast cytoplasmic tRNA Asp incubated at 37°C with cell-free extracts of the WT, ⌬pus9, ⌬rib2/pus8, and ⌬rib2/pus8/⌬pus9 yeast strains. The uniformly [␣-32 P]UTP-labeled tRNA Asp transcript was incubated with the extracts, phenol-extracted, and completely digested by RNase T2. The resulting [3Ј-␣-32 P]mononucleotides were separated by two-dimensional TLC on cellulose plates. Quantification was done by using a PhosphorImager, and the amount of formed ⌿ residue was calculated taking into account the nucleotide composition of the transcript.
FIG. 5. Mapping of residue ⌿32 in the mitochondrial tRNA Ser (A) and cytoplasmic tRNA Gly (B) from the WT and mutated S. cerevisiae BMA64A strains. Total RNA was extracted from the WT, ⌬pus9, ⌬rib2/pus8, and ⌬rib2/pus8/⌬pus9 S. cerevisiae strains and modified by CMCT, for 2, 10, and 20 min with (ϩ) or without (Ϫ) subsequent alkaline treatment (OH Ϫ ). For both tRNAs a control experiment was performed in the absence of CMCT treatment. Lanes U, G, C, and A correspond to the sequencing ladders obtained with the same oligonucleotide. The reverse transcription stops, corresponding to residue ⌿32 or to residues ⌿31 and ⌿38, are indicated. acid (Asp) to alanine (Ala) amino acid substitution in the GRLD motif of the active site. The RNA:⌿-synthase activities of the four recombinant proteins were tested by the nearest neighbor approach using an in vitro transcribed S. cerevisiae tRNA Asp labeled by [␣-32 P]UTP incorporation. Thin layer chromatography (TLC) analysis demonstrated that the recombinant WT Pus9p and Rib2/Pus8p proteins both had a tRNA:⌿32-synthase activity (Fig. 7, B and D), whereas the mutated proteins were inactive (Fig. 7, C and E). Using the CMCT/RT approach, we confirmed that the U to ⌿ conversion occurred only at position 32 in tRNA Asp (Fig. 7F). Taken together, our data confirmed the activity of both Rib2/Pus8p and Pus9p RNA:⌿-synthases at position 32 in tRNAs.
Simultaneous Inactivation of the RIB2/PUS8 and PUS9 ORFs Impairs ⌿32 Formation in Both Cytoplasmic and Mitochondrial tRNAs-For a complete demonstration of the requirement of both Rib2/Pus8p and Pus9p for ⌿32 formation in all tRNAs, we disrupted the RIB2/PUS8 ORF by Kan r marker in the pus9⌬::TRP1 strain. The resulting strain, which will be designated as ⌬rib2/pus8/⌬pus9, was viable only in the presence of 15 mg/liter riboflavin in the growth medium. The absence of tRNA:⌿32-synthase activity in the cell extract was demonstrated by the in vitro activity test based on the use of [␣-32 P]UTP-labeled tRNA Asp , as described above (Fig. 4). In addition, the absence of U to ⌿ conversion at position 32 in the cytoplasmic tRNA Gly and the mitochondrial tRNA Ser extracted from the ⌬rib2/pus8/⌬pus9 strain was confirmed by CMCT/RT analysis (Fig. 5A, lanes 9 and 10 and Fig. 5B, lanes 15 and 16). Altogether the data clearly demonstrated that two enzymes (Rib2/Pus8p and Pus9p) are required for ⌿32 formation in both cytoplasmic and mitochondrial tRNAs. Also, as expected, expression of the WT, but not of the D112A-mutated Rib2/Pus8p protein, restored ⌿32 formation in the cytoplasmic tRNA Gly of the ⌬rib2/pus8/⌬pus9 strain (Fig. 8).
As demonstrated by CMCT/RT analyses of the cytoplasmic tRNA Val (Fig. 8, lanes 15 and 16), and the mitochondrial tRNA Ser (data not shown), expression of the N-terminal RNA:⌿-synthase domain of Rib2/Pus8p (p416GalS-RIB2/PUS8⌬DRAP) was also able to restore ⌿32 formation in cytoplasmic tRNAs, but not in mitochondrial tRNAs. This observed complementation demonstrates that the presence of the deaminase domain of Rib2/ Pus8p is required neither for folding of the N-terminal RNA: ⌿-synthase domain nor for its activity.
Because the tRNA:⌿1-and tRNA:⌿72-synthase activities of extract from the ⌬rib2/pus8/⌬pus9 strain were found to be identical to those of the WT strain (Table I), we concluded that some RNA:⌿-synthases, other than Rib2/Pus8p and Pus9p, should be responsible for the formation of these two posttranscriptional modifications.
Cellular Localization of Pus9p in S. cerevisiae-Based on the presence of a characteristic Lys/Arg-rich sequence at its N terminus, a mitochondrial localization was predicted for Pus9p by the PSORTII and NNPSL software (29,30). To test these predictions, we built a yeast plasmid (p416GalS-PUS9⌬N) expressing a Pus9p variant lacking 23 amino acids (amino acids   After RNase T2 digestion, the released 3Ј-mononucleotides were separated by two-dimensional TLC, and the autoradiograms of the plates are shown. A control digestion without prior incubation with recombinant enzyme is also shown (A). The ⌿MP spot is indicated by an arrow. The N/R chromatographic system was described previously (45). To control that the ⌿MP spot observed on the plates corresponds to modification at position 32 (F), CMCT/RT experiments were performed with unlabeled tRNA Asp transcripts incubated or not with the recombinant WT His 6 -Rib2/Pus8p protein.
from the ⌬rib2/pus8/⌬pus9 strain transformed with p416GalS-PUS9⌬N construct revealed the formation of ⌿32 in cytoplasmic (Fig. 8, lanes 19 and 20), but not mitochondrial tRNAs (Fig.  9, lanes 15 and 16). These data demonstrated that the Nterminal sequence of Pus9p is required for its mitochondrial targeting and confirmed the capacity of Pus9p to modify cytoplasmic tRNAs in vivo. DISCUSSION Two unusual features of the yeast S. cerevisiae tRNA modification machinery are revealed in this work: (i) two members of the RluA family of RNA:⌿-synthases are required for U to ⌿ conversion at position 32 in cytoplasmic and mitochondrial tRNAs and (ii) the cytoplasmic enzyme Rib2/Pus8p contains an additional domain with an unrelated activity.

Bacterial Members of the RluA Family Have Different RNA Specificities Compared with Their Yeast Counterparts-The
RluA family of RNA:⌿-synthases seems to be the most complex of the five identified RNA:⌿-synthase families. Four members of this family are present in both E. coli (RluA, RluC, RluD, and TruC) and S. cerevisiae (Pus5p, Pus6p, Rib2/Pus8p, and Pus9p). However, while three of the four E. coli enzymes modify 23 S rRNA (RluC (32), RluD (33), and RluA, which also acts at position 32 in tRNAs (34)), only one of the four S. cerevisiae enzymes modifies rRNAs (Pus5p, which modifies the mitochondrial 21 S rRNA (13)). Indeed, despite its sequence homology with RluA, RluC, and RluD, the yeast Pus6p enzyme is responsible for pseudouridylation at position 31 in both cytoplasmic and mitochondrial tRNAs (14). This tRNA position is not modified in E. coli. We demonstrate here that the two remaining unidentified members of the RluA family in S. cerevisiae, Rib2/ Pus8p and Pus9p, have the same activity as the E. coli RluA enzyme; they both act at position 32 in tRNAs. Accordingly, Rib2/Pus8p and Pus9p are more similar to RluA, than to TruC, which is responsible for ⌿65 formation in E. coli tRNAs (35). Formation of ⌿65 in S. cerevisiae is catalyzed by Pus1p, a member of the TruA family (7). Unexpectedly, presently identified Pus9p shows a higher similarity with the RluD E. coli rRNA:⌿-synthase than with the RluA E. coli tRNA:⌿32-synthase. These observations suggest that RNA-substrate specificity of S. cerevisiae RluA-related RNA:⌿-synthases is likely related to a different mechanism of rRNA modification in bacteria and eukarya. In eukarya, a unique RNA:⌿-synthase Cbf5p (in yeast)/dyskerin (in human) associated with the guide H/ACA small nucleolar RNAs (11), is expected to form most of the pseudouridylations of cytoplasmic rRNAs. The Cbf5p/dyskerin enzyme belongs to the TruB family, so that members of the RluA family are not expected to be required for pseudouridylation of cytoplasmic rRNAs. However, it was recently proposed that the formation of one of the highly phylogenetically conserved 2Ј-O-methylation in yeast 25 S rRNA is catalyzed by both the Spb1 2Ј-O-methylase and the C/D snR52 small nucleolar ribonucleoprotein (36). Thus, one cannot exclude the possibility that some of the highly phylogenetically conserved ⌿ residues in S. cerevisiae 25 S rRNA (namely ⌿2257 and ⌿2259) are formed both by an H/ACA small nucleolar ribonucleoprotein and/or one of the Pus5p, Pus6p, Rib2/Pus8p, or Pus9p enzymes. However, our data clearly show that the activities of the S. cerevisiae RluA-like enzymes are largely dedicated to tRNA modifications, whereas their E. coli counterparts are mainly involved in rRNA modification (32)(33)(34)37). This extended activity on tRNAs in S. cerevisiae may be explained by the emergence of additional pseudouridylation sites in tRNAs compared with E. coli (position 31) and the need of distinct enzymes for the cytoplasmic and mitochondrial compartments. Taken together, these observations suggest that the evolution of RNA:⌿-synthase substrate specificity is a rather rapid process at the scale of long term evolution. Appearance of the RNA-guided system during evolution probably resulted both in changes of RNA:⌿-synthase specificities and disappearance of some of the protein catalysts.
Two Distinct tRNA:⌿32-synthases Are Present in the Cytoplasmic and Mitochondrial Compartments of S. cerevisiae-Disruption of the PUS9 ORF led to complete disappearance of ⌿32 formation in mitochondrial tRNAs, which is in perfect agreement with the computer prediction of a mitochondrial targeting signal in Pus9p and the preferential cytoplasmic localization of Rib2/Pus8p. Also in accord with the prediction of only a partial localization of Pus9p in mitochondria, cytoplas-FIG. 8. The RNA:⌿32-synthase activity of Rib2/Pus8p depends upon residue Asp-211 and is not linked to the deaminase domain. The yeast S. cerevisiae strain ⌬rib2/pus8/⌬pus9 was transformed with plasmids coding the WT (lanes 9 -12) or mutated (D211A) Rib2/ Pus8p protein (lanes 5-8) or its truncated version (⌬DRAP lacking C-terminal DRAP-deaminase domain, lanes [13][14][15][16]. Transformation of the same strain was also performed with the p416GalS-PUS9⌬N plasmid, coding the N-terminally truncated (⌬N) Pus9p variant (lanes [17][18][19][20]. The presence of residue ⌿32 in the cytoplasmic tRNA Val (UAC) extracted from the transformed strains was analyzed by the CMCT/RT approach (as described in the legend for Fig. 5). The reverse transcription stops, corresponding to residues ⌿27 and ⌿32 are indicated.
FIG. 9. The N-terminal sequence of protein Pus9p is required for its mitochondrial targeting. The ⌬rib2/pus8/⌬pus9 strain was transformed with plasmid p416GalS expressing the WT (lanes 9 -12) or ⌬N variant (lanes 13-16) of Pus9p protein. The presence of residue ⌿32 in the mitochondrial tRNA Ser of the transformed strain was analyzed by the CMCT/RT approach by using conditions described in the legend for Fig. 5. The reverse transcription stops, corresponding to residues ⌿31 and ⌿32 are indicated.