A previously unidentified activity of yeast and mouse RNA:pseudouridine synthases 1 (Pus1p) on tRNAs
Abstract
Mouse pseudouridine synthase 1 (mPus1p) was the first vertebrate RNA:pseudouridine synthase that was cloned and characterized biochemically. The mPus1p was previously found to catalyze Ψ formation at positions 27, 28, 34, and 36 in in vitro produced yeast and human tRNAs. On the other hand, the homologous Saccharomyces cerevisiae scPus1p protein was shown to modify seven uridine residues in tRNAs (26, 27, 28, 34, 36, 65, and 67) and U44 in U2 snRNA. In this work, we expressed mPus1p in yeast cells lacking scPus1p and studied modification of U2 snRNA and several yeast tRNAs. Our data showed that, in these in vivo conditions, the mouse enzyme efficiently modifies yeast U2 snRNA at position 44 and tRNAs at positions 27, 28, 34, and 36. However, a tRNA:Ψ26-synthase activity of mPus1p was not observed. Furthermore, we found that both scPus1p and mPus1p, in vivo and in vitro, have a previously unidentified activity at position 1 in cytoplasmic tRNAArg(ACG). This modification can take place in mature tRNA, as well as in pre-tRNAs with 5′ and/or 3′ extensions. Thus, we identified the protein carrying one of the last missing yeast tRNA:Ψ synthase activities. In addition, our results reveal an additional activity of mPus1p at position 30 in tRNA that scPus1p does not possess.
Keywords
INTRODUCTION
Pseudouridine (Ψ), is one of the two most abundant modified nucleotides found in stable RNA species, including tRNAs (Bjork et al. 1987), rRNAs (for review, see Ofengand and Fournier 1998), and UsnRNAs (Massenet et al. 1999; for review, see Massenet et al. 1998). Pseudouridine residues in RNAs are formed post-transcriptionally by a group of enzymes called RNA:Ψ-synthases (Kammen et al. 1988; Koonin 1996). The isomerization reaction requires no cofactor and no energy source (Kammen et al. 1988).
On the basis of sequence comparisons, four distinct RNA:Ψ-synthase families (TruA, TruB, RluA, and RsuA) were defined (Gustafsson et al. 1996; Koonin 1996) and a fifth family (TruD) was recently discovered (Behm-Ansmant et al. 2003; Kaya and Ofengand 2003; Ma et al. 2003). RNA:Ψ-synthases can function alone, by direct interaction with their RNA substrates, or after incorporation into ribonucleoprotein complexes (RNPs) that contain a guide RNA. The guide RNAs ensure the specific recognition of the substrate. In vertebrates, scaRNAs located in Cajal bodies are expected to guide Ψ formation in U1, U2, U4, and U5 snRNAs (Ganot et al. 1999; Jady and Kiss 2001; Darzacq et al. 2002; Zhao et al. 2002; Vitali et al. 2003; Kiss et al. 2004). The RNA-guided RNA:Ψ-synthases (dyskerin/Cbf5) belong to the TruB family.
Although Saccharomyces cerevisiae RNA:Ψ-synthases have been extensively studied (for reviews, see Ansmant and Motorin 2001; Ofengand 2002), two S. cerevisiae tRNA:pseudouridine synthase activities acting at position 1 in the cytoplasmic tRNAArg(ACG) and tRNALys(UUU) and at position 72 in the mitochondrial tRNAMet i(CAU) (see Fig. 1C,E) remain to be identified (Behm-Ansmant et al. 2004). In addition, only a limited number of RNA:Ψ-synthases have been studied in Eukarya: (1) the mouse Pus3p enzyme specific for positions 38 and 39 in tRNAs (Chen and Patton 2000) and (2) the homologs of the S. cerevisiae Pus1p enzyme (scPus1p) from Schizosaccharomyces pombe (spPus1p) (Hellmuth et al. 2000), Caenorhabditis elegans (cePus1p) (Patton and Padgett 2003), and mouse (mPus1p) (Chen and Patton 1999). The Pus1p enzymes have a broad RNA recognition specificity. In yeast, scPus1p was shown to modify seven distinct positions in cytoplasmic tRNAs (26, 27, 28, 34, 36, 65, and 67) (Simos et al. 1996; Motorin et al. 1998; Behm-Ansmant et al. 2003) and at position 44 in U2 snRNA (Massenet et al. 1999). The mPus1p specificity was partially characterized by in vitro experiments using synthetic yeast tRNA transcripts and a human pre-tRNASer transcript as the substrates (Chen and Patton 1999). These in vitro experiments showed that, like scPus1p, mPus1p catalyzes Ψ formation at positions 27, 28, 34, and 36 in tRNAs (Chen and Patton 1999).
Sequences and secondary structures of the S. cerevisiae tRNA substrates used in this work. The various panels represent, respectively, the tRNATrp(CCA) (A), the pre-tRNAIle(UAU) (B), the mature tRNAArg(ACG) (C), the 5′p3′p tRNAArg(ACG) with 5′ and 3′ extensions mimicking a tRNA precursor (D), mitochondrial tRNAMet i(CAU) (E), tRNAVal(CAC) (F), and tRNAGly(GCC) (G). All tRNAs (except tRNAArg(ACG) precursor) are drawn with all their identified post-transcriptional modifications (tRNA database; Sprinzl et al. 1998; see, also, http://www.uni-bayreuth.de/departments/biochemie/sprinzl/trna/). The intronic sequence in pre-tRNAIle is shown in small characters and small arrows indicate the exon–intron borders. The Ψ residues studied during this work are circled.
The interest to complete the analysis of the vertebrate Pus1p specificity was recently strengthened by two important findings. One observation arose from the study of patients suffering from mitochondrial myopathy and sideroblastic anemia (MLASA). Indeed, this rare autosomal recessive disorder of oxidative phosphorylation and iron metabolism was found to be a consequence of the inactivation of the Pus1p activity by substitution of a highly conserved R116 residue in human Pus1p (hPus1p) with a W116 residue (Bykhovskaya et al. 2004; Zeharia et al. 2005). The absence of tRNA modification at positions 27 and 28 in patients with MLASA was demonstrated (Patton et al. 2005). The second recent important finding was the unexpected discovery of the mPus1p coactivator activity on class I and class II nuclear receptors (NR) (Zhao et al. 2004). Coactivation of the mouse retinoic acid receptor γ (mRARγ) requires the formation of a triple interaction between mPus1p, the steroid receptor activator (SRA) RNA, and a mRARγ receptor. Furthermore, the mPus1p RNA:Ψ-synthase activity is needed for activation. Accordingly, in vitro experiments demonstrated that mPus1p catalyzes Ψ formation in the SRA RNA (Zhao et al. 2004). Thus, mPus1p seems to act on more than one type of RNA substrate as is the case for scPus1p.
To complete the study of the mPus1p specificity, we took advantage of the existence of a S. cerevisiae strain lacking scPus1p expression (Δpus1 cells), which was previously used to study the scPus1p specificity in vivo (Motorin et al. 1998; Massenet et al. 1999). The mPus1p activity was tested in Δpus1 cells and compared with in vitro results. Modification of both tRNAs and U2 snRNA was tested and we investigated whether scPus1p and mPus1p exhibit one or both of the yet unidentified yeast tRNA:Ψ-synthase activities, namely, pseudouridylation at position 1 in the cytoplasmic tRNAArg(ACG) and tRNALys(UUU) (Madison and Boguslawski 1974; Weissenbach et al. 1975) and pseudouridylation at position 72 in the mitochondrial tRNAMet i(CAU) (Canaday et al. 1980).
Here we describe an extensive comparison of the mPus1p and scPus1p activities in vivo and in vitro. Our data demonstrate that mPus1p modifies position 44 in yeast U2 snRNA and that both mPus1p and scPus1p modify the cytoplasmic tRNAArg(ACG) at position 1.
RESULTS
In yeast, mPus1p modifies U2 snRNA at position 44 and tRNAs at positions 26, 27, 28, 34, and 36
To test whether mPus1p can functionally replace its yeast homolog, we expressed mPus1p in a S. cerevisiae strain with a PUS1 ORF disruption (Δpus1) (see Materials and Methods). To accomplish this, we cloned the wild-type and a mutated version of the mPUS1 ORF in the p416GalS yeast expression plasmid, downstream from the GalS promoter. In the variant mPus1p, the essential Asp112 residue from the conserved ARTD motif of the active site was substituted with an Ala residue (D112A). The resulting recombinant plasmids were used for complementation assays in the Δpus1 strain.
U2 snRNA pseudouridine residues were identified by the CMCT/RT approach as previously described, using total RNA from extracts as templates (Massenet et al. 1999). As expected, only residue Ψ44 disappeared in the Δpus1 strain (Fig. 2B, lanes 5,6), and the modification was recovered upon expression of the wild-type mPus1p (Fig. 2B, lanes 7,8), but not of the D112A variant protein (Fig. 2B, lanes 9,10). Analysis of the 110-nt segment at the 5′ extremity of U2 snRNA (Fig. 2A) showed that mPus1p modified only position 44 in this fragment.
The mPus1p enzyme is able to modify yeast U2 snRNA at position 44 in vivo. (A) The 5′-terminal part of U2 snRNA containing three identified Ψ residues (Massenet et al. 1999). The oligonucleotide (O-U2) used for primer extension of U2 snRNA is indicated by an arrow. (B) Identification of in vivo pseudouridylation of U2 snRNA by CMCT/RT approach. The yeast S. cerevisiae Δpus1 strain was transformed with p416GalS plasmids bearing either the wild-type or the mutated mPUS1 ORF. Total RNAs were extracted from the wild-type, Δpus1 (Δ1), Δpus1+p416GalS-mPUS1D112 (Δ1+mPus1p), and Δpus1+p416GalS-mPUS1D112A (Δ1+mPus1p D112A) cells and modified by CMCT, for 1, 10, and 20 min with (+) or without (−) subsequent alkaline treatment (OH−), as previously described (Bakin and Ofengand 1993; Massenet et al. 1999). A control experiment was performed in the absence of CMCT treatment. The pseudouridylated positions were identified by extension of oligonucleotide O-U2 with reverse transcriptase. Lanes U, G, C, and A correspond to the sequencing ladders obtained with the same oligonucleotide. The reverse transcription stops, corresponding to residue Ψ44 and to residues Ψ35 and Ψ42, are indicated.
To study the in vivo activity of mPus1p at positions 26, 27, and 28 in cytoplasmic tRNAs, we chose the yeast tRNATrp(CCA) (Fig. 1A; Keith et al. 1972), which is modified by scPus1p at positions 26, 27, and 28 (Motorin et al. 1998). As expected, no Ψ residue was detected at these three positions in the tRNATrp from the untransformed Δpus1 strain (Fig. 3, lanes 7,8). The mPus1p expression predominantly restored Ψ formation at position 27 (Fig. 3, lanes 11,12). Reverse transcription stops were detected at the adjacent U26 and U28 residues, but the intensity of the bands was only slightly higher than the one observed at the same positions with the Δpus1 strain (Fig. 3, cf. lanes 7,8 and 11,12).
Analysis of in vivo mPus1p activity on yeast tRNATrp. Total RNAs were extracted from the wild-type, Δpus1 (Δ1), and Δpus1+p416GalS-mPUS1 (Δ1+mPus1p) cells and were modified by CMCT, for 1, 10, and 20 min with (+) or without (−) subsequent alkaline treatment (OH−), in conditions previously described (Massenet et al. 1999). A control experiment was performed in the absence of CMCT treatment. Pseudouridylated positions were identified by extension of oligonucleotide O-tRNATrp by reverse transcriptase. Lanes U, G, C, and A correspond to the sequencing ladders obtained with the same oligonucleotide. The reverse transcription stops, corresponding to residues Ψ26, Ψ27, Ψ28, and Ψ39, are indicated.
Mouse Pus1p was previously shown to form residues Ψ27, Ψ34, and Ψ36 in an in vitro transcribed yeast pre-tRNAIle(UAU) (Fig. 1B; Chen and Patton 1999). By CMCT/RT analysis of the pre-tRNAIle(UAU) extracted from the control Δpus1 strain and the Δpus1 strain expressing mPus1p, we showed that the in vivo expressed mPus1p modifies the pre-tRNAIle(UAU) at positions 27, 34, and 36 (Fig. 4). However, modification by mPus1p at position 36 was less efficient than modification by scPus1p (Fig. 4, lanes 3,4 and 7,8). Since the conversion of the pre-tRNAIle into mature tRNAIle is dependent on the presence of the intron and takes place in the nucleus, Ψ34 and Ψ36 formation by mPus1p in vivo strongly suggests that mPus1p was efficiently transported to the yeast nucleus.
Analysis of in vivo mPus1p activity on yeast pre-tRNAIle. Total RNAs were extracted from the wild-type, Δpus1 (Δ1), and Δpus1+p416GalS-mPUS1 (Δ1+mPus1p) cells and modified by CMCT, for 1, 10, and 20 min with (+) or without (−) subsequent alkaline treatment (OH−), in conditions previously described (Massenet et al. 1999). A control experiment was performed in the absence of CMCT treatment. Pseudouridylated positions were identified by extension of oligonucleotide O-pre-tRNAIle with reverse transcriptase. Lanes U, G, C, and A correspond to the sequencing ladders obtained with the same oligonucleotide. The reverse transcription stops, corresponding to residues Ψ27, Ψ30, Ψ34, and Ψ36, are indicated. (B) Sequence comparison of the yeast pre-tRNAIle(UAU), mouse tRNAIle(AAU), and mouse pre-tRNAIle(UAU) anticodon stem-loops. Positions of introns in the pre-tRNAs are indicated.
mPus1p may have a tRNA:Ψ30-synthase activity
Interestingly, the CMCT/RT analysis of the pre-tRNAIle(UAU) modified in Δpus1 cells expressing mPus1p revealed an unexpected reverse transcription stop at position 30 (Fig. 4, lanes 7,8). This stop was not detected when the pre-tRNAIle was extracted from wild-type cells or from untransformed Δpus1 cells. The dependence of the detected RT stop on CMCT modification and its resistance to alkaline treatment demonstrate that it corresponds to a U to Ψ conversion. We conclude that mPus1p can modify residue U30 in yeast pre-tRNAIle in vivo. This result was confirmed in vitro by incubation of total RNA extracted from the Δpus1 strain, which was lacking the modification at position 30, with the recombinant mPus1p (data not shown). Interestingly, when the same in vitro experiment was performed with scPus1p, no significant modification was detected at position 30 of the pre-tRNAIle(UAU) (data not shown).
Both scPus1p and mPus1p have a tRNA:Ψ1-synthase activity
We then tested whether scPus1p and mPus1p may have a tRNA:Ψ1-synthase activity on cytoplasmic tRNAArg(ACG) (Fig. 1C). The presence of a Ψ residue at position 1 in tRNAs can easily be detected by nuclease P1 digestion of 5′-end-labeled tRNAs since, depending on the nature of residue 1, a [5′-32P]ΨMP or a [5′-32P]UMP will be released. The in vitro transcribed cytoplasmic tRNAArg(ACG) was 5′-end labeled using [γ-32P]ATP and T4 polynucleotide kinase and then incubated with a wild-type or Δpus1 cellular extract. After nuclease P1 digestion, the resulting 5′-NMPs were fractionated by 2D-TLC. As shown in Figure 5A, Ψ1 formation was detected after incubation with the wild-type extract, but not with the Δpus1 cell extract. These data were further confirmed by analysis of cellular RNA fractions highly enriched in tRNAs (see Materials and Methods), that were prepared from wild-type cells, Δpus1 cells, or Δpus1 cells expressing mPus1p. As above, to test whether the tRNAArg(ACG) and tRNALys(UUU) present in these fractions had a Ψ residue at position 1, RNAs from these fractions were 5′-end labeled and analyzed by nuclease P1 digestion, followed by 2D-TLC. As shown in Figure 5B, only [5′-32P]UMP was detected in the digestion products of RNAs isolated from the Δpus1 cells, whereas both [5′-32P]ΨMP and [5′-32P]UMP were observed for RNAs isolated from both the wild-type cells and Δpus1 cells expressing mPus1p. Thus, when expressed in yeast, both scPus1p and mPus1p have a tRNA:Ψ1-synthase activity.
Analyses of the tRNA:Ψ1-synthase activity in vitro and in vivo. (A) In vitro transcribed yeast tRNAArg was 5′ labeled with [γ-32P]ATP and incubated with S10 extracts from the wild-type or Δpus1 cells in conditions described in Materials and Methods. After incubation, the transcripts were digested with nuclease P1 and 5′-NMPs were fractionated by 2D-TLC as previously described (Jiang et al. 1997). The autoradiograms of the TLC plates are shown. Positions of the 5′-NMPs (pA, pC, pU, pG, and pΨ) were identified according to Keith (1995). (B) The tRNA enriched fractions from the wild-type, Δpus1, and Δpus1+p416GalS-mPUS1(p) (Δ1+mPus1p) strains were dephosphorylated and subsequently 5′-end labeled with [γ-32P]ATP. The identity of the nucleotide at the 5′ extremity of tRNAs was then analyzed by nuclease P1 digestion, followed by 2D-TLC as in A. Panel (C) [5′-32P]-labeled tRNAArg transcript incubated (+scPus1p) or not (control) with the recombinant scPus1p was digested with nuclease P1. Positions of nonradioactive nucleotide marquers (pA, pC, pG) are indicated by dashed circles.
To further confirm the site of scPus1p activity on tRNAArg(ACG), the in vitro transcribed tRNAArg(ACG) was 5′-end labeled and we studied the identity of its terminal nucleotide after incubation with the recombinant scPus1p. As shown in Figure 5C, residue U1 in tRNAArg is almost completely transformed into a Ψ1 residue. This clearly demonstrated the tRNA:Ψ1-activity of scPus1p.
Like all other yeast tRNAs, tRNAArg(ACG) and tRNALys(UUU) are transcribed as pre-tRNAs in yeast and it was interesting to know whether scPus1p and mPus1p can act at position 1 (as referred to the mature tRNAs) in the pre-tRNAs and their maturation intermediates. To answer this question, RNAs that mimic the pre-tRNAArg(ACG) transcript, its maturation intermediates, and the mature tRNA were produced by in vitro transcription and then incubated with the recombinant scPus1p and mPus1p enzymes. Since the transcription initiation and termination sites of the tRNAArg(ACG) gene have not been identified, we produced a tRNAArg transcript containing a 40-nt-long 5′ extension and a 29-nt-long 3′ extension to mimic the authentic transcript (Fig. 1D). This transcript was designated as 5′p3′p tRNAArg. Typically, pre-tRNA maturation in yeast starts by the elimination of the 5′ extension by RNase P (for review, see Schon 1999). Therefore in order to mimic this partially mature tRNA we also produced a tRNAArg transcript containing only the 3′ extension (5′m3′p tRNAArg). However, in a few cases, as for tRNATrp, maturation of yeast tRNA starts by elimination of the 3′ extension (Kufel and Tollervey 2003). Therefore we also produced a tRNAArg transcript with only the 5′ extension (5′p3′m tRNAArg). In all these transcripts a U residue was present immediately downstream from the targeted U1 residue, so that Ψ1 formation was tested by the nearest neighbor approach, using transcripts labeled by [α-32P]UTP incorporation. Another potential target of Pus1p modification in this tRNA (U27) is followed by a C residue, so that its modification could not detected by the nearest neighbor approach. As shown in Figure 6, all the tested transcripts were efficiently modified upon incubation with recombinant mPusp1 or scPusp1 enzymes, and the mole Ψ/mole tRNA ratio was always ≤1, indicating that the only modification detected was that at position 1. Since several UU dinucleotides were present in transcripts mimicking the precursor tRNA and its maturation intermediates, we used the CMCT/RT approach to show for one of them (the 5′p3′m transcript) that the modification detected by the nearest neighbor approach indeed occurred at position 1. Figure 7 (lanes 5 and 6) demonstrates that this is the case. Within the 90 nt examined in the CMCT/RT analysis, only position 1 was found to be significantly converted into a Ψ residue. These results clearly indicate that both scPus1p and mPus1p can catalyze Ψ1 formation in the mature tRNAArg, the pre-tRNAArg, and its maturation intermediates.
Analyses of the in vitro tRNA:Ψ1-synthase activity of recombinant scPus1p and mPus1p enzymes on yeast tRNAArg variants. In vitro transcribed RNA substrates, corresponding to yeast mature tRNAArg (A), tRNAArg with a 3′ extension (5′m3′p) (B), tRNAArg with a 5′ extension (5′p3′m) (C), and tRNAArg with both 5′ and 3′ extensions (5′p3′p) (D) were labeled by incorporation of [α-32P]UTP and incubated with or without the recombinant His6-scPus1p (scPus1p) or His6-mPus1p (mPus1p) enzyme. After incubation, the transcripts were digested with RNase T2, and 3′-NMPs were fractionated on 2D-TLC. The autoradiograms of the TLC plates are shown. Positions of the 3′-NMPs (Ap, Cp, Up, Gp, and Ψp) were identified according to Keith (1995). Quantification of the formed Ψ residue was done by measuring the radioactivity in each spot with a PhosphoImager, using the ImageQuant software and is indicated below the panel.
CMCT/RT mapping of Ψ residues formed in a 5′-extended tRNAArg transcript (5′p3′m). An unlabeled 5′p3′m tRNAArg transcript (5′p3′m tr) was incubated with or without the recombinant His6-scPus1p (scPus1p) and analyzed by the CMCT/RT approach. RNAs were modified by CMCT, for 1, 10, and 20 min with (+) or without (−) subsequent alkaline treatment (OH−). A control experiment was performed in the absence of CMCT treatment. Pseudouridylated positions were identified by extension of oligonucleotide O-tRNAArg by reverse transcriptase. Lanes U, G, C, and A correspond to the sequencing ladders obtained with the same oligonucleotide. The reverse transcription stop corresponding to residue Ψ1 is indicated.
Both scPus1p and mPus1p lack tRNA:Ψ72-synthase activity
Finally, we tested whether scPus1p and mPus1p may act at position 72 in the mitochondrial tRNAMet i(CAU) (Fig. 1E), which is the last position in yeast tRNAs without attributed RNA:pseudouridylation activity. To this end, in vitro transcribed yeast mitochondrial tRNAMet i was incubated with cell-free extracts from wild-type cells and Δpus1 cells, either with or without the plasmid expressing mPus1p. The data showed that the tRNA:Ψ72-synthase activity was still present in the Δpus1 strain (Table 1). Moreover, neither the recombinant scPus1p nor the recombinant mPus1p were able to modify a yeast mitochondrial tRNAMet i transcript at position 72 (Table 1). Thus, it seems unlikely that either scPus1p or mPus1p has a tRNA:Ψ72-synthase activity.
Analysis of Ψ72 formation in an in vitro transcribed yeast mitochondrial tRNAMet i(CAU) after incubation either with extracts from wild-type, Δpus1, and Δpus1+p416GalS-mPUS1 yeast cells or with recombinant scPus1p and mPus1p
DISCUSSION
mPus1p has the same activity on yeast U2 snRNA as scPus1p
Here we demonstrate that, like scPus1p, mPus1p acts at position 44 in yeast U2 snRNA in vivo. Previous studies on C. elegans showed that the disruption of the cePus1p gene results in the absence of Ψ27 formation in tRNAs, but has no effect on Ψ45 formation in U2 snRNA (the counterpart of Ψ44 in yeast U2 snRNA) (Patton and Padgett 2003). It was thus proposed that this modification may be catalyzed by an RNA-guided enzyme in eukaryotes other than yeasts. However, with respect to human scaRNAs, which are the best studied among vertebrate scaRNAs, only 8 out of 13 Ψ residues present in human U2 snRNA can be guided by the presently identified scaRNAs (Vitali et al. 2003; Kiss et al. 2004), and position 43, which is the counterpart of the yeast U2 snRNA position 44, is not one of these. In addition, as previously proposed (Ma et al. 2005), one cannot exclude the possible existence of two distinct systems for generation of critical post-transcriptional modifications in UsnRNAs. This may be the case of Ψ44 in U2 snRNA since Ψ formation at this position in yeast is phylogenetically conserved in all studied eukaryal species. In addition, it is found in a segment of U2 snRNA located upstream of the branch site complementary sequence (Fig. 8), which is involved in the binding of several important splicing factors: an SF3b component Cus1p (Wells et al. 1996; Yan et al. 1998), the protein complex of Prp9p, Prp11p, and Prp21p (Wells and Ares 1994), and RNA helicase Prp5p (Wells and Ares 1994; Gozani et al. 1996). Moreover, the modified U2 snRNA segment is involved in formation of Helix III with U6 snRNA (Fig. 8; Sun and Manley 1995).
The Ψ residue formed by Pus1p in U2 snRNA is highly conserved and involved in a U2–U6 interaction. (A) The branch site (BS) consensus sequences of the major introns from vertebrates and the yeast introns are shown. The branch site recognition sequences (BSRS) of U2 snRNAs are indicated, as well as the modified nucleotides present in this U2 snRNA segment (for review, see Massenet et al. 1998). The philogenetically conserved Ψ residue formed by Pus1p in yeast is circled. (B) The heterologous helices I, II, and III formed between the vertebrate U2 and U6 snRNAs are represented (Sun and Manley 1995; for review, see Madhani and Guthrie 1994). The post-transcriptional modifications are indicated, and the philogenetically conserved Ψ residue formed by Pus1p in yeast is circled (for review, see Massenet et al. 1998).
Comparison of yeast and mouse Pus1p activities on tRNAs
In the previous study, the recombinant mPus1p enzyme was found to modify residue U32 at low yield in an in vitro transcribed S. cerevisiae tRNAVal(UAC) (Chen and Patton 1999). Putative tRNA:Ψ32-synthase activity of mPus1p in yeast was verified in a strain carrying disruptions of both the PUS8 and PUS9 ORFs, encoding cytoplasmic and mitochondrial tRNA:Ψ32-synthases, respectively (Δpus8Δpus9 strain) (Behm-Ansmant et al. 2004). This strain was transformed with the p416GalS-mPUS1 plasmid and the CMCT/RT method was used to analyze the conversion of residue U32 into a pseudouridine in tRNAGly(GCC) and tRNAVal(CAC) (Fig. 1F, G). For both tRNAs, expression of mPus1p did not restore Ψ32 formation at a detectable level (not shown).
Our data show that the scPus1p and mPus1p enzymes show several common tRNA:Ψ-synthase activities. In yeast cells, they both act at positions 27, 34, and 36 in tRNAs and the previously proposed difference of specificity of scPus1p and mPus1p at position 32 in tRNAs is not confirmed by the present data. One of the positions (U27) modified by both enzymes is frequently modified in tRNAs. In contrast, in all the species studied until now, the presence of Ψ34 and Ψ36 residues was only detected in one tRNA (tRNAIle(UAU)) (Szweykowska-Kulinska et al. 1994). Although no clear experimental demonstration was obtained, the presence of these two Ψ residues in the tRNAIle anticodon may participate in translational fidelity (Senger et al. 1997; Auffinger and Westhof 1998). Formation of residues 34 and 36 in yeast tRNAIle(UAU) occurs in the nucleus and was found to be strictly dependent on the presence of the intron (Szweykowska-Kulinska et al. 1994). Hence, our observation of an mPus1p activity at these two positions in yeast suggests a strong conservation of the complex maturation process of tRNAIle(UAU). Unfortunately, knowledge on post-transcriptional modification in mouse tRNAs is rather limited (Sprinzl et al. 1998) and modifications in tRNAIle(UAU) were not studied in this species.
In yeast cells, in contrast to scPus1p, mPus1p was not found to act efficiently at position 28 in tRNATrp(CCA). However, it was active at this position in vitro. As pseudouridylation at both positions 27 and 28 was found to be absent in the cytoplasmic tRNASer(UGA) and mitochondrial tRNALys(UUU) from patients suffering from MLASA (Patton et al. 2005), hPus1p is expected to act at position 28 in human cells. The low activity of mPus1p at position 28 in the yeast tRNATrp(CCA) may be due to an unfavorable sequence of the anticodon stem-loop of this tRNA. Interestingly, previous experiments based on tRNA aminoacylation experiments suggested that residue Ψ28 in the yeast tRNATrp(CCA) is needed for stabilization of the anticodon stem, which is less stable compared to the corresponding stem-loop in bovine tRNA (Carnicelli et al. 2001). Therefore, one can imagine that in each of these species, the activity of Pus1p at position 28 in tRNAs is sensitive to the sequence of the anticodon stems of the tRNAs that are modified. On the other hand, tRNA modification is a complex multistep process that is accomplished by independently acting tRNA(RNA)-modification enzymes. In a few cases, the interdependence between tRNA modification at distinct positions has been demonstrated (Pintard et al. 2002). Thus, the observed difference between in vitro and in vivo activities of both enzymes may also be explained by the fact that, in a living cell, both enzymes act on partially modified pre-tRNA transcripts that are the real substrates of modification enzymes in vivo.
A second difference between scPus1p and mPus1p concerns position 26. Indeed, whereas scPus1p has an efficient in vivo tRNA:Ψ26 activity toward the tRNATrp(CCA) (Motorin et al. 1998; present data), mPus1p was not found to modify this position efficiently. In connection with this observation, it should be pointed out that pseudouridylation at position 26 is not a frequent modification in vertebrate tRNAs. In fact, only one sequenced human tRNA (tRNAMet(CAU)) has a Ψ26 residue, while five others bear unmodified U at this position (Sprinzl et al. 1998).
The third difference detected between the two enzymes concerns the possible additional tRNA:Ψ30-synthase activity of mPus1p. Obviously, other experiments, like a mPUS1 gene knockout, are required to confirm this activity in mouse. However, in agreement with our data, residue U30 is never pseudouridylated in yeast tRNAs, whereas a Ψ30 residue is found in the mouse tRNAIle(AAU) (Fig. 4B; Shinriki et al. 1981; Sprinzl et al. 1998; the tRNA database: http://www.uni-bayreuth.de/departments/biochemie/trna). Here again, this may be explained by divergence of the tRNA sequences in the course of evolution. In fact, the anticodon stems of the yeast tRNAIle(UAU) and mouse tRNAIle(AAU) are more similar than are the anticodon stems of yeast and mouse tRNAsIle(UAU) (Fig. 4B).
Apart from the above-mentioned differences, we demonstrated that scPus1p and mPus1p both carry the yet unassigned yeast tRNA:Ψ1-synthase activity and that these two proteins can act at this position in both precursor and mature tRNA molecules. The existence of a Ψ1 residue in mouse tRNAs has not been reported yet, but, as already mentioned, the knowledge on post-transcriptional modifications in mouse tRNAs is rather limited (Sprinzl et al. 1998). According to the mouse genomic sequence, 22 tRNAs (7 tRNAAsp, 4 tRNAGln, 8 tRNAGlu, 1 tRNASer, 1 tRNATyr, and 1 tRNAVal) are expected to have a U residue at position 1, and post-transcriptional modifications were only studied for one of them, namely, tRNAGlu(CUC), which lacks the modification. Thus, there are still 21 tRNA candidates for a possible modification at position 1.
Taken together, our data show a remarkable conservation of the RNA specificities of the Pus1p enzymes, with a few differences that are likely explained by their coevolution with tRNA sequences.
RNA substrate recognition by scPus1p and mPus1p
Both yeast and mouse RNA:Ψ-synthases Pus1p are multisite-specific modification enzymes acting at multiple sites in tRNAs and are also able to modify pre-tRNAs and U2 snRNA. Based on the previous data on the scPus1p and mPus1p, it was generally thought that both enzymes recognize a relatively simple structural domain that consists of an RNA helix with an internal bulge bearing the target uridine. The present observation of a Pus1p activity at position 1 in tRNA modifies the view of scPus1p and mPus1p specificities, since a simple RNA helix with an accessible uridine residue at its 5′ extremity is also a good substrate for both enzymes. Thus, both enzymes have very relaxed substrate specificities. Indeed, any accessible U residue seems to be a substrate for Pus1p modification, provided that an RNA helix is present in its vicinity. This relaxed substrate specificity does not exclude the possible existence of well-defined point identity determinants in the proximal RNA stem. Indeed, four yeast tRNAs bear U1 residue and thus are potential sc Pus1p substrates. However, only two out of these four tRNAs undergo in vivo conversion U to Ψ at position 1.
MATERIALS AND METHODS
Yeast strains
The haploid S. cerevisiae BY4742 strain (denoted wild-type in this paper) and the haploid strain carrying a disruption of the PUS1 gene (pus1Δ::Kanr, denoted Δpus1) have been obtained from the EUROSCARF collection (Germany). The S. cerevisiae BMA64-derived strain with deletion of the PUS8 and PUS9 genes was previously described (Behm-Ansmant et al. 2004).
mPus1p expression in yeast
For mPus1p expression in yeast, the mPus1p ORF was recovered by digestion of plasmid pET16b-mPUS1 (described by Chen and Patton 1999) with the XbaI and XhoI endonucleases and cloned into yeast p416GalS expression plasmid (Mumberg et al. 1995). The junction regions and the entire insert sequence of the recombinant plasmid (p416-mPUS1) were sequenced. In this construction, expression of the mouse ORF is under the control of the inducible GalS promoter. To produce the D112A mPus1p variant, a point mutation (GAC → GCC; Asp → Ala) was introduced by PCR-mediated site-directed mutagenesis using the Quick Change Kit (Stratagene). By using the standard lithium acetate procedure (Adams et al. 1997), the haploid BY4742-Δpus1 and BMA64 Δpus8-Δpus9 strains were transformed with the p416GalS plasmids containing either the wild-type or the mutated mPUS1 ORF. Transformed and untransformed strains were grown for 24 h in an Ura-Gal medium to induce expression of the transgene. Preparations of yeast cell-free S10 extracts for in vitro tRNA:Ψ-synthase activity assays and for preparation of total RNA extracts were done as described previously (Auxilien et al. 1996; Behm-Ansmant et al. 2003).
Analysis of in vivo pseudouridylations by CMCT/RT mapping
Total RNAs from the wild-type, Δpus1, Δpus1+p416-mPUS1, Δpus1+p416-mPUS1(D112A), Δpus8-Δpus9 and Δpus8-Δpus9+p416-mPUS1 yeast strains were prepared as described previously (Massenet et al. 1999). The Ψ residues in tRNAs and U2 snRNA were analyzed by modification with 1-cyclohexyl-3-[2-morpholinoethyl] carbodiimide metho-p-toluenesulfonate (CMCT) followed by reverse transcription with specific primer (CMCT-RT approach) (Bakin and Ofengand 1993; Massenet et al. 1999). To test for tRNA:Ψ−synthase activities at positions 26, 27, and 28 in yeast tRNATrp(CCA) (Keith et al. 1972) and 27, 34, and 36 in pre-tRNAIle(UAU), the oligonucleotides used as primers for reverse transcription were complementary to nucleotides 40–57 in tRNATrp(CCA) (O-tRNATrp) and to the pre-tRNAIle(UAU) intron (O-pre-tRNAIle), respectively. The tRNA:Ψ32-synthase activity was tested on cytoplasmic tRNAGly(GCC) and tRNAVal(CAC), using oligonucleotides complementary to positions 59–76. Sequences of tRNA substrates used in this study are presented in Figure 1.
Expression and purification of the recombinant mPus1p and scPus1p
ScPus1p and mPus1p were prepared as previously described (Simos et al. 1996; Chen and Patton 1999), except that the Escherichia coli strain BL21-CodonPlus(DE3)-RIL was used instead of the BL21 (DE3) pLysS strain.
Production of tRNA transcripts
Plasmid pT7U2Sc, kindly provided by P. Fabrizio (Max-Planck-Institute of Biophysical Chemistry, Göttingen, Germany), was used for in vitro transcription of S. cerevisiae U2 snRNA. To produce in vitro transcribed yeast cytoplasmic tRNAArg(ACG) and mitochondrial tRNAMeti(CAU), the coding sequences were amplified by polymerase chain reaction (PCR) using genomic DNA from the S. cerevisiae BMA64 strain. The sense oligonucleotide was designed in order to introduce a T7 RNA polymerase promoter. DNA templates for production of 5′− and 3′-extended tRNAArg transcripts were prepared by PCR amplification using oligonucleotides containing corresponding extensions. The amplified fragments were inserted at the SmaI restriction site of plasmid pUC18. The pTFM-Val plasmid used for in vitro transcription of cytoplasmic tRNAVal(UAC) was kindly provided by C. Florentz (IBMC, Strasbourg, France). Prior to in vitro transcription, the pUC18-tRNAArg, pUC19-tRNAMet i, and pTFM-Val plasmids were all cleaved with the BstNI nuclease. In vitro T7 RNA polymerase transcription with or without incorporation of the appropriate [α-32P]NTP and purification of the resulting RNA transcripts by electrophoresis on denaturing gel were performed as previously described (Jiang et al. 1997).
In vitro RNA:Ψ-synthase activity assays
The tRNA:Ψ-synthase activities of recombinant His6-mPus1p, His6-scPus1p, and of total cellular extracts were tested by incubation of the purified proteins or the S10 extracts with total RNA fractions extracted from Δpus1 strain or with in vitro produced yeast tRNAs and U2 snRNA transcripts. Total RNA (10 μg) or tRNA transcript (50–100 fmol) were incubated for 2 h with ~0.2 μg of the recombinant protein or 10 μL of S10 cellular extract (final concentration of about 0.5–1.0 mg of total protein/mL), in the following buffer: 100 mM Tris-HCl (pH 8.0), 100 mM ammonium acetate, 5 mM MgCl2, 2 mM DTT, 0.1 mM EDTA. After incubation, the modified RNAs were phenol extracted, precipitated by ethanol, and analyzed by either the CMCT/RT method or the nearest neighbor approach.
To study the tRNA:Ψ-synthase activities at position 1 in yeast cytoplasmic tRNAArg(ACG) and its precursors, the in vitro transcribed RNAs were labeled with [α-32P]UTP. For analysis of the tRNA:Ψ72-synthase activity by the nearest neighbor approach, the yeast mitochondrial tRNAMet i(CAU) was labeled with [α-32P]ATP. After modification, the tRNA transcripts were totally digested with RNase T2 and the produced 3′ NMPs were fractionated by two-dimensional thin layer chromatography (2D-TLC), as previously described (Jiang et al. 1997).
Analysis of Ψ1 residue formation by 5′ labeling of RNAs
The presence of Ψ1 residues in in vivo or in vitro modified tRNAs was analyzed by complete digestion of 5′-labeled tRNA fractions with nuclease P1, followed by 2D-TLC analysis of the resulting 5′-NMPs. Specific labeling of 5′-terminal nucleotides was achieved by dephosphorylation of the total tRNA fraction (∼20 μg) with alkaline phosphatase (1 U/μg) and subsequent 5′ labeling with [γ-32P]ATP and T4 polynucleotide kinase (2U) for 45 min at 37°C. To verify that the ΨMP detected in the 5′-extended tRNAArg(ACG) corresponded to modification at position 1, the modified transcript was analyzed by reverse transcription using an oligonucleotide complementary to bases 55–76 (O-tRNAArg).
Purification of a highly enriched fraction of yeast tRNAs was accomplished by high-performance anion-exchange chromatography on a MonoQ HR5/5 column (Amersham Biosciences) followed by PAGE on denaturing urea gels. The region of the gel corresponding to tRNAs was excised and the RNAs were eluted and precipitated by ethanol.
ACKNOWLEDGMENTS
We thank C. Florentz and R. Giegé (CNRS, Strasbourg, France) for helpful discussions and for providing plasmid containing the yeast tRNAVal coding region, P. Fabrizio (Max-Planck-Institute of Biophysical Chemistry, Göttingen, Germany), for providing plasmid pT7U2, and J. Ugolini for technical assistance. This work was supported by laboratory funds from the CNRS and the “Ministère de la jeunesse, de l’éducation nationale et de la recherche” and a Research and Productive Scholarship Grant from the University of South Carolina to J.R.P. I. Behm-Ansmant was a pre-doctoral fellow from the “Ministère de la jeunesse, de l’éducation nationale et de la recherche.”
Footnotes
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Reprint requests to: Yuri Motorin, Laboratoire Maturation des ARN et Enzymologie Moléculaire, UMR 7567 CNRS-UHP Nancy I, Faculté des Sciences, BP 239, 54506 Vandoeuvre-les-Nancy Cedex, France; e-mail: iouri.motorine{at}maem.uhp-nancy.fr; fax: 33.3.83.68.43.07.
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Article published online ahead of print. Article and publication date are at http://www.rnajournal.org/cgi/doi/10.1261/rna.100806.
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- Received March 30, 2006.
- Accepted May 18, 2006.
- Copyright © 2006 RNA Society