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 Psi 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:Psi26-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:Psi synthase activities. In addition, our results reveal an additional activity of mPus1p at position 30 in tRNA that scPus1p does not possess.

FIGURE 1.

Sequences and secondary structures of the S. cerevisiae tRNA substrates used in this work. The various panels represent, respectively, the tRNA^Trp(CCA) (A), the pre-tRNA^Ile(UAU) (B), the mature tRNA^Arg(ACG) (C), the 5′p3′p tRNA^Arg(ACG) with 5′ and 3′ extensions mimicking a tRNA precursor (D), mitochondrial tRNA^Met _i(CAU) (E), tRNA^Val(CAC) (F), and tRNA^Gly(GCC) (G). All tRNAs (except tRNA^Arg(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-tRNA^Ile is shown in small characters and small arrows indicate the exon–intron borders. The Ψ residues studied during this work are circled.

FIGURE 2.

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.

FIGURE 3.

Analysis of in vivo mPus1p activity on yeast tRNA^Trp. 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-tRNA^Trp 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.

FIGURE 4.

Analysis of in vivo mPus1p activity on yeast pre-tRNA^Ile. 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-tRNA^Ile 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-tRNA^Ile(UAU), mouse tRNA^Ile(AAU), and mouse pre-tRNA^Ile(UAU) anticodon stem-loops. Positions of introns in the pre-tRNAs are indicated.

FIGURE 5.

Analyses of the tRNA:Ψ1-synthase activity in vitro and in vivo. (A) In vitro transcribed yeast tRNA^Arg 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 tRNA^Arg 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.

FIGURE 6.

Analyses of the in vitro tRNA:Ψ1-synthase activity of recombinant scPus1p and mPus1p enzymes on yeast tRNA^Arg variants. In vitro transcribed RNA substrates, corresponding to yeast mature tRNA^Arg (A), tRNA^Arg with a 3′ extension (5′m3′p) (B), tRNA^Arg with a 5′ extension (5′p3′m) (C), and tRNA^Arg 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.

FIGURE 7.

CMCT/RT mapping of Ψ residues formed in a 5′-extended tRNA^Arg transcript (5′p3′m). An unlabeled 5′p3′m tRNA^Arg 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-tRNA^Arg 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.

FIGURE 8.

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).