The yeast tRNA:pseudouridine synthase Pus1p displays a multisite substrate specificity.

We have previously shown that the yeast gene PUS1 codes for a tRNA:pseudouridine synthase and that recombinant Pus1p catalyzes, in an intron-dependent way, the formation of psi34 and psi36 in the anticodon loop of the yeast minor tRNA(Ile) in vitro (Simos G et al., 1996, EMBO J 15:2270-2284). Using a set of T7 transcripts of different tRNA genes, we now demonstrate that yeast pseudouridine synthase 1 catalyzes in vitro pseudouridine formation at positions 27 and/or 28 in several yeast cytoplasmic tRNAs and at position 35 in the intron-containing tRNA(Tyr) (anticodon GUA). Thus, Pus1p not only displays a broad specificity toward the RNA substrates, but is also capable of catalyzing the pseudouridine (psi) formation at distinct noncontiguous sites within the same tRNA molecule. The cell-free extract prepared from the yeast strain bearing disrupted gene PUS1 is unable to catalyze the formation of psi27, psi28, psi34, and psi36 in vitro, however, psi35 formation in the intron-containing tRNA(Tyr)(GUA) remains unaffected. Thus, in yeast, only one gene product accounts for tRNA pseudouridylation at positions 27, 28, 34, and 36, whereas for position 35 in tRNA(Tyr), another site-specific tRNA:pseudouridine synthase with overlapping specificity exists. Mapping of pseudouridine residues present in various tRNAs extracted from the PUS1-disrupted strain confirms the in vitro data obtained with the recombinant Pus1p. In addition, they suggest that Pus1p is implicated in modification at positions U26, U65, and U67 in vivo.

[1]  V. Arluison,et al.  Transfer RNA-pseudouridine synthetase Pus1 of Saccharomyces cerevisiae contains one atom of zinc essential for its native conformation and tRNA recognition. , 1998, Biochemistry.

[2]  M. Fornerod,et al.  Identification of a nuclear export receptor for tRNA , 1998, Current Biology.

[3]  D. Tollervey,et al.  The box H + ACA snoRNAs carry Cbf5p, the putative rRNA pseudouridine synthase. , 1998, Genes & development.

[4]  G. Lipowsky,et al.  Identification of a tRNA-specific nuclear export receptor. , 1998, Molecular cell.

[5]  E. Hurt,et al.  Characterization of Yeast Protein Deg1 as Pseudouridine Synthase (Pus3) Catalyzing the Formation of Ψ38 and Ψ39 in tRNA Anticodon Loop* , 1998, The Journal of Biological Chemistry.

[6]  D. Santi,et al.  A Conserved Aspartate of tRNA Pseudouridine Synthase Is Essential for Activity and a Probable Nucleophilic Catalyst , 1998 .

[7]  D. Santi,et al.  Molecular recognition of tRNA by tRNA pseudouridine 55 synthase. , 1998, Biochemistry.

[8]  Maurille J. Fournier,et al.  The Pseudouridine Residues of rRNA: Number, Location, Biosynthesis, and Function , 1998 .

[9]  C. Branlant,et al.  Posttranscriptional Modifications in the U Small Nuclear RNAs , 1998 .

[10]  E. Hurt,et al.  A conserved domain within Arc1p delivers tRNA to aminoacyl-tRNA synthetases. , 1998, Molecular cell.

[11]  D. Santi,et al.  A conserved aspartate of tRNA pseudouridine synthase is essential for activity and a probable nucleophilic catalyst. , 1998, Biochemistry.

[12]  C. Florentz,et al.  Major identity determinants for enzymatic formation of ribothymidine and pseudouridine in the T psi-loop of yeast tRNAs. , 1997, Journal of molecular biology.

[13]  H. Beier,et al.  Characterization of nuclear tRNATyr introns: their evolution from red algae to higher plants , 1997, FEBS letters.

[14]  R. Planta,et al.  The yeast gene YNL292w encodes a pseudouridine synthase (Pus4) catalyzing the formation of psi55 in both mitochondrial and cytoplasmic tRNAs. , 1997, Nucleic acids research.

[15]  M. Caizergues-Ferrer,et al.  A small nucleolar RNP protein is required for pseudouridylation of eukaryotic ribosomal RNAs , 1997, The EMBO journal.

[16]  Y. Motorin,et al.  Pleiotropic effects of intron removal on base modification pattern of yeast tRNAPhe: an in vitro study. , 1997, Nucleic acids research.

[17]  Tamás Kiss,et al.  Site-Specific Pseudouridine Formation in Preribosomal RNA Is Guided by Small Nucleolar RNAs , 1997, Cell.

[18]  J. Steitz,et al.  Sno Storm in the Nucleolus: New Roles for Myriad Small RNPs , 1997, Cell.

[19]  J. Ni,et al.  Small Nucleolar RNAs Direct Site-Specific Synthesis of Pseudouridine in Ribosomal RNA , 1997, Cell.

[20]  F. Fasiolo,et al.  Intron-dependent enzymatic formation of modified nucleosides in eukaryotic tRNAs: a review. , 1997, Biochimie.

[21]  S. Auxilien,et al.  Mechanism, specificity and general properties of the yeast enzyme catalysing the formation of inosine 34 in the anticodon of transfer RNA. , 1996, Journal of molecular biology.

[22]  D. Santi,et al.  Identification of new RNA modifying enzymes by iterative genome search using known modifying enzymes as probes. , 1996, Nucleic acids research.

[23]  E V Koonin,et al.  Pseudouridine synthases: four families of enzymes containing a putative uridine-binding motif also conserved in dUTPases and dCTP deaminases. , 1996, Nucleic acids research.

[24]  D. Tollervey,et al.  Nuclear pore proteins are involved in the biogenesis of functional tRNA. , 1996, The EMBO journal.

[25]  J. Krajewski,et al.  Mutations of Arabidopsis thaliana pre-tRNA(Tyr) affecting pseudouridylation of U35. , 1995, Biochimica et biophysica acta.

[26]  Andrei V Bakin,et al.  Mapping of the 13 pseudouridine residues in Saccharomyces cerevisiae small subunit ribosomal RNA to nucleotide resolution. , 1995, Nucleic acids research.

[27]  J. Ofengand,et al.  A dual-specificity pseudouridine synthase: an Escherichia coli synthase purified and cloned on the basis of its specificity for psi 746 in 23S RNA is also specific for psi 32 in tRNA(phe). , 1995, RNA.

[28]  J. Ofengand,et al.  Purification, cloning, and properties of the tRNA psi 55 synthase from Escherichia coli. , 1995, RNA.

[29]  B. Senger,et al.  Intron‐dependent formation of pseudouridines in the anticodon of Saccharomyces cerevisiae minor tRNA(Ile). , 1994, The EMBO journal.

[30]  J. Kowalak,et al.  The single pseudouridine residue in Escherichia coli 16S RNA is located at position 516. , 1994, Nucleic acids research.

[31]  J Ofengand,et al.  Four newly located pseudouridylate residues in Escherichia coli 23S ribosomal RNA are all at the peptidyltransferase center: analysis by the application of a new sequencing technique. , 1993, Biochemistry.

[32]  A. Hopper,et al.  The Saccharomyces cerevisiae LOS1 gene involved in pre-tRNA splicing encodes a nuclear protein that behaves as a component of the nuclear matrix. , 1993, The Journal of biological chemistry.

[33]  Mathias Sprinzl,et al.  Compilation of tRNA sequences and sequences of tRNA genes , 1993, Nucleic Acids Res..

[34]  B. Senger,et al.  The anticodon triplet is not sufficient to confer methionine acceptance to a transfer RNA. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[35]  G. Keith,et al.  Rapid transfer of small RNAs from a polyacrylamide gel onto a nylon membrane using a gel dryer. , 1992, Nucleic acids research.

[36]  Z. Szweykowska-Kulinska,et al.  Sequence and structure requirements for the biosynthesis of pseudouridine (psi 35) in plant pre‐tRNA(Tyr). , 1992, The EMBO journal.

[37]  L. Marechal-Drouard,et al.  Use of a dot blot hybridization method for identification of pure tRNA species on different membranes. , 1992, Biochimica et biophysica acta.

[38]  J. Ebel,et al.  Conformation in solution of yeast tRNA(Asp) transcripts deprived of modified nucleotides. , 1990, Biochimie.

[39]  T. Samuelsson,et al.  Transfer RNA pseudouridine synthases in Saccharomyces cerevisiae. , 1990, The Journal of biological chemistry.

[40]  O. Uhlenbeck,et al.  Role of the tertiary nucleotides in the interaction of yeast phenylalanine tRNA with its cognate synthetase. , 1990, Biochemistry.

[41]  G. Keith.  Chapter 3 Nucleic Acid Chromatographic Isolation and Sequence Methods , 1990 .

[42]  C. C. Marvel,et al.  Purification, structure, and properties of Escherichia coli tRNA pseudouridine synthase I. , 1988, The Journal of biological chemistry.

[43]  H. Swerdlow,et al.  Structure of intron-containing tRNA precursors. Analysis of solution conformation using chemical and enzymatic probes. , 1984, The Journal of biological chemistry.

[44]  J. Abelson,et al.  The yeast tRNATyr gene intron is essential for correct modification of its tRNA product , 1983, Nature.

[45]  K. Waltersson,et al.  The crystal structure of Cs[VOF3] · 12H2O , 1979 .

[46]  H. Feldmann,et al.  Mapping of yeast tRNAs by two‐dimensional electrophoresis on polyacrylamide gels , 1975, FEBS letters.

[47]  W. Wooster,et al.  Crystal structure of , 2005 .

[48]  M. Stephenson,et al.  The preparation and some properties of a low molecular weight ribonucleic acid from baker's yeast. , 1960, Biochimica et biophysica acta.