Publications

• The Saccharomyces cerevisiae U2 snRNA:pseudouridine-synthase Pus7p is a novel multisite-multisubstrate RNA:Psi-synthase also acting on tRNAs.

  Behm-Ansmant I., Urban A., Ma X., Yu Y.T., Motorin Y., Branlant C.

  The Saccharomyces cerevisiae Pus7 protein was recently characterized as a novel RNA:pseudouridine (Psi)-synthase acting at position 35 in U2 snRNA. However, U2 snRNA was the only potential substrate tested for this enzyme. In this work, we demonstrated that although Pus7p is responsible for the fo ... >> More

  The Saccharomyces cerevisiae Pus7 protein was recently characterized as a novel RNA:pseudouridine (Psi)-synthase acting at position 35 in U2 snRNA. However, U2 snRNA was the only potential substrate tested for this enzyme. In this work, we demonstrated that although Pus7p is responsible for the formation of only one of the six Psi residues present in yeast UsnRNAs, it catalyzes U to Psi conversion at position 13 in cytoplasmic tRNAs and at position 35 in pre-tRNA(Tyr). Sites of RNA modification by Pus7p were identified by analysis of the in vivo RNA modification defects resulting from the absence of active Pus7p production and by in vitro tests using extracts from WT and genetically modified yeast cells. For demonstration of the direct implication of Pus7p in RNA modification, the activity of the WT and mutated Pus7p recombinant proteins was tested on in vitro produced tRNA and pre-tRNA transcripts. Mutation of an aspartic acid residue (D256) that is conserved in all Pus7 homologs abolishes the enzymatic activity both in vivo and in vitro. This suggests the direct involvement of D256 in catalysis. Target sites of Pus7p in RNAs share a common sequence Pu(G/C)UNPsiAPu (Pu = purine, N = any nucleotide), which is expected to be important for substrate recognition. Modification of tRNAs by Pus7p explains the presence of Pus7p homologs in archaea and some bacteria species, which do not have U2 snRNA, and in vertebrates, where Psi34 (equivalent to Psi35 in yeast) formation in U2 snRNA is an H/ACA snoRNA guided process. Our results increase the number of known RNA modification enzymes acting on different types of cellular RNAs. << Less

  RNA 9:1371-1382(2003) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• RNA sequence and two-dimensional structure features required for efficient substrate modification by the Saccharomyces cerevisiae RNA:{Psi}-synthase Pus7p.

  Urban A., Behm-Ansmant I., Branlant C., Motorin Y.

  The RNA:pseudouridine (Psi) synthase Pus7p of Saccharomyces cerevisiae is a multisite-specific enzyme that is able to modify U(13) in several yeast tRNAs, U(35) in the pre-tRNA(Tyr) (GPsiA), U(35) in U2 small nuclear RNA, and U(50) in 5 S rRNA. Pus7p belongs to the universally conserved TruD-like ... >> More

  The RNA:pseudouridine (Psi) synthase Pus7p of Saccharomyces cerevisiae is a multisite-specific enzyme that is able to modify U(13) in several yeast tRNAs, U(35) in the pre-tRNA(Tyr) (GPsiA), U(35) in U2 small nuclear RNA, and U(50) in 5 S rRNA. Pus7p belongs to the universally conserved TruD-like family of RNA:Psi-synthases found in bacteria, archaea, and eukarya. Although several RNA substrates for yeast Pus7p have been identified, specificity of their recognition and modification has not been studied. However, conservation of a 7-nt-long sequence, including the modified U residue, in all natural Pus7p substrates suggested the importance of these nucleotides for Pus7p recognition and/or catalysis. Using site-directed mutagenesis, we designed a set of RNA variants derived from the yeast tRNA(Asp)(GUC), pre-tRNA(Tyr)(GPsiA), and U2 small nuclear RNA and tested their ability to be modified by Pus7p in vitro. We demonstrated that the highly conserved U(-2) and A(+1) residues (nucleotide numbers refer to target U(0)) are crucial identity elements for efficient modification by Pus7p. Nucleotide substitutions at other surrounding positions (-4, -3, +2, +3) have only a moderate effect. Surprisingly, the identity of the nucleotide immediately 5' to the target U(0) residue (position -1) is not important for efficient modification. Alteration of tRNA three-dimensional structure had no detectable effect on Pus7p activity at position 13. However, our results suggest that the presence of at least one stem-loop structure including or close to the target U nucleotide is required for Pus7p-catalyzed modification. << Less

  J. Biol. Chem. 284:5845-5858(2009) [PubMed] [EuropePMC]

• X-ray structure of tRNA pseudouridine synthase TruD reveals an inserted domain with a novel fold.

  Ericsson U.B., Nordlund P., Hallberg B.M.

  Pseudouridine synthases catalyse the isomerisation of uridine to pseudouridine in structural RNA. The pseudouridine synthase TruD, that modifies U13 in tRNA, belongs to a recently identified and large family of pseudouridine synthases present in all kingdoms of life. We report here the crystal str ... >> More

  Pseudouridine synthases catalyse the isomerisation of uridine to pseudouridine in structural RNA. The pseudouridine synthase TruD, that modifies U13 in tRNA, belongs to a recently identified and large family of pseudouridine synthases present in all kingdoms of life. We report here the crystal structure of Escherichia coli TruD at 2.0 A resolution. The structure reveals an overall V-shaped molecule with an RNA-binding cleft formed between two domains: a catalytic domain and an insertion domain. The catalytic domain has a fold similar to that of the catalytic domains of previously characterised pseudouridine synthases, whereas the insertion domain displays a novel fold. << Less

  FEBS Lett. 565:59-64(2004) [PubMed] [EuropePMC]

• Enzymatic characterization and mutational studies of TruD--the fifth family of pseudouridine synthases.

  Chan C.M., Huang R.H.

  Pseudouridine (Psi) is formed through isomerization of uridine (U) catalyzed by a class of enzymes called pseudouridine synthases (PsiS). TruD is the fifth family of PsiS. Studies of the first four families (TruA, TruB, RsuA, and RluA) of PsiS reveal a conserved Asp and Tyr are critical for cataly ... >> More

  Pseudouridine (Psi) is formed through isomerization of uridine (U) catalyzed by a class of enzymes called pseudouridine synthases (PsiS). TruD is the fifth family of PsiS. Studies of the first four families (TruA, TruB, RsuA, and RluA) of PsiS reveal a conserved Asp and Tyr are critical for catalysis. However, in TruD family, the tyrosine is not conserved. In this study, we measured the enzymatic parameters for TruD in Escherichia coli, and carried out enzymatic assays for a series of single, double, and triple TruD mutants. Our studies indicate that a Glu, strictly conserved in only TruD family is likely to be the general base in TruD. We also proposed a possible distinct mechanism of TruD-catalyzed Psi formation compared to the first four families. << Less

  Arch. Biochem. Biophys. 489:15-19(2009) [PubMed] [EuropePMC]

• A novel unanticipated type of pseudouridine synthase with homologs in bacteria, archaea, and eukarya.

  Kaya Y., Ofengand J.

  Putative pseudouridine synthase genes are members of a class consisting of four subgroups that possess characteristic amino acid sequence motifs. These genes have been found in all organisms sequenced to date. In Escherichia coli, 10 such genes have been identified, and the 10 synthase gene produc ... >> More

  Putative pseudouridine synthase genes are members of a class consisting of four subgroups that possess characteristic amino acid sequence motifs. These genes have been found in all organisms sequenced to date. In Escherichia coli, 10 such genes have been identified, and the 10 synthase gene products have been shown to function in making all of the pseudouridines found in tRNA and ribosomal RNA except for tRNA(Glu) pseudouridine13. In this work, a protein able to make this pseudouridine was purified by standard biochemical procedures. Amino-terminal sequencing of the isolated protein identified the synthase as YgbO. Deletion of the ygbO gene caused the loss of tRNA(Glu) pseudouridine13 and plasmid-borne restoration of the structural gene restored pseudouridine13. Reaction of the overexpressed gene product, renamed TruD, with a tRNA(Glu) transcript made in vitro also yielded only pseudouridine13. A search of the database detected 58 homologs of TruD spanning all three phylogenetic domains, including ancient organisms. Thus, we have identified a new wide-spread class of pseudouridine synthase with no sequence homology to the previously known four subgroups. The only completely conserved sequence motif in all 59 organisms that contained aspartate was GXKD, in motif II. This aspartate was essential for in vitro activity. << Less

  RNA 9:711-721(2003) [PubMed] [EuropePMC]