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James Ofengand - One of the best experts on this subject based on the ideXlab platform.
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the Pseudouridine synthase rlud is required for normal ribosome assembly and function in escherichia coli
RNA, 2005Co-Authors: Nancy S Gutgsell, Murray P Deutscher, James OfengandAbstract:RluD is the Pseudouridine synthase responsible for the formation of Psi1911, Psi1915, and Psi1917 in Escherichia coli 23S rRNA. Previous work from our laboratory demonstrated that disruption of the rluD gene and/or loss of the Pseudouridine residues for which it is responsible resulted in a severe growth phenotype. In the current work we have examined further the effect of the loss of the RluD protein and its product Pseudouridine residues in a deletion strain lacking the rluD gene. This strain exhibits defects in ribosome assembly, biogenesis, and function. Specifically, there is a deficit of 70S ribosomes, an increase in 50S and 30S subunits, and the appearance of new 62S and 39S particles. Analysis of the 39S particles indicates that they are immature precursors of the 50S subunits, whereas the 62S particles are derived from the breakdown of unstable 70S ribosomes. In addition, purified mutant 70S ribosomes were found to be somewhat less efficient than wild type in protein synthesis. The defect in ribosome assembly and resulting growth phenotype of the mutant could be restored by expression of wild-type RluD and synthesis of Psi1911, Psi1915, and Psi1917 residues, but not by catalytically inactive mutant RluD proteins, incapable of Pseudouridine formation. The data suggest that the loss of the Pseudouridine residues can account for all aspects of the mutant phenotype; however, a possible second function of the RluD synthase is also discussed.
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number position and significance of the Pseudouridines in the large subunit ribosomal rna of haloarcula marismortui and deinococcus radiodurans
RNA, 2005Co-Authors: Mark Del Campo, Claudia Recinos, Giscard Yanez, Steven C Pomerantz, Rebecca Guymon, Pamela F Crain, James A Mccloskey, James OfengandAbstract:The number and position of the Pseudouridines of Haloarcula marismortui and Deinococcus radiodurans large subunit RNA have been determined by a combination of total nucleoside analysis by HPLC-mass spectrometry and Pseudouridine sequencing by the reverse transcriptase method and by LC/MS/MS. Three Pseudouridines were found in H. marismortui, located at positions 1956, 1958, and 2621 corresponding to Escherichia coli positions 1915, 1917, and 2586, respectively. The three Pseudouridines are all in locations found in other organisms. Previous reports of a larger number of Pseudouridines in this organism were incorrect. Three Pseudouridines and one 3-methyl Pseudouridine (m 3 ) were found in D. radiodurans 23S RNA at positions 1894, 1898 (m 3 ), 1900, and 2584, the m 3 site being determined by a novel application of mass spectrometry. These positions correspond to E. coli positions 1911, 1915, 1917, and 2605, which are also Pseudouridines in E. coli (1915 is m 3 ). The Pseudouridines in the helix 69 loop, residues 1911, 1915, and 1917, are in positions highly conserved among all phyla. Pseudouridine 2584 in D. radiodurans is conserved in eubacteria and a chloroplast but is not found in archaea or eukaryotes, whereas Pseudouridine 2621 in H. marismortui is more conserved in eukaryotes and is not found in eubacteria. All the pseudoridines are near, but not exactly at, nucleotides directly involved in various aspects of ribosome function. In addition, two D. radiodurans synthases responsible for the four were identified.
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a novel unanticipated type of Pseudouridine synthase with homologs in bacteria archaea and eukarya
RNA, 2003Co-Authors: Yusuf Kaya, James OfengandAbstract: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.
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ribosomal rna Pseudouridines and Pseudouridine synthases
FEBS Letters, 2002Co-Authors: James OfengandAbstract:Pseudouridines are found in virtually all ribosomal RNAs but their function is unknown. There are four to eight times more Pseudouridines in eukaryotes than in eubacteria. Mapping 19 Haloarcula marismortui Pseudouridines on the three-dimensional 50S subunit does not show clustering. In bacteria, specific enzymes choose the site of Pseudouridine formation. In eukaryotes, and probably also in archaea, selection and modification is done by a guide RNA-protein complex. No unique specific role for ribosomal Pseudouridines has been identified. We propose that Pseudouridine's function is as a molecular glue to stabilize required RNA conformations that would otherwise be too flexible.
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a second function for Pseudouridine synthases a point mutant of rlud unable to form Pseudouridines 1911 1915 and 1917 in escherichia coli 23s ribosomal rna restores normal growth to an rlud minus strain
RNA, 2001Co-Authors: Nancy S Gutgsell, Saumya Raychaudhuri, Mark Del Campo, James OfengandAbstract:This laboratory previously showed that truncation of the gene for RluD, the Escherichia coli Pseudouridine synthase responsible for synthesis of 23S rRNA Pseudouridines 1911, 1915, and 1917, blocks Pseudouridine formation and inhibits growth. We now show that RluD mutants at the essential aspartate 139 allow these two functions of RluD to be separated. In vitro, RluD with aspartate 139 replaced by threonine or asparagine is completely inactive. In vivo, the growth defect could be completely restored by transformation of an RluD-inactive strain with plasmids carrying genes for RluD with aspartate 139 replaced by threonine or asparagine. Pseudouridine sequencing of the 23S rRNA from these transformed strains demonstrated the lack of these Pseudouridines. Pseudoreversion, which has previously been shown to restore growth without Pseudouridine formation by mutation at a distant position on the chromosome, was not responsible because transformation with empty vector under identical conditions did not alter the growth rate.
Henri Grosjean - One of the best experts on this subject based on the ideXlab platform.
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formation of the conserved Pseudouridine at position 55 in archaeal trna
Nucleic Acids Research, 2006Co-Authors: Martine Roovers, Henri Grosjean, Caryn Hale, Catherine Tricot, Michael P Terns, Rebecca M Terns, Louis DroogmansAbstract:Pseudouridine (Psi) located at position 55 in tRNA is a nearly universally conserved RNA modification found in all three domains of life. This modification is catalyzed by TruB in bacteria and by Pus4 in eukaryotes, but so far the Psi55 synthase has not been identified in archaea. In this work, we report the ability of two distinct Pseudouridine synthases from the hyperthermophilic archaeon Pyrococcus furiosus to specifically modify U55 in tRNA in vitro. These enzymes are (pfu)Cbf5, a protein known to play a role in RNA-guided modification of rRNA, and (pfu)PsuX, a previously uncharacterized enzyme that is not a member of the TruB/Pus4/Cbf5 family of Pseudouridine synthases. (pfu)PsuX is hereafter renamed (pfu)Pus10. Both enzymes specifically modify tRNA U55 in vitro but exhibit differences in substrate recognition. In addition, we find that in a heterologous in vivo system, (pfu)Pus10 efficiently complements an Escherichia coli strain deficient in the bacterial Psi55 synthase TruB. These results indicate that it is probable that (pfu)Cbf5 or (pfu)Pus10 (or both) is responsible for the introduction of Pseudouridine at U55 in tRNAs in archaea. While we cannot unequivocally assign the function from our results, both possibilities represent unexpected functions of these proteins as discussed herein.
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lack of Pseudouridine 38 39 in the anticodon arm of yeast cytoplasmic trna decreases in vivo recoding efficiency
Journal of Biological Chemistry, 2002Co-Authors: Francois Lecointe, Olivier Namy, Isabelle Hatin, George Simos, Jeanpierre Rousset, Henri GrosjeanAbstract:Many different modified nucleotides are found in naturally occurring tRNA, especially in the anticodon region. Their importance for the efficiency of the translational process begins to be well documented. Here we have analyzed the in vivo effect of deleting genes coding for yeast tRNA-modifying enzymes, namely Pus1p, Pus3p, Pus4p, or Trm4p, on termination readthrough and +1 frameshift events. To this end, we have transformed each of the yeast deletion strains with a lacZ-luc dual-reporter vector harboring selected programmed recoding sites. We have found that only deletion of the PUS3 gene, encoding the enzyme that introduces Pseudouridines at position 38 or 39 in tRNA, has an effect on the efficiency of the translation process. In this mutant, we have observed a reduced readthrough efficiency of each stop codon by natural nonsense suppressor tRNAs. This effect is solely due to the absence of Pseudouridine 38 or 39 in tRNA because the inactive mutant protein Pus3[D151A]p did not restore the level of natural readthrough. Our results also show that absence of Pseudouridine 39 in the slippery tRNA(UAG)(Leu) reduces +1 frameshift efficiency. Therefore, the presence of Pseudouridine 38 or 39 in the tRNA anticodon arm enhances misreading of certain codons by natural nonsense tRNAs as well as promotes frameshifting on slippery sequences in yeast.
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Pseudouridine Mapping in the Saccharomyces cerevisiae Spliceosomal U Small Nuclear RNAs (snRNAs) Reveals that Pseudouridine Synthase Pus1p Exhibits a Dual Substrate Specificity for U2 snRNA and tRNA
Molecular and Cellular Biology, 1999Co-Authors: Séverine Massenet, Henri Grosjean, Yuri Motorin, Denis Lafontaine, Eduard Hurt, Christiane BranlantAbstract:Pseudouridine (Psi) residues were localized in the Saccharomyces cerevisiae spliceosomal U small nuclear RNAs (UsnRNAs) by using the chemical mapping method. In contrast to vertebrate UsnRNAs, S. cerevisiae UsnRNAs contain only a few Psi residues, which are located in segments involved in intermolecular RNA-RNA or RNA-protein interactions. At these positions, UsnRNAs are universally modified. When yeast mutants disrupted for one of the several Pseudouridine synthase genes (PUS1, PUS2, PUS3, and PUS4) or depleted in rRNA-Pseudouridine synthase Cbf5p were tested for UsnRNA Psi content, only the loss of the Pus1p activity was found to affect Psi formation in spliceosomal UsnRNAs. Indeed, Psi44 formation in U2 snRNA was abolished. By using purified Pus1p enzyme and in vitro-produced U2 snRNA, Pus1p is shown here to catalyze Psi44 formation in the S. cerevisiae U2 snRNA. Thus, Pus1p is the first UsnRNA Pseudouridine synthase characterized so far which exhibits a dual substrate specificity, acting on both tRNAs and U2 snRNA. As depletion of rRNA-Pseudouridine synthase Cbf5p had no effect on UsnRNA Psi content, formation of Psi residues in S. cerevisiae UsnRNAs is not dependent on the Cbf5p-snoRNA guided mechanism.
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the yeast trna Pseudouridine synthase pus1p displays a multisite substrate specificity
RNA, 1998Co-Authors: Yuri Motorin, George Simos, Eduard C Hurt, G Keith, C Simon, D Foiret, Henri GrosjeanAbstract: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.
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characterization of yeast protein deg1 as Pseudouridine synthase pus3 catalyzing the formation of psi 38 and psi 39 in trna anticodon loop
Journal of Biological Chemistry, 1998Co-Authors: Francois Lecointe, George Simos, Yuri Motorin, Anke Sauer, Eduard C Hurt, Henri GrosjeanAbstract:Abstract The enzymatic activity of yeast gene product Deg1 was identified using both disrupted yeast strain and cloned recombinant protein expressed in yeast and in Escherichia coli. The results show that the DEG1-disrupted yeast strain lacks synthase activity for the formation of Pseudouridines Ψ38and Ψ39 in tRNA whereas the other activities, specific for Ψ formation at positions 13, 27, 28, 32, 34, 35, 36, and 55 in tRNA, remain unaffected. Also, the His6-tagged recombinant yeast Deg1p expressed in E. coli as well as a protein fusion with protein A in yeast display the enzymatic activity only toward Ψ38 and Ψ39 formation in different tRNA substrates. Therefore, Deg1p is the third tRNA:Pseudouridine synthase (Pus3p) characterized so far in yeast. Disruption of theDEG1 gene is not lethal but reduces considerably the yeast growth rate, especially at an elevated temperature (37 °C). Deg1p localizes both in the nucleus and in the cytoplasm, as shown by immunofluorescence microscopy. Identification of the Pseudouridine residues present (or absent) in selected naturally occurring cytoplasmic and mitochondrial tRNAs from DEG1-disrupted strain points out a common origin of Ψ38- and Ψ39-synthesizing activity in both of these two cellular compartments. The sensitivity of Pus3p (Deg1p) activity to overall three-dimensional tRNA architecture and to a few individual mutations in tRNA was also studied. The results indicate the existence of subtle differences in the tRNA recognition by yeast Pus3p and by its homologous tRNA:Pseudouridine synthase truA from E. coli (initially called hisT or PSU-I gene product).
Eugene G. Mueller - One of the best experts on this subject based on the ideXlab platform.
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the Pseudouridine synthases proceed through a glycal intermediate
Journal of the American Chemical Society, 2016Co-Authors: Govardhan Reddy Veerareddygari, Sanjay K Singh, Eugene G. MuellerAbstract:The Pseudouridine synthases isomerize (U) in RNA to Pseudouridine (Ψ), and the mechanism that they follow has long been a question of interest. The recent elucidation of a product of the mechanistic probe 5-fluorouridine that had been epimerized to the arabino isomer suggested that the Ψ synthases might operate through a glycal intermediate formed by deprotonation of C2′. When that position in substrate U is deuterated, a primary kinetic isotope effect is observed, which indisputably indicates that the proposed deprotonation occurs during the isomerization of U to Ψ and establishes the mechanism followed by the Ψ synthases.
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The Pseudouridine Synthases Proceed through a Glycal Intermediate
2016Co-Authors: Govardhan Reddy Veerareddygari, Sanjay K. Singh, Eugene G. MuellerAbstract:The Pseudouridine synthases isomerize (U) in RNA to Pseudouridine (Ψ), and the mechanism that they follow has long been a question of interest. The recent elucidation of a product of the mechanistic probe 5-fluorouridine that had been epimerized to the arabino isomer suggested that the Ψ synthases might operate through a glycal intermediate formed by deprotonation of C2′. When that position in substrate U is deuterated, a primary kinetic isotope effect is observed, which indisputably indicates that the proposed deprotonation occurs during the isomerization of U to Ψ and establishes the mechanism followed by the Ψ synthases
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The products of 5-fluorouridine by the action of the Pseudouridine synthase TruB disfavor one mechanism and suggest another.
Journal of the American Chemical Society, 2011Co-Authors: Edward J. Miracco, Eugene G. MuellerAbstract:The Pseudouridine synthase TruB handles 5-fluorouridine in RNA as a substrate, converting it into two isomeric hydrated products. Unexpectedly, the two products differ not in the hydrated pyrimidine ring but in the pentose ring, which is epimerized to arabinose in the minor product. This inversion of stereochemistry at C2' suggests that Pseudouridine generation may proceed by a mechanism involving a glycal intermediate or that the previously proposed mechanism involving an acylal intermediate operates but with an added reaction manifold for 5-fluorouridine versus uridine. The arabino product strongly disfavors a mechanism involving a Michael addition to the pyrimidine ring.
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mechanistic investigations of the Pseudouridine synthase rlua using rna containing 5 fluorouridine
Biochemistry, 2006Co-Authors: Christopher S Hamilton, Todd M Greco, Caroline A Vizthum, Joy M Ginter, Murray V Johnston, Eugene G. MuellerAbstract:The pseuoduridine synthases (Ψ synthases) isomerize uridine (U) to Pseudouridine (Ψ) in RNA, and they fall into five families that share very limited sequence similarity but have the same overall fold and active-site architecture, including an essential Asp. The mechanism by which the Ψ synthases operate remains unknown, and mechanistic work has largely made use of RNA containing 5-fluorouridine (f5U) in place of U. The Ψ synthase TruA forms a covalent adduct with such RNA, and heat disruption of the adduct generates a hydrated product of f5U, which was reasonably concluded to result from the hydrolysis of an ester linkage between the essential Asp and f5U. In contrast, the Ψ synthase TruB, which is a member of a different family, does not form an adduct with f5U in RNA but catalyzes the rearrangement and hydration of the f5U, which labeling studies with [18O]water showed does not result from ester hydrolysis. To extend the line of mechanistic investigation to another family of Ψ synthases and an enzyme t...
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the Pseudouridine synthases revisiting a mechanism that seemed settled
Journal of the American Chemical Society, 2004Co-Authors: Christopher J. Spedaliere, Joy M Ginter, Murray V Johnston, Eugene G. MuellerAbstract:RNA containing 5-fluorouridine, [f 5U]RNA, has been used as a mechanistic probe for the Pseudouridine synthases, which convert uridine in RNA to its C-glycoside isomer, Pseudouridine. Hydrated products of f 5U were attributed to ester hydrolysis of a covalent complex between an essential aspartic acid residue and f 5U, and the results were construed as strong support for a mechanism involving Michael addition by the aspartic acid residue. Labeling studies with [18O]water are now reported that rule out such ester hydrolysis in one Pseudouridine synthase, TruB. The aspartic acid residue does not become labeled, and the hydroxyl group in the hydrated product of f 5U derives directly from solvent. The hydrated product, therefore, cannot be construed to support Michael addition during the conversion of uridine to Pseudouridine, but the results do not rule out such a mechanism. A hypothesis is offered for the seemingly disparate behavior of different Pseudouridine synthases toward [f 5U]RNA.
Michael W. Gray - One of the best experts on this subject based on the ideXlab platform.
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evolutionary appearance of genes encoding proteins associated with box h aca snornas cbf5p in euglena gracilis an early diverging eukaryote and candidate gar1p and nop10p homologs in archaebacteria
Nucleic Acids Research, 2000Co-Authors: Yohichi Watanabe, Michael W. GrayAbstract:A reverse transcription–polymerase chain reaction (RT–PCR) approach was used to clone a cDNA encoding the Euglena gracilis homolog of yeast Cbf5p, a protein component of the box H/ACA class of snoRNPs that mediate Pseudouridine formation in eukaryotic rRNA. Cbf5p is a putative Pseudouridine synthase, and the Euglena homolog is the first full-length Cbf5p sequence to be reported for an early diverging unicellular eukaryote (protist). Phylogenetic analysis of putative Pseudouridine synthase sequences confirms that archaebacterial and eukaryotic (including Euglena) Cbf5p proteins are specifically related and are distinct from the TruB/Pus4p clade that is responsible for formation of Pseudouridine at position 55 in eubacterial (TruB) and eukaryotic (Pus4p) tRNAs. Using a bioinformatics approach, we also identified archaebacterial genes encoding candidate homologs of yeast Gar1p and Nop10p, two additional proteins known to be associated with eukaryotic box H/ACA snoRNPs. These observations raise the possibility that Pseudouridine formation in archaebacterial rRNA may be dependent on analogs of the eukaryotic box H/ACA snoRNPs, whose evolutionary origin may therefore predate the split between Archaea (archaebacteria) and Eucarya (eukaryotes). Database searches further revealed, in archaebacterial and some eukaryotic genomes, two previously unrecognized groups of genes (here designated ‘PsuX’ and ‘PsuY’) distantly related to the Cbf5p/TruB gene family.
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Pseudouridine in rna what where how and why
Iubmb Life, 2000Co-Authors: Michael Charette, Michael W. GrayAbstract:Pseudouridine (5-ribosyluracil) is a ubiquitous yet enigmatic constituent of structural RNAs (transfer, ribosomal, small nuclear, and small nucleolar). Although Pseudouridine (psi) was the first modified nucleoside to be discovered in RNA, and is the most abundant, its biosynthesis and biological roles have remained poorly understood since its identification as a "fifth nucleoside" in RNA. Recently, a combination of biochemical, biophysical, and genetic approaches has helped to illuminate the structural consequences of psi in polyribonucleotides, the biochemical mechanism of U-->psi isomerization in RNA, and the role of modification enzymes (psi synthases) and box H/ACA snoRNAs, a class of eukaryotic small nucleolar RNAs, in the site-specific biosynthesis of psi. Through its unique ability to coordinate a structural water molecule via its free N1-H, psi exerts a subtle but significant "rigidifying" influence on the nearby sugar-phosphate backbone and also enhances base stacking. These effects may underlie the biological role of most (but perhaps not all) of the psi residues in RNA. Certain genetic mutants lacking specific psi residues in tRNA or rRNA exhibit difficulties in translation, display slow growth rates, and fail to compete effectively with wild-type strains in mixed culture. In particular, normal growth is severely compromised in an Escherichia coli mutant deficient in a Pseudouridine synthase responsible for the formation of three closely spaced psi residues in the mRNA decoding region of the 23S rRNA. Such studies demonstrate that pseudouridylation of RNA confers an important selective advantage in a natural biological context.
Christiane Branlant - One of the best experts on this subject based on the ideXlab platform.
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Comparative Study of Two Box H/ACA Ribonucleoprotein Pseudouridine-Synthases: Relation between Conformational Dynamics of the Guide RNA, Enzyme Assembly and Activity
2016Co-Authors: Jean-baptiste Fourmann. ¤a, Annesophie Tillault, Christiane Branlant, Magali Blaud. ¤b, Bruno CharpentierAbstract:Multiple RNA-guided Pseudouridine synthases, H/ACA ribonucleoprotein particles (RNPs) which contain a guide RNA and four proteins, catalyze site-specific post-transcriptional isomerization of uridines into Pseudouridines in substrate RNAs. In archaeal particles, the guide small RNA (sRNA) is anchored by the Pseudouridine synthase aCBF5 and the ribosomal protein L7Ae. Protein aNOP10 interacts with both aCBF5 and L7Ae. The fourth protein, aGAR1, interacts with aCBF5 and enhances catalytic efficiency. Here, we compared the features of two H/ACA sRNAs, Pab21 and Pab91, from Pyrococcus abyssi. We found that aCBF5 binds much more weakly to Pab91 than to Pab21. Surprisingly, the Pab91 sRNP exhibits a higher catalytic efficiency than the Pab21 sRNP. We thus investigated the molecular basis of the differential efficiencies observed for the assembly and catalytic activity of the two enzymes. For this, we compared profiles of the extent of lead-induced cleavages in these sRNAs during a stepwise reconstitution of the sRNPs, and analyzed the impact of the absence of the aNOP10–L7Ae interaction. Such probing experiments indicated that the sRNAs undergo a series of conformational changes upon RNP assembly. These changes were also evaluated directly by circular dichroism (CD) spectroscopy, a tool highly adapted to analyzing RNA conformational dynamics. In addition, our results reveal that the conformation of helix P1 formed at the base of the H/ACA sRNAs is optimized in Pab21 for efficient aCBF5 binding and RNP assembly. Moreover, P1 swapping improved the assembly of the Pab91 sRNP. Nonetheless, efficient aCBF5 binding probably also relies on the pseudouridylation pocke
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combining native ms approaches to decipher archaeal box h aca ribonucleoprotein particle structure and activity
Proteomics, 2015Co-Authors: Jeanmichel Saliou, Xavier Manival, Annesophie Tillault, Cedric Atmanene, Claude Bobo, Christiane Branlant, Alain Van DorsselaerAbstract:Site-specific isomerization of uridines into Pseudouridines in RNAs is catalyzed either by stand-alone enzymes or by box H/ACA ribonucleoprotein particles (sno/sRNPs). The archaeal box H/ACA sRNPs are five-component complexes that consist of a guide RNA and the aCBF5, aNOP10, L7Ae, and aGAR1 proteins. In this study, we performed pairwise incubations of individual constituents of archaeal box H/ACA sRNPs and analyzed their interactions by native MS to build a 2D-connectivity map of direct binders. We describe the use of native MS in combination with ion mobility-MS to monitor the in vitro assembly of the active H/ACA sRNP particle. Real-time native MS was used to monitor how box H/ACA particle functions in multiple-turnover conditions. Native MS also unambiguously revealed that a substrate RNA containing 5-fluorouridine (f(5) U) was hydrolyzed into 5-fluoro-6-hydroxy-Pseudouridine (f(5) ho(6) Ψ). In terms of enzymatic mechanism, box H/ACA sRNP was shown to catalyze the pseudouridylation of a first RNA substrate, then to release the RNA product (S22 f(5) ho(6) ψ) from the RNP enzyme and reload a new substrate RNA molecule. Altogether, our native MS-based approaches provide relevant new information about the potential assembly process and catalytic mechanism of box H/ACA RNPs.
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comparative study of two box h aca ribonucleoprotein Pseudouridine synthases relation between conformational dynamics of the guide rna enzyme assembly and activity
PLOS ONE, 2013Co-Authors: Jeanbaptiste Fourmann, Annesophie Tillault, Christiane Branlant, Magali Blaud, Fabrice Leclerc, Bruno CharpentierAbstract:Multiple RNA-guided Pseudouridine synthases, H/ACA ribonucleoprotein particles (RNPs) which contain a guide RNA and four proteins, catalyze site-specific post-transcriptional isomerization of uridines into Pseudouridines in substrate RNAs. In archaeal particles, the guide small RNA (sRNA) is anchored by the Pseudouridine synthase aCBF5 and the ribosomal protein L7Ae. Protein aNOP10 interacts with both aCBF5 and L7Ae. The fourth protein, aGAR1, interacts with aCBF5 and enhances catalytic efficiency. Here, we compared the features of two H/ACA sRNAs, Pab21 and Pab91, from Pyrococcus abyssi. We found that aCBF5 binds much more weakly to Pab91 than to Pab21. Surprisingly, the Pab91 sRNP exhibits a higher catalytic efficiency than the Pab21 sRNP. We thus investigated the molecular basis of the differential efficiencies observed for the assembly and catalytic activity of the two enzymes. For this, we compared profiles of the extent of lead-induced cleavages in these sRNAs during a stepwise reconstitution of the sRNPs, and analyzed the impact of the absence of the aNOP10-L7Ae interaction. Such probing experiments indicated that the sRNAs undergo a series of conformational changes upon RNP assembly. These changes were also evaluated directly by circular dichroism (CD) spectroscopy, a tool highly adapted to analyzing RNA conformational dynamics. In addition, our results reveal that the conformation of helix P1 formed at the base of the H/ACA sRNAs is optimized in Pab21 for efficient aCBF5 binding and RNP assembly. Moreover, P1 swapping improved the assembly of the Pab91 sRNP. Nonetheless, efficient aCBF5 binding probably also relies on the pseudouridylation pocket which is not optimized for high activity in the case of Pab21.
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Pseudouridine Mapping in the Saccharomyces cerevisiae Spliceosomal U Small Nuclear RNAs (snRNAs) Reveals that Pseudouridine Synthase Pus1p Exhibits a Dual Substrate Specificity for U2 snRNA and tRNA
Molecular and Cellular Biology, 1999Co-Authors: Séverine Massenet, Henri Grosjean, Yuri Motorin, Denis Lafontaine, Eduard Hurt, Christiane BranlantAbstract:Pseudouridine (Psi) residues were localized in the Saccharomyces cerevisiae spliceosomal U small nuclear RNAs (UsnRNAs) by using the chemical mapping method. In contrast to vertebrate UsnRNAs, S. cerevisiae UsnRNAs contain only a few Psi residues, which are located in segments involved in intermolecular RNA-RNA or RNA-protein interactions. At these positions, UsnRNAs are universally modified. When yeast mutants disrupted for one of the several Pseudouridine synthase genes (PUS1, PUS2, PUS3, and PUS4) or depleted in rRNA-Pseudouridine synthase Cbf5p were tested for UsnRNA Psi content, only the loss of the Pus1p activity was found to affect Psi formation in spliceosomal UsnRNAs. Indeed, Psi44 formation in U2 snRNA was abolished. By using purified Pus1p enzyme and in vitro-produced U2 snRNA, Pus1p is shown here to catalyze Psi44 formation in the S. cerevisiae U2 snRNA. Thus, Pus1p is the first UsnRNA Pseudouridine synthase characterized so far which exhibits a dual substrate specificity, acting on both tRNAs and U2 snRNA. As depletion of rRNA-Pseudouridine synthase Cbf5p had no effect on UsnRNA Psi content, formation of Psi residues in S. cerevisiae UsnRNAs is not dependent on the Cbf5p-snoRNA guided mechanism.
