Publications (15)78.84 Total impact

  • [Show abstract] [Hide abstract] ABSTRACT: Degradation of RNA as an intermediate message between genes and corresponding proteins is important for rapid attenuation of gene expression and maintenance of cellular homeostasis. This process is controlled by ribonucleases that have different target specificities. In the bacterial pathogen Helicobacter pylori, an exo- and endoribonuclease RNase J is essential for growth. To explore the role of RNase J in H. pylori, we identified its putative targets at a global scale using next generation RNA sequencing. We found that strong depletion for RNase J led to a massive increase in the steady-state levels of non-ribosomal RNAs. Messenger RNAs and RNAs antisense to open reading frames were most affected with over 80% increased more than two-fold. Non-coding RNAs expressed in the intergenic regions are much less affected by RNase J depletion. Northern blotting of selected messenger and non-coding RNAs validated these results. Globally, our data suggest that RNase J of H. pylori is a major RNase involved in degradation of most cellular RNAs.
    No preview · Article · Jan 2016 · RNA biology
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    [Show abstract] [Hide abstract] ABSTRACT: Protein complexes directing messenger RNA (mRNA) degradation are present in all kingdoms of life. In Escherichia coli, mRNA degradation is performed by an RNA degradosome organized by the major ribonuclease RNase E. In bacteria lacking RNase E, the existence of a functional RNA degradosome is still an open question. Here, we report that in the bacterial pathogen Helicobacter pylori, RNA degradation is directed by a minimal RNA degradosome consisting of Hp-RNase J and the only DExD-box RNA helicase of H. pylori, RhpA. We show that the protein complex promotes faster degradation of double-stranded RNA in vitro in comparison with Hp-RNase J alone. The ATPase activity of RhpA is stimulated in the presence of Hp-RNase J, demonstrating that the catalytic capacity of both partners is enhanced upon interaction. Remarkably, both proteins are associated with translating ribosomes and not with individual 30S and 50S subunits. Moreover, Hp-RNase J is not recruited to ribosomes to perform rRNA maturation. Together, our findings imply that in H. pylori, the mRNA-degrading machinery is associated with the translation apparatus, a situation till now thought to be restricted to eukaryotes and archaea.
    Full-text · Article · Oct 2012 · Nucleic Acids Research
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    Yulia Redko · Ciarán Condon
    [Show abstract] [Hide abstract] ABSTRACT: 23S rRNA maturation in Bacillus subtilis is catalyzed by the recently characterized enzyme Mini-RNase-III. Mini-III is dispensable, however, and 23S rRNA is matured by other ribonucleases in strains lacking this enzyme. Here we show that these RNases are the 5′-to-3′ exoribonuclease RNase J1 and the 3′-to-5′ exoribonucleases, principally RNase PH and YhaM.
    Preview · Article · Oct 2009 · Journal of bacteriology
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    Yulia Redko · Ciarán Condon
    [Show abstract] [Hide abstract] ABSTRACT: Ribosomal RNAs (rRNAs) are processed from larger primary transcripts in every living system known. The maturation of 23S rRNA in Bacillus subtilis is catalysed by Mini-III, a member of the RNase III family of enzymes that lacks the characteristic double-stranded RNA binding domain of its relatives. We have previously shown that Mini-III processing of 23S precursor rRNA in assembled 50S ribosomal subunits is much more efficient than a substrate with no ribosomal proteins bound, suggesting that one or more large subunit proteins act as a cofactor for Mini-III cleavage. Here we show that this cofactor is ribosomal protein L3. Stimulation of the Mini-III cleavage reaction is through L3 binding to its normal site at the 3' end of 23S rRNA. We present indirect evidence that suggests that L3 acts at the level of substrate, rather than enzyme conformation. We also discuss the potential implication of using ribosomal protein cofactors in rRNA processing for ribosome quality control.
    Preview · Article · Feb 2009 · Molecular Microbiology
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    Yulia Redko · David H Bechhofer · Ciarán Condon
    [Show abstract] [Hide abstract] ABSTRACT: The late steps of both 16S and 5S ribosomal RNA maturation in the Gram-positive bacterium Bacillus subtilis have been shown to be catalysed by ribonucleases that are not present in the Gram-negative paradigm, Escherichia coli. Here we present evidence that final maturation of the 5' and 3' extremities of B. subtilis 23S rRNA is also performed by an enzyme that is absent from the Proteobacteria. Mini-III contains an RNase III-like catalytic domain, but curiously lacks the double-stranded RNA binding domain typical of RNase III itself, Dicer, Drosha and other well-known members of this family of enzymes. Cells lacking Mini-III accumulate precursors and alternatively matured forms of 23S rRNA. We show that Mini-III functions much more efficiently on precursor 50S ribosomal subunits than naked pre-23S rRNA in vitro, suggesting that maturation occurs primarily on assembled subunits in vivo. Lastly, we provide a model for how Mini-III recognizes and cleaves double-stranded RNA, despite lacking three of the four RNA binding motifs of RNase III.
    Preview · Article · Jul 2008 · Molecular Microbiology
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    [Show abstract] [Hide abstract] ABSTRACT: The mode of abscisic acid (ABA) action, and its relations to drought adaptive responses in particular, has been a captivating area of plant hormone research for much over a decade. The hormone triggers stomatal closure to limit water loss through transpiration, as well as mobilizes a battery of genes that presumably serve to protect the cells from the ensuing oxidative damage in prolonged stress. The signaling network orchestrating these various responses is, however, highly complex. This review summarizes several significant advances made within the last few years. The biosynthetic pathway of the hormone is now almost completely elucidated, with the latest identification of the ABA4 gene encoding a neoxanthin synthase, which seems essential for de novo ABA biosynthesis during water stress. This leads to the interesting question on how ABA is then delivered to perception sites. In this respect, regulated transport has attracted renewed focus by the unexpected finding of a shoot-to-root translocation of ABA during drought response, and at the cellular level, by the identification of a beta-galactosidase that releases biologically active ABA from inactive ABA-glucose ester. Surprising candidate ABA receptors were also identified in the form of the Flowering Time Control Protein A (FCA) and the Chloroplastic Magnesium Protoporphyrin-IX Chelatase H subunit (CHLH) in chloroplast-nucleus communication, both of which have been shown to bind ABA in vitro. On the other hand, the protein(s) corresponding to the physiologically detectable cell-surface ABA receptor(s) is (are) still not known with certainty. Genetic and physiological studies based on the guard cell have reinforced the central importance of reversible phosphorylation in modulating rapid ABA responses. Sucrose Non-Fermenting Related Kinases (SnRK), Calcium-Dependent Protein Kinases (CDPK), Protein Phosphatases (PP) of the 2C and 2A classes figure as prominent regulators in this single-cell model. Identifying their direct in vivo targets of regulation, which may include H(+)-ATPases, ion channels, 14-3-3 proteins and transcription factors, will logically be the next major challenge. Emerging evidence also implicates ABA as a repressor of innate immune response, as hinted by the highly similar roster of genes elicited by certain pathogens and ABA. Undoubtedly, the most astonishing revelation is that ABA is not restricted to plants and mosses, but overwhelming evidence now indicates that it also exists in metazoans ranging from the most primitive to the most advance on the evolution scale (sponges to humans). In metazoans, ABA has healing properties, and plays protective roles against both environmental and pathogen related injuries. These cross-kingdom comparisons have shed light on the surprising ancient origin of ABA and its attendant mechanisms of signal transduction.
    Full-text · Article · Mar 2008 · Molecular Plant
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    [Show abstract] [Hide abstract] ABSTRACT: Significant progress has been made recently regarding the identification of the ribonucleases involved in RNA maturation and degradation in Bacillus subtilis. More than half of these enzymes have no ortholog in Escherichia coli. To confirm that the in vivo effects of mutations in genes encoding RNases are direct, it is often necessary to purify the enzymes and assay their activity in vitro. Development of such assays is also necessary for detailed biochemical analysis of enzyme properties. In this chapter, we describe the purification and assay of 12 RNases of B. subtilis thought to be involved in stable RNA maturation or RNA degradation.
    Full-text · Article · Feb 2008 · Methods in enzymology
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    [Show abstract] [Hide abstract] ABSTRACT: Significant progress has been made recently regarding the identification of the ribonucleases involved in RNA maturation and degradation in Bacillus subtilis. More than half of these enzymes have no ortholog in Echerichia coli. To confirm that the in vivo effects of mutations in genes encoding RNases are direct, it is often necessary to purify the enzymes and assay their activity in vitro. Development of such assays is also necessary for detailed biochemical analysis of enzyme properties. In this chapter, we describe the purification and assay of 12 RNases of B. subtilis thought to be involved in stable RNA maturation or RNA degradation.
    Full-text · Article · Jan 2008
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    [Show abstract] [Hide abstract] ABSTRACT: YjgB is one of five peptidoglycan hydrolases previously identified in Lactococcus lactis. Analysis of its amino acid sequence revealed that YjgB contains an NlpC/P60 domain, whereas no specific cell wall binding domain or motif could be identified. The NlpC/P60 family is characterized by three conserved residues, a cysteine, a histidine, and a polar residue. In agreement with the presence of a Cys residue in the catalytic site of YjgB, its enzymatic activity was enhanced in the presence of dithiothreitol. Peptidoglycan-hydrolyzing activity of YjgB was detected in growing cells of an L. lactis strain overexpressing YjgB, as revealed by the presence of disaccharide (DS)-dipeptide in the muropeptide composition of the overexpressing strain. YjgB hydrolyzes the peptide chains of L. lactis muropeptides between γ-d-Gln and l-Lys residues. Its hydrolytic activity was detected on DSs with tetra- and pentapeptide chains, whereas hydrolytic activity was very low on DS-tripeptides. Thus, we demonstrated that YjgB is an endopeptidase which cleaves γ-d-Gln-l-Lys bonds in peptide chains of L. lactis peptidoglycan.
    Full-text · Article · Oct 2007 · Applied and Environmental Microbiology
  • [Show abstract] [Hide abstract] ABSTRACT: RNase Z is a widely distributed and often essential endoribonuclease that is responsible for the maturation of the 3'-end of a large family of transfer RNAs (tRNAs). Although it has been the subject of study for more than 25 years, interest in this enzyme intensified dramatically with the identification of the encoding gene in 2002. This led to the discovery of RNase Z in bacteria, in which the final step in the generation of the mature 3'-end of tRNAs had previously been assumed to be catalysed by exoribonucleases. It also led inevitably to structural studies, and the recent resolution of the structure of RNase Z in complex with tRNA has provided a detailed understanding of the molecular mechanisms of RNase Z substrate recognition and cleavage. The identification of the RNase Z gene also allowed the search for alternative substrates for this enzyme to begin in earnest. In this Review, we outline the important recent developments that have contributed to our understanding of this enzyme, particularly in prokaryotes.
    No preview · Article · May 2007 · Nature Reviews Microbiology
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    Marta Riera · Yulia Redko · Jeffrey Leung
    [Show abstract] [Hide abstract] ABSTRACT: The Arabidopsis thaliana RNA binding protein UBA2a is the closest homologue of the Vicia faba AKIP1 (56% identity). Like AKIP1, UBA2a is a constitutively-expressed nuclear protein and in response to ABA it is also reorganized within the nucleus in "speckles" suggesting a possible role of this protein in the regulation of mRNA metabolism during ABA signaling. AKIP1 interacts with, and is phosphorylated by, the upstream ABA-activated protein kinase AAPK. We have investigated if a pathway similar to that described in Vicia faba also exists in Arabidopsis. Our results showed that despite the resemblance between the corresponding Vicia and Arabidopsis proteins, it appears that the function of UBA2a is independent of OST1 phosphorylation.
    Full-text · Article · Aug 2006 · FEBS Letters
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    [Show abstract] [Hide abstract] ABSTRACT: Ribonuclease E is an essential hydrolytic endonuclease in Escherichia coli, and it plays a central role in maintaining the balance and composition of the messenger RNA population. The enzyme is also required for rRNA and tRNA processing. We have shown earlier that the highly conserved catalytic domain of E. coli RNase E is a homotetramer [Callaghan, A. J. et al. (2003) Biochemistry 42, 13848-13855]. Here, we report that this quaternary organization requires zinc. Two protomers share a single zinc ion, and quantitative analysis indicates that each protein contributes two cysteine thiols toward the coordination of the metal. The candidate cysteines are part of a motif that is conserved in the RNase E protein family, and mutation of these residues causes the partial loss of zinc, the complete disruption of the tetramer into dimers, and effective catalytic inactivation. However, these mutations do not affect RNA binding. The tetramer can be artificially maintained by disulfide bond formation, which fully displaces the zinc but largely preserves the catalytic activity. Thus, catalytic activity does not require zinc directly but does require the quaternary structure, for which the metal is essential. We propose that the RNase E tetramer has two nonequivalent subunit interfaces, one of which is mediated by a single, tetrathiol-zinc complex, which we refer to as a "Zn-link" motif. One or both interfaces organize the active site, which is distinct from the primary site of RNA binding.
    Full-text · Article · Apr 2005 · Biochemistry
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    [Show abstract] [Hide abstract] ABSTRACT: The peptidoglycan hydrolase (PGH) complement of Lactococcus lactis was identified by amino acid sequence similarity searching of the L. lactis IL-1403 complete genome sequence. Five PGHs that are not encoded by prophages were detected, including the previously characterized AcmA and AcmB proteins. Four of these PGHs, AcmA to AcmD, contain a catalytic domain homologous to that of enterococcal muramidase, but they have different domain structures. The fifth one (YjgB) has sequence similarity with the active-site domain of peptidoglycan-specific endopeptidases. The three new PGH-encoding genes identified in this study are all actively transcribed in L. lactis subsp. cremoris MG1363. The relative abundance of their transcripts varied during growth and was maximal during the early exponential growth phase. The three encoded proteins have peptidoglycan-hydrolyzing activities which are detected only at acidic pHs by zymography. Like AcmA and AcmB, AcmC has N-acetylglucosaminidase activity rather than the N-acetylmuramidase activity predicted by sequence similarity.
    Full-text · Article · Jul 2004 · Applied and Environmental Microbiology
  • [Show abstract] [Hide abstract] ABSTRACT: RNase E is an essential endoribonuclease that plays a central role in the processing and degradation of RNA in Escherichia coli and other bacteria. Most endoribonucleases have been shown to act distributively; however, Feng et al. [(2002) Proc. Natl. Acad. Sci. U.S.A. 99, 14746-14751] have recently found that RNase E acts via a scanning mechanism. A structural explanation for the processivity of RNase E is provided here, with our finding that the conserved catalytic domain of E. coli RNase E forms a homotetramer. Nondissociating nanoflow-electrospray mass spectrometry suggests that the tetramer binds up to four molecules of a specific substrate RNA analogue. The tetrameric assembly of the N-terminal domain of RNase E is consistent with crystallographic analyses, which indicate that the tetramer possesses approximate D(2) dihedral symmetry. Using X-ray solution scattering data and symmetry restraints, a solution shape is calculated for the tetramer. This shape, together with limited proteolysis data, suggests that the S1-RNA binding domains of RNase E lie on the periphery of the tetramer. These observations have implications for the structure and function of the RNase E/RNase G ribonuclease family and for the assembly of the E. coli RNA degradosome, in which RNase E is the central component.
    No preview · Article · Jan 2004 · Biochemistry
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    [Show abstract] [Hide abstract] ABSTRACT: Ribonuclease E is required for the rapid decay and correct processing of RNA in Escherichia coli. A detailed understanding of the hydrolysis of RNA by this and related enzymes will require the integration of structural and molecular data with quantitative measurements of RNA hydrolysis. Therefore, an assay for RNaseE that can be set up to have relatively high throughput while being sensitive and quantitative will be advantageous. Here we describe such an assay, which is based on the automated high pressure liquid chromatography analysis of fluorescently labeled RNA samples. We have used this assay to optimize reaction conditions, to determine for the first time the catalytic parameters for a polypeptide of RNaseE, and to investigate the RNaseE-catalyzed reaction through the modification of functional groups within an RNA substrate. We find that catalysis is dependent on both protonated and unprotonated functional groups and that the recognition of a guanosine sequence determinant that is upstream of the scissile bond appears to consist of interactions with the exocyclic 2-amino group, the 7N of the nucleobase and the imino proton or 6-keto group. Additionally, we find that a ribose-like sugar conformation is preferred in the 5'-nucleotide of the scissile phosphodiester bond and that a 2'-hydroxyl group proton is not essential. Steric bulk at the 2' position in the 5'-nucleotide appears to be inhibitory to the reaction. Combined, these observations establish a foundation for the functional interpretation of a three-dimensional structure of the catalytic domain of RNaseE when solved.
    Preview · Article · Dec 2003 · Journal of Biological Chemistry