title: Identification of the RNA Pyrophosphohydrolase RppH of Helicobacter pylori and Global Analysis of Its RNA Targets.
abstract: RNA degradation is crucial for regulating gene expression in all organisms. Like the decapping of eukaryotic mRNAs, the conversion of the 5'-terminal triphosphate of bacterial transcripts to a monophosphate can trigger RNA decay by exposing the transcript to attack by 5'-monophosphate-dependent ribonucleases. In both biological realms, this deprotection step is catalyzed by members of the Nudix hydrolase family. The genome of the gastric pathogen Helicobacter pylori, a Gram-negative epsilonproteobacterium, encodes two proteins resembling Nudix enzymes. Here we present evidence that one of them, HP1228 (renamed HpRppH), is an RNA pyrophosphohydrolase that triggers RNA degradation in H. pylori, whereas the other, HP0507, lacks such activity. In vitro, HpRppH converts RNA 5'-triphosphates and diphosphates to monophosphates. It requires at least two unpaired nucleotides at the 5' end of its substrates and prefers three or more but has only modest sequence preferences. The influence of HpRppH on RNA degradation in vivo was examined by using RNA-seq to search the H. pylori transcriptome for RNAs whose 5'-phosphorylation state and cellular concentration are governed by this enzyme. Analysis of cDNA libraries specific for transcripts bearing a 5'-triphosphate and/or monophosphate revealed at least 63 potential HpRppH targets. These included mRNAs and sRNAs, several of which were validated individually by half-life measurements and quantification of their 5'-terminal phosphorylation state in wild-type and mutant cells. These findings demonstrate an important role for RppH in post-transcriptional gene regulation in pathogenic Epsilonproteobacteria and suggest a possible basis for the phenotypes of H. pylori mutants lacking this enzyme.
authors.affiliation: From the Research Center for Infectious Diseases, University of Würzburg, Josef-Schneider-Strasse 2/D15, 97080 Würzburg, Germany; the Institute of Molecular Infection Biology, University of Würzburg, Josef-Schneider-Strasse 2/D15, 97080 Würzburg, Germany and.
authors.forename: Thorsten
authors.initials: T
authors.lastname: Bischler
authors.affiliation: the Kimmel Center for Biology and Medicine at the Skirball Institute and the Department of Microbiology, New York University School of Medicine, New York, New York 10016.
authors.forename: Ping-Kun
authors.initials: PK
authors.lastname: Hsieh
authors.affiliation: From the Research Center for Infectious Diseases, University of Würzburg, Josef-Schneider-Strasse 2/D15, 97080 Würzburg, Germany; the Institute of Molecular Infection Biology, University of Würzburg, Josef-Schneider-Strasse 2/D15, 97080 Würzburg, Germany and.
authors.forename: Marcus
authors.initials: M
authors.lastname: Resch
authors.affiliation: the Kimmel Center for Biology and Medicine at the Skirball Institute and the Department of Microbiology, New York University School of Medicine, New York, New York 10016.
authors.forename: Quansheng
authors.initials: Q
authors.lastname: Liu
authors.affiliation: the Institute of Molecular Infection Biology, University of Würzburg, Josef-Schneider-Strasse 2/D15, 97080 Würzburg, Germany and.
authors.forename: Hock Siew
authors.initials: HS
authors.lastname: Tan
authors.affiliation: the Kimmel Center for Biology and Medicine at the Skirball Institute and the Department of Microbiology, New York University School of Medicine, New York, New York 10016.
authors.forename: Patricia L
authors.initials: PL
authors.lastname: Foley
authors.affiliation: From the Research Center for Infectious Diseases, University of Würzburg, Josef-Schneider-Strasse 2/D15, 97080 Würzburg, Germany; the Institute of Molecular Infection Biology, University of Würzburg, Josef-Schneider-Strasse 2/D15, 97080 Würzburg, Germany and.
authors.forename: Anika
authors.initials: A
authors.lastname: Hartleib
authors.affiliation: From the Research Center for Infectious Diseases, University of Würzburg, Josef-Schneider-Strasse 2/D15, 97080 Würzburg, Germany; the Institute of Molecular Infection Biology, University of Würzburg, Josef-Schneider-Strasse 2/D15, 97080 Würzburg, Germany and. Electronic address: cynthia.sharma@uni-wuerzburg.de.
authors.forename: Cynthia M
authors.initials: CM
authors.lastname: Sharma
authors.affiliation: the Kimmel Center for Biology and Medicine at the Skirball Institute and the Department of Microbiology, New York University School of Medicine, New York, New York 10016. Electronic address: joel.belasco@med.nyu.edu.
authors.forename: Joel G
authors.initials: JG
authors.lastname: Belasco
mesh_terms: D017766:Acid Anhydride Hydrolases / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D016480:Helicobacter pylori / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity; D020871:RNA Stability / Q000502:physiology*; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D012329:RNA, Bacterial; D012333:RNA, Messenger; D017766:Acid Anhydride Hydrolases
keywords: Helicobacter pylori; Nudix; RNA degradation; RNA modification; RNA turnover; RNA-protein interaction; deep sequencing; gene regulation