Identification of the RNA Pyrophosphohydrolase RppH of Helicobacter pylori and Global Analysis of Its RNA Targets

Abstract

Edited by Patrick SungRNA 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 Gramnegative 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.

Review of current diagnostic methods and advances inHelicobacter pyloridiagnostics in the era of next generation sequencing

“…The literature search mostly yielded basic research studies investigating a broad spectrum of fundamental mechanism in H. pylori like the presence of genes associated with bacterial virulence[99-104] and biofilm formation[105,106], the characterization of methylases[107], nudix hydrolases[108], restriction-modification (R-M) systems[109], exo- and endo-ribonucleases[110] ; and the transcriptional response of H. pylori to exposition to different salt concentrations[111], different pH conditions[112], heat shock[113] and chemical agents like bismuth[114] and nickel[115]. Most of these studies initially depleted ribosomal RNA, either using RiboZero eukaryotic rRNA depletion treatment (Epicentre, Illumina)[112,113,116] or a rRNA modified capture hybridization approach[110], and performed sequencing on an Illumina platform (Illumina, United States).…”

Regulatory Tools for Controlling Gene Expression in Cyanobacteria

“…All RppH proteins identified so far have been part of the Nudix family, but it is difficult to determine the substrates of these protein family members based on amino acid sequence. RppH homologs have been identified previously by purifying these proteins and testing their activities in vitro 112 . Cyanobacteria possess many proteins annotated as members of the Nudix family and it is yet to be seen if one may have the activity of RppH.…”

DapF stabilizes the substrate-favoring conformation of RppH to stimulate its RNA-pyrophosphohydrolase activity in Escherichia coli

“…In the absence of BsRppH, the average measured half-life of the yhxA-glpP transcript increased from 3.7 ± 0.6 min to 8.3 ± 0.6 min, accompanied by an elevation in monophosphorylated RNA ( 19 ). In the gastric pathogen Helicobacter pylori , HpRppH has a similar function to EcRppH and triggers at least 63 potential targets, including mRNA and sRNA, for degradation ( 20 ). Given the determinant role of RppH in mRNA decay, leveraging activity of this enzyme could be an economic strategy by which microbes can rapidly adjust their cellular mRNA in response to changed nutrient availability or external stimuli.…”