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

Thorsten Bischler,Ping-Kun Hsieh,Marcus Resch,Quansheng Liu,Hock Siew Tan,Patricia L Foley,Anika Hartleib,Cynthia M Sharma,Joel G Belasco

doi:10.1074/jbc.m116.761171

Thorsten Bischler, Ping-Kun Hsieh + Show 7 more

Open Access

https://doi.org/10.1074/jbc.m116.761171

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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.

Highlights

RNA degradation is crucial for regulating gene expression in all organisms
The sequence of HP1228, which is well conserved in other Epsilonproteobacteria, is 34% identical to that of E. coli RppH (EcRppH) and contains each of the 23 amino acid residues that are strictly conserved in virtually all proteobacterial orthologs of EcRppH (Fig. 1A) [16]
We have identified the Nudix protein HP1228 as an RNA pyrophosphohydrolase important for RNA degradation in H. pylori, characterized its biochemical activity and substrate specificity in vitro, and identified several of its mRNA and sRNA targets in vivo by employing a global strategy based on high-throughput sequencing

Summary

Edited by Patrick Sung

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. Ments 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. It enables these enzymes to rapidly degrade monophosphosphorylated intermediates generated by prior ribonuclease cleavage [12] It can assist them in attacking full-length transcripts whose 5Ј-triphosphate has been converted to a monophosphate by an RNA pyrophosphohydrolase [11, 13]. The genomes of most species encode multiple Nudix enzymes, which can be identified by a characteristic sequence motif (the Nudix motif) [27] that usually is well conserved [17] Protein domains containing this motif typically fold so as to form a central four-stranded mixed ␤ sheet (␤ strands 1, 3, 4, and 5) and an antiparallel ␤ sheet (␤ strands 2 and 6) sandwiched between three ␣ helices (␣1, ␣2, and ␣3) [27].

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