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-rRNAs. mRNAs and RNAs antisense to open reading frames were most affected with over 80% increased more than 2-fold. Non-coding RNAs expressed in the intergenic regions were 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.

Present in every kingdom of life, generally in multiple copies, DEAD-box RNA helicases are specialized enzymes that unwind RNA secondary structures. They play major roles in mRNA decay, ribosome biogenesis, and adaptation to cold temperatures. Most bacteria have multiple DEAD-box helicases that present both specialized and partially redundant functions. By using phylogenomics, we revealed that the Helicobacter genus, including the major gastric pathogen H. pylori, is among the exceptions, as it encodes a sole DEAD-box RNA helicase. In H. pylori, this helicase, designated RhpA, forms a minimal RNA degradosome together with the essential RNase, RNase J, a major player in the control of RNA decay. Here, we used H. pylori as a model organism with a sole DEAD-box helicase and investigated the role of this helicase in H. pylori physiology, ribosome assembly, and during in vivo colonization. Our data showed that RhpA is dispensable for growth at 37°C but crucial at 33°C, suggesting an essential role of the helicase in cold adaptation. Moreover, we found that a ΔrhpA mutant was impaired in motility and deficient in colonization of the mouse model. RhpA is involved in the maturation of 16S rRNA at 37°C and is associated with translating ribosomes. At 33°C, RhpA is, in addition, recruited to individual ribosomal subunits. Finally, via its role in the RNA degradosome, RhpA directs the regulation of the expression of its partner, RNase J. RhpA is thus a multifunctional enzyme that, in H. pylori, plays a central role in gene regulation and in the control of virulence.

Posttranscriptional regulation is a major level of gene expression control in any cell. In bacteria, multiprotein machines called RNA degradosomes are central for RNA processing and degradation, and some were reported to be compartmentalized inside these organelleless cells. The minimal RNA degradosome of the important gastric pathogen Helicobacter pylori is composed of the essential ribonuclease RNase J and RhpA, its sole DEAD box RNA helicase, and plays a major role in the regulation of mRNA decay and adaptation to gastric colonization. Here, the subcellular localization of the H. pylori RNA degradosome was investigated using cellular fractionation and both confocal and superresolution microscopy. We established that RNase J and RhpA are peripheral inner membrane proteins and that this association was mediated neither by ribosomes nor by RNA nor by the RNase Y membrane protein. In live H. pylori cells, we observed that fluorescent RNase J and RhpA protein fusions assemble into nonpolar foci. We identified factors that regulate the formation of these foci without affecting the degradosome membrane association. Flotillin, a bacterial membrane scaffolding protein, and free RNA promote focus formation in H. pylori. Finally, RNase J-GFP (RNase J-green fluorescent protein) molecules and foci in cells were quantified by three-dimensional (3D) single-molecule fluorescence localization microscopy. The number and size of the RNase J foci were found to be scaled with growth phase and cell volume as previously reported for eukaryotic ribonucleoprotein granules. In conclusion, we propose that membrane compartmentalization and the regulated clustering of RNase J-based degradosome hubs represent important levels of control of their activity and specificity. IMPORTANCE Helicobacter pylori is a bacterial pathogen that chronically colonizes the stomach of half of the human population worldwide. Infection by H. pylori can lead to the development of gastric pathologies such as ulcers and adenocarcinoma, which causes up to 800,000 deaths in the world each year. Persistent colonization by H. pylori relies on regulation of the expression of adaptation-related genes. One major level of such control is posttranscriptional regulation, which, in H. pylori, largely relies on a multiprotein molecular machine, an RNA degradosome, that we previously discovered. In this study, we established that the two protein partners of this machine are associated with the membrane of H. pylori. Using cutting-edge microscopy, we showed that these complexes assemble into hubs whose formation is regulated by free RNA and scaled with bacterial size and growth phase. Organelleless cellular compartmentalization of molecular machines into hubs emerges as an important regulatory level in bacteria.

Importance 46Helicobacter pylori is a bacterial pathogen that chronically colonizes the stomach of 47 half of the human population worldwide. Infection by H. pylori can lead to the 48 development of gastric pathologies such as ulcers and adenocarcinoma, that causes 49 up to 800.000 deaths in the world each year. Persistent colonization by H. pylori relies 50 on regulation of the expression of adaptation-related genes. One major level of such 51 control is post-transcriptional regulation that, in H. pylori, largely relies on a multi-52 protein molecular machine, an RNA-degradosome, that we previously discovered. In 53 this study, we established that the two protein partners of this machine are associated 54 to the membrane of H. pylori. Using cutting-edge microscopy, we showed that these 55 complexes assemble into hubs whose formation is regulated by free RNA and scaled 56 with bacterial size and growth phase. Cellular compartmentalization of molecular 57 machines into hubs emerges as an important regulatory level in the organelle-less 58 bacteria. 59 61Post-transcriptional regulation is one of the most important levels of control of gene 62 expression in every kingdom of life. Ribonucleases (RNases) are key enzymes in post-63 transcriptional regulation, involved in RNA maturation and degradation. RNases often 64 act in multi-protein complexes that are designated exosomes in Eukarya and Archaea 65 and RNA degradosomes in bacteria and chloroplasts [for a review, see (1)]. RNA-66 degradosomes were established in several bacterial species and are defined by two 67 core components, an RNase and an RNA helicase (2). These RNA helicases belong 68 to the DEAD-box family and act by unwinding RNA, thereby allowing access of the 69 ribonucleases to some of their target sites on RNAs. RNA degradosomes are 70 widespread and vary in composition, although only few have been described in detail 71(1). Most RNA degradosomes reported so far are assembled on the essential 72 endoribonuclease RNase E, like in Escherichia coli, Caulobacter crescentus or 73 Mycobacterium tuberculosis (3-5). In the E. coli degradosome, RNase E serves as a 74 scaffold for the binding of the DEAD-box RNA helicase RhlB, the metabolic enzyme 75 enolase and the 3'-5' exoribonuclease PNPase (2, 6). Nevertheless, our recent 76 analysis on a representative set of 1,535 bacterial genomes revealed that RNase E is 77 absent from about half of the bacterial species (1). Most of the remaining bacteria 78 (47%), that lack RNase E, have either RNase Y or RNase J enzymes or both. RNase 79 J and RNase Y, first identified in Bacillus subtilis, both display endoribonucleolytic 80 activities but RNase J acts in addition as a 5'-3' exoribonuclease (7, 8). Although being 81 unrelated proteins, these enzymes constitute functional homologues of RNase E. In 82 the Gram-positive bacteria Staphylococcus aureus and B. subtilis, RNA 83 degradosomes comprising RNase Y and RNase J have been proposed, but to date 84 20). Interestingly, similarly to the situation in H. pylori, the E. ...

Ribonucleases are central players in post-transcriptional regulation, a major level of gene expression regulation in all cells. Here, we characterized the 3′-5′ exoribonuclease RNase R from the bacterial pathogen Helicobacter pylori. The ‘prototypical’ Escherichia coli RNase R displays both exoribonuclease and helicase activities, but whether this latter RNA unwinding function is a general feature of bacterial RNase R had not been addressed. We observed that H. pylori HpRNase R protein does not carry the domains responsible for helicase activity and accordingly the purified protein is unable to degrade in vitro RNA molecules with secondary structures. The lack of RNase R helicase domains is widespread among the Campylobacterota, which include Helicobacter and Campylobacter genera, and this loss occurred gradually during their evolution. An in vivo interaction between HpRNase R and RhpA, the sole DEAD-box RNA helicase of H. pylori was discovered. Purified RhpA facilitates the degradation of double stranded RNA by HpRNase R, showing that this complex is functional. HpRNase R has a minor role in 5S rRNA maturation and few targets in H. pylori, all included in the RhpA regulon. We concluded that during evolution, HpRNase R has co-opted the RhpA helicase to compensate for its lack of helicase activity.