The RNase J-Based RNA Degradosome Is Compartmentalized in the Gastric Pathogen Helicobacter pylori

Abstract

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.

RNase R is associated in a functional complex with the RhpA DEAD-box RNA helicase inHelicobacter pylori

“…However, we found, that independently of RhpA, Hp RNase R localizes to the inner membrane of H. pylori just like the two RNA degradosome partners, RhpA and RNase J ( 11 ). Interestingly, one of the putative RNase R interactors identified by AP-MS is the membrane scaffolding protein flotillin FloA (HPB8_1315) which we previously found to promote the compartmentalization of the RNA degradosome of H. pylori ( 11 ). Altogether, these data are in favor of an ‘RNA degradation hub’ that would be compartmentalized at the membrane of H. pylori .…”

“…We recently reported that the two partners of the RNA degradosome, RhpA and RNase J, localize to the inner membrane of H. pylori ( 11 ). Just like RhpA and RNase J, the Hp RNase R protein does not present any transmembrane segment or signal peptide ( Supplementary Figure S5 ).…”

“…We used the protocol as described in ( 11 ). Briefly, H. pylori cells were grown to an OD 600 nm of 0.7–1, harvested by centrifugation, resuspended in 10 mM Tris–HCl pH 7.4 and cOmplete Protease Inhibitor cocktail (Roche) (buffer A) to an OD 600 nm of 5.…”

“…These two proteins interact and stimulate each other's activity in vitro ( 2 ). In addition, they copurify with ribosomes ( 2 ), localize at the inner membrane of H. pylori and form foci that have been hypothesized to be active degradation hubs ( 11 ).…”

Riboregulation in the Major Gastric Pathogen Helicobacter pylori

“…The membrane localization of RNA degradosomes has been linked to the rate of degradation of different categories of transcripts including regulatory sRNAs (Moffitt et al, 2016), indicating a compartmentalization of RNA degradation and maturation that plays a role in transcriptome dynamics in prokaryotes. In H. pylori, we showed that the RNA degradosome, composed of the essential RNase J protein and of RhpA, the sole DEAD-box RNA helicase of this bacterium (Redko et al, 2013;El Mortaji et al, 2018), is compartmentalized at the inner membrane where it is assembled into foci whose formation is regulated and likely represent RNA degradation hubs (Tejada-Arranz et al, 2020b;Figures 1A,C). In addition, the 3 -5 exoribonuclease RNase R was also associated to the H. pylori inner membrane (Tejada-Arranz et al, 2021; Figure 1A).…”