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.

中文翻译：

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基于RNase J的RNA降解体在胃病原体幽门螺杆菌中区室化。

转录后调控是任何细胞中基因表达控制的主要水平。在细菌中，称为RNA降解体的多蛋白机器对于RNA加工和降解至关重要，据报道其中一些机器在这些无细胞的细胞内被分隔。重要的胃病原体幽门螺杆菌的最小RNA降解体由必需的核糖核酸酶RNase J和RhpA（其唯一的DEAD box RNA解旋酶）组成，并且在调节mRNA衰变和适应胃定植中起主要作用。在这里，幽门螺杆菌的亚细胞定位使用细胞分级分离，共聚焦和超分辨率显微镜研究了RNA降解体。我们确定RNase J和RhpA是外周内膜蛋白，并且这种结合既不由核糖体，RNA也不由RNase Y膜蛋白介导。在活幽门螺杆菌细胞中，我们观察到荧光RNase J和RhpA蛋白融合体组装成非极性病灶。我们确定了调节这些灶的形成而又不影响降解体膜结合的因素。细菌膜支架蛋白Flotillin和游离RNA促进幽门螺杆菌形成病灶。最后，通过三维（3D）单分子荧光定位显微镜对细胞中的RNase J-GFP（RNase J-绿色荧光蛋白）分子和病灶进行定量。发现RNase J灶的数量和大小与生长期和细胞体积成正比，如先前报道的真核核糖蛋白颗粒。总之，我们建议膜分隔和基于RNase J的降解体中枢的调控簇代表了对其活性和特异性的重要控制水平。