Ribonucleoprotein (RNP) granules play an important role in organizing eukaryotic mRNA metabolism via liquid-liquid phase separation (LLPS) of mRNA decay factors into membrane-less organelles in the cytoplasm. Here we show that the bacterium Caulobacter crescentus Ribonuclease (RNase) E assembles RNP LLPS condensates that we term bacterial RNP-bodies (BR-bodies), similar to eukaryotic P-bodies and stress granules. RNase E requires RNA to assemble a BR-body, and disassembly requires RNA cleavage, suggesting BR-bodies provide localized sites of RNA degradation. The unstructured C-terminal domain of RNase E is both necessary and sufficient to assemble the core of the BR-body, is functionally conserved in related α-proteobacteria, and influences mRNA degradation. BR-bodies are rapidly induced under cellular stresses and provide enhanced cell growth under stress. To our knowledge, Caulobacter RNase E is the first bacterial protein identified that forms LLPS condensates, providing an effective strategy for subcellular organization in cells lacking membrane-bound compartments.

Ribonucleoprotein (RNP) granules play an important role in organizing eukaryotic mRNA metabolism via liquid-liquid phase separation (LLPS) of mRNA decay factors into membrane-less “droplet” organelles in the cytoplasm. Here we show that the bacterium Caulobacter crescentus Ribonuclease (RNase) E assembles RNP LLPS droplets that we term bacterial RNP-bodies (BR-bodies) similar to eukaryotic P-bodies and stress granules. RNase E requires RNA to assemble a BR-body, and disassembly requires RNA cleavage, suggesting BR-bodies provide localized sites of RNA degradation. The unstructured C-terminal domain of RNase E is both necessary and sufficient to assemble the core of the BR-body, is functionally conserved in related α-proteobacteria, and influences mRNA degradation. BR-bodies are rapidly induced under cellular stresses and provide enhanced cell growth under stress. To our knowledge, Caulobacter RNase E is the first bacterial protein identified that forms LLPS droplets, providing an effective strategy for subcellular organization in cells lacking membrane bound compartments.

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AbstractBiomolecular condensates play a key role in organizing RNAs and proteins into membraneless organelles. Bacterial RNP-bodies (BR-bodies) are a biomolecular condensate containing the RNA degradosome mRNA decay machinery, but the biochemical function of such organization remains poorly defined. Here we define the RNA substrates of BR-bodies through enrichment of the bodies followed by RNA-seq. We find that long, poorly translated mRNAs, small RNAs, and antisense RNAs are the main substrates, while rRNA, tRNA, and other conserved ncRNAs are excluded from these bodies. BR-bodies stimulate the mRNA decay rate of enriched mRNAs, helping to reshape the cellular mRNA pool. We also observe that BR-body formation promotes complete mRNA decay, avoiding the build-up of toxic endo-cleaved mRNA decay intermediates. The combined selective permeability of BR-bodies for both, enzymes and substrates together with the stimulation of the sub-steps of mRNA decay provide an effective organization strategy for bacterial mRNA decay.

In bacteria, mRNA decay is controlled by megadalton scale macromolecular assemblies called, "RNA degradosomes," composed of nucleases and other RNA decay associated proteins. Recent advances in bacterial cell biology have shown that RNA degradosomes can assemble into phase-separated structures, termed bacterial ribonucleoprotein bodies (BR-bodies), with many analogous properties to eukaryotic processing bodies and stress granules. This review will highlight the functional role that BR-bodies play in the mRNA decay process through its organization into a membraneless organelle in the bacterial cytoplasm. This review will also highlight the phylogenetic distribution of BRbodies across bacterial species, which suggests that these phase-separated structures are broadly distributed across bacteria, and in evolutionarily related mitochondria and chloroplasts.

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Bacterial Ribonucleoprotein bodies (BR-bodies) play an essential role in organizing RNA degradation via liquid-liquid phase separation in the cytoplasm of bacteria. BR-bodies mediate multi-step mRNA decay through the concerted activity of the endoribonuclease RNase E coupled with the 3'-5' exonuclease Polynucleotide Phosphorylase (PNPase). Our past in vivo studies indicated that the loss of PNPase recruitment into BR-bodies led to a significant build-up of RNA decay intermediates in Caulobacter crescentus. We reconstituted RNase E's C-terminal domain together with PNPase to understand how RNase E biomolecular condensates can tailor the functions of PNPase. We found that PNPase catalytic activity is accelerated when colocalized with the RNase E biomolecular condensates. In contrast, disruption of the RNase E-PNPase protein-protein interaction led to a loss of PNPase recruitment into the BR-bodies and a loss of ribonuclease rate enhancement. We also found that BR-bodies could enhance the decay of select RNA substrates, as we observed a 3.4-fold enhancement of polyadenylic acid (poly(A)) degradation and no impact upon poly(U) degradation. Our investigation into the origins of the 3.4-fold rate enhancement for poly(A) decay indicates a combination of scaffolding and mass action effects impact due to the concentrated biomolecular condensate environment accelerating RNA decay. Consistent with our past in vivo work, these studies suggest BR-bodies are sites of accelerated RNA decay that can shape the available transcriptome.

Bacterial Ribonucleoprotein bodies (BR-bodies) play an essential role in organizing RNA degradation via phase separation in the cytoplasm of bacteria. BR-bodies mediate multi-step mRNA decay through the concerted activity of the endoribonuclease RNase E coupled with the 3′-5′ exoribonuclease Polynucleotide Phosphorylase (PNPase). In vivo, studies indicated that the loss of PNPase recruitment into BR-bodies led to a significant build-up of RNA decay intermediates in Caulobacter crescentus. However, it remained unclear whether this is due to a lack of colocalized PNPase and RNase E within BR-bodies or whether PNPase’s activity is stimulated within the BR-body. We reconstituted RNase E’s C-terminal domain with PNPase towards a minimal BR-body in vitro to distinguish these possibilities. We found that PNPase’s catalytic activity is accelerated when colocalized within the RNase E biomolecular condensates, partly due to scaffolding and mass action effects. In contrast, disruption of the RNase E-PNPase protein–protein interaction led to a loss of PNPase recruitment into the RNase E condensates and a loss of ribonuclease rate enhancement. We also found that RNase E’s unique biomolecular condensate environment tuned PNPase’s substrate specificity for poly(A) over poly(U). Intriguingly, a critical PNPase reactant, phosphate, reduces RNase E phase separation both in vitro and in vivo. This regulatory feedback ensures that under limited phosphate resources, PNPase activity is enhanced by recruitment into RNase E’s biomolecular condensates.