Proteomic composition of eukaryotic and bacterial RNA decay condensates suggests convergent evolution.

Bacterial cells have a unique challenge to organize their cytoplasm without the use of membrane-bound organelles. Biomolecular condensates (henceforth BMCs) are a class of nonmembrane-bound organelles, which, through the physical process of phase separation, can form liquid-like droplets with proteins/nucleic acids. BMCs have been broadly characterized in eukaryotic cells, and BMCs have been recently identified in bacteria, with the first and best studied example being bacterial ribonucleoprotein bodies (BR-bodies). BR-bodies contain the RNA decay machinery and show functional parallels to eukaryotic P-bodies (PBs) and stress granules (SGs). Due to the finding that mRNA decay machinery is compartmentalized in BR-bodies and in eukaryotic PBs/SGs, we will explore the functional similarities in the proteins, which are known to enrich in these structures based on recent proteomic studies. Interestingly, despite the use of different mRNA decay and post-transcriptional regulatory machinery, this analysis has revealed evolutionary convergence in the classes of enriched enzymes in these structures.

The BR-body proteome contains a complex network of protein-protein and protein-RNA interactions.

Bacterial RNP bodies (BR-bodies) are non-membrane-bound structures that facilitate mRNA decay by concentrating mRNA substrates with RNase E and the associated RNA degradosome machinery. However, the full complement of proteins enriched in BR-bodies has not been defined. Here we define the protein components of BR-bodies through enrichment of the bodies followed by mass spectrometry-based proteomic analysis. We found 111 BR-body enriched proteins, including several RNA binding proteins, many of which are also recruited directly to in vitro reconstituted RNase E droplets, showing BR-bodies are more complex than previously assumed. While most BR-body enriched proteins that were tested cannot phase separate, we identified five that undergo RNA-dependent phase separation in vitro, showing other RNP condensates interface with BR-bodies. RNA degradosome protein clients are recruited more strongly to RNase E droplets than droplets of other RNP condensates, implying that client specificity is largely achieved through direct protein-protein interactions. We observe that some RNP condensates assemble with preferred directionally, suggesting that RNA may be trafficked through RNP condensates in an ordered manner to facilitate mRNA processing/decay, and that some BR-body associated proteins have the capacity to dissolve the condensate. Finally, we find that RNA dramatically stimulates the rate of RNase E phase separation in vitro, explaining the dissolution of BR-bodies after cellular mRNA depletion observed previously. Altogether, these results suggest that a complex network of protein-protein and protein-RNA interactions controls BR-body phase separation and RNA processing.