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 substrates of BR-bodies through enrichment of the bodies followed by liquid chromatography-mass spec (LC-MS) proteomic analysis. We found 111 BR-body enriched proteins, including many RNA binding proteins, many of which are also recruited directly to in vitro reconstituted RNase E droplets, suggesting BR-bodies are more complex than previously assumed. While most BR-body enriched proteins cannot phase separate, we identified five which can undergo RNA dependent phase separation in vitro, suggesting other RNP condensates interface with BR-bodies. The RNA degradosome protein clients are recruited more strongly to RNase E droplets than that of the other RNP condensates, suggesting that client specificity is largely achieved through direct protein-protein interactions. We observe that some RNP condensates assemble unidirectionally, 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 observed 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 while coordinating this mode of organization with RNA processing.
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