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

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Abstract
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. Highlights: BR-body proteomics identified 111 proteins enriched in BR-bodies. BR-bodies associate with an interconnected network of RNP condensates. BR-body condensation is modulated by its interaction network. RNA is required for rapid BR-body condensation. Graphical Summary of the BR-body protein interactome. Lines between two protein circles represent a direct interaction.

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