I. W. Rathnayaka-Mudiyanselage

The RNA helicases from the DEAD-box family participate in several biochemical processes, with special relevance in the assembly of ribosome ribonucleoparticles and in RNA turnover as part of the multi-enzymatic RNA degradosome complex. Caulobacter crescentus has three DEAD-box RNA helicases, including RhlB, the main RNA helicase associated with the RNA degradosome. In this work, we evaluated the impact of RhlB on C. crescentus gene expression both at normal and low temperatures and its role in global RNA decay. The rhlB null mutant showed a freezing-sensitive phenotype after pre-incubation at low temperature, indicating a failure in assembling a proper cryotolerance response. A global transcriptomic profile of the wild-type and rhlB mutants was carried out at 30°C and 10°C and showed 73 and 477 differentially expressed genes, with the most affected gene categories being translation and posttranslational modifications. The rhlB mutant is more sensitive to streptonigrin, suggesting that the downregulation of iron-regulated genes may be due to a higher intracellular iron concentration. A high throughput screening for RhlB-binding RNAs identified 220 transcripts at 30°C, and global RNA decay analyses identified several mRNAs and sRNAs that displayed altered profiles of RNA decay rates. Analyses of the transcripts’ 5′-ends from the C. crescentus rhlB mutant and from Pseudomonas aeruginosa PAO1 rhlE1 and rhlE2 mutants confirm that in the absence of the DEAD-box RNA helicase associated with RNase E, there is an accumulation of RNA degradation intermediates. IMPORTANCE: One of the most important control points in gene regulation is RNA stability, which determines the half-life of a transcript from its transcription until its degradation. Bacteria have evolved a sophisticated multi-enzymatic complex, the RNA degradosome, which is dedicated mostly to RNA turnover. The combined activity of RNase E and the other RNA degradosome enzymes provides an efficient pipeline for the complete degradation of RNAs. The DEAD-box RNA helicases are very often found in RNA degradosomes from phylogenetically distant bacteria, confirming their importance in unwinding structured RNA for subsequent degradation. This work showed that the absence of the RNA helicase RhlB in the free-living Alphaproteobacterium Caulobacter crescentus causes important changes in gene expression and cell physiology. These are probably due, at least in part, to inefficient RNA processing by the RNA degradosome, particularly at low-temperature conditions.

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.