Critical functions and key interactions mediated by the RNase E scaffolding domain in Pseudomonas aeruginosa

The RNA degradosome, a multi-protein complex regulating mRNA levels in bacteria, assembles in Pseudomonadota (Proteobacteria) on the RNase E C-terminal domain (CTD) via short linear motifs (SLiMs) that bind other RNA degradosome components and RNA. The composition of Pseudomonas aeruginosa RNA degradosome remains unknown, and its RNase E CTD shows limited similarity to those in well-studied Proteobacteria models like Escherichia coli or Caulobacter crescentus. Our study identified and characterized the SLiMs in P. aeruginosa RNase E, revealing a large duplicated sequence termed the 'REER-repeats' region. This region, along with AR1 and AR4 SLiMs, mediates RNase E CTD RNA binding and is necessary for the subcellular localization of the RNA degradosome in foci. Pull-down and bacterial two-hybrid assays identified PNPase and RhlB as RNase E-interacting proteins. We confirmed through protein-protein binding assays that PNPase and RhlB directly interact with RNase E, and additionally show that the interactions are mediated by the NDPR and AR1 SLiMs, respectively. Additionally, we confirm that the RhlE2 RNA helicase interacts with RNase E, but this interaction involves RNase E N-terminal domain. Finally, we show that RNase E CTD truncations are impaired in growth on cold and mutations affecting CTD RNA binding impaired twitching motility and virulence in a Galleria mellonella infection model. This study elucidates and highlights the critical role of RNase E CTD-mediated RNA binding and RNA degradosome assembly in the virulence and adaptability of P. aeruginosa. Overall design: PAO1 wild type and mutants were grown in 50 mL Erlenmeyer flasks (10 mL NYB broth) until OD600nm= 1.0-1.2. RNA was purified using the Monarch Total RNA Miniprep kit, following manufacturer instruction. The absence of residual gDNA contamination was verified by performing a PCR for 35 cycles using primer pairs targeting rpoD. Ribosomal RNA was depleted with Ribo-Zero rRNA Removal Kit (Illumina). Then, libraries were prepared and sequenced using the Illumina HiSeq 2000, 150 bp double end read at Novogene.