Global identification of RsmA/N binding sites in Pseudomonas aeruginosa by in vivo UV CLIP-seq

Posttranscriptional regulation of gene expression in bacteria is performed by a complex and hierarchical signaling cascade. Pseudomonas aeruginosa harbors two redundant RNA-binding proteins RsmA/RsmN (RsmA/N), which play a critical role in balancing acute and chronic infections. However, in vivo binding sites on target transcripts and the overall impact on the physiology remains unclear. In this study, we applied in vivo UV crosslinking immunoprecipitation followed by RNA-sequencing (UV CLIP-seq) to detect RsmA/N binding sites at single-nucleotide resolution and mapped more than 500 peaks to approximately 400 genes directly bound by RsmA/N in P. aeruginosa. This also demonstrated the ANGGA sequence in apical loops skewed towards 5UTRs as a consensus motif for RsmA/N binding. Genetic analysis combined with CLIP-seq results identified previously unrecognized RsmA/N targets involved in LPS modification. Moreover, the small non-coding RNAs RsmY/RsmZ, which sequester RsmA/N away from target mRNAs, are positively regulated by the RsmA/N-mediated translational repression of hptB, encoding a histidine phosphotransfer protein, and cafA, encoding a cytoplasmic axial filament protein, thus providing a possible mechanistic explanation for homeostasis of the Rsm system. Our findings present the global RsmA/N-RNA interaction network that exerts pleiotropic effects on gene expression in P. aeruginosa. IMPORTANCEThe ubiquitous bacterium Pseudomonas aeruginosa is notorious as an opportunistic pathogen causing life-threatening acute and chronic infections in immunocompromised patients. P. aeruginosa infection processes are governed by two major gene regulatory systems, namely, the GacA/GacS (GacAS) two-component system and the RNA-binding proteins RsmA/RsmN (RsmA/N). RsmA/N basically function as a translational repressor or activator directly by competing with the ribosome. In this study, we identified hundreds of RsmA/N regulatory target RNAs and the consensus motifs for RsmA/N bindings by UV crosslinking in vivo. Moreover, our CLIP-seq revealed that RsmA/N posttranscriptionally regulate cell wall organization and exert feedback control on GacAS-RsmA/N systems. Many genes including small regulatory RNAs identified in this study are attractive targets for further elucidating the regulatory mechanisms of RsmA/N in P. aeruginosa.