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

Abstract

<i>Pseudomonas aeruginosa</i> harbours two redundant RNA-binding proteins RsmA/RsmN (RsmA/N), which play a critical role in balancing acute and chronic infections. However, <i>in vivo</i> binding sites on target transcripts and the overall impact on the physiology remains unclear. In this study, we applied <i>in vivo</i> 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 binding sites to approximately 400 genes directly bound by RsmA/N in <i>P. aeruginosa</i>. This also verified the ANGGA sequence in apical loops skewed towards 5'UTRs as a consensus motif for RsmA/N binding. Genetic analysis combined with CLIP-seq results suggested previously unrecognized RsmA/N targets involved in LPS modification. Moreover, the RsmA/N-titrating RNAs RsmY/RsmZ may be positively regulated by the RsmA/N-mediated translational repression of their upstream regulators, thus providing a possible mechanistic explanation for homoeostasis of the Rsm system. Thus, our study provides a detailed view of RsmA/N-RNA interactions and a resource for further investigation of the pleiotropic effects of RsmA/N on gene expression in <i>P. aeruginosa</i>.