SummaryIn bacteria, the highly conserved RsmA/CsrA family of RNA-binding proteins functions as global posttranscriptional regulators acting on mRNA translation and stability. Through phenotypic complementation of an rsmA mutant in Pseudomonas aeruginosa, we discovered a family member, termed RsmN. Elucidation of the RsmN crystal structure and that of the complex with a hairpin from the sRNA, RsmZ, reveals a uniquely inserted α helix, which redirects the polypeptide chain to form a distinctly different protein fold to the domain-swapped dimeric structure of RsmA homologs. The overall β sheet structure required for RNA recognition is, however, preserved with compensatory sequence and structure differences, allowing the RsmN dimer to target binding motifs in both structured hairpin loops and flexible disordered RNAs. Phylogenetic analysis indicates that, although RsmN appears unique to P. aeruginosa, homologous proteins with the inserted α helix are more widespread and arose as a consequence of a gene duplication event.
Pseudomonads typically carry multiple non-identical alleles of the post-transcriptional regulator rsmA. In Pseudomonas aeruginosa, RsmN is notable in that its structural rearrangement confers distinct and overlapping functions with RsmA. However, little is known about the specificities of RsmN for its target RNAs and overall impact on the biology of this pathogen. We purified and mapped 503 transcripts directly bound by RsmN in P. aeruginosa. About 200 of the mRNAs identified encode proteins of demonstrated function including some determining acute and chronic virulence traits. For example, RsmN reduces biofilm development both directly and indirectly via multiple pathways, involving control of Pel exopolysaccharide biosynthesis and c-di-GMP levels. The RsmN targets identified are also shared with RsmA, although deletion of rsmN generally results in less pronounced phenotypes than those observed for ΔrsmA or ΔrsmArsmNind mutants, probably as a consequence of different binding affinities. Targets newly identified for the Rsm system include the small non-coding RNA CrcZ involved in carbon catabolite repression, for which differential binding of RsmN and RsmA to specific CrcZ regions is demonstrated. The results presented here provide new insights into the intricacy of riboregulatory networks involving multiple but distinct RsmA homologues.
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