Genome-wide mapping of the RNA targets of the Pseudomonas aeruginosa riboregulatory protein RsmN

Pseudomonads typically carry multiple non-identical alleles of the post-transcriptional regulator rsmA. In P. aeruginosa, RsmN is notable in that its structural rearrangement confers distinct and overlapping functions with RsmA. However, little is known about the specificities 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 rsmA or rsmAN mutants, probably as a consequence of different binding affinities. New targets for the Rsm system, previously masked by the presence of RsmN were identified. These include the small non-coding RNA CrcZ involved in carbon catabolite repression, for which differential binding of the two proteins to specific CrcZ target sequences is demonstrated. The results presented here provide new insights into the intricacy of riboregulatory networks involving multiple but distinct RsmA homologues. To identify direct RsmN targets in P. aeruginosa and genes that are post-transcriptionally regulated by RsmN through interaction with their corresponding transcripts, RsmN was expressed in strain PAO1-L and co-purified with its binding RNAs. These were extracted and subjected to RNASeq to characterize the RsmN regulon under the growth conditions used. Total RNA was also extracted from the same culture samples to determine specific RNA abundances and to compare these to those of the RsmN-bound fractions as enrichment ratios. Applying a stringent 3.5-fold minimum enrichment cut-off, a total of 503 transcripts was identified as transcripts bound by RsmN, several of which were experimentally validated. The Preliminary data set is of two conditions, total RNA and RsmN-bound RNA both in duplicate. The final study is of the same two conditions but in triplicate.