Conditional Hfq association with small non-coding RNAs in Pseudomonas aeruginosa revealed through comparative UV crosslinking immunoprecipitation followed by high-throughput sequencing [RNA-Seq]

Bacterial small non-coding RNAs (sRNAs) play post-transcriptional regulatory roles in cellular responses to changing environmental cues and in adaptation to harsh conditions. Generally, the RNA-binding protein Hfq helps sRNAs associate with target mRNAs to modulate their translation and to modify global RNA pools depending on physiological state. Here, a combination of in vivo UV crosslinking immunoprecipitation followed by high-throughput sequencing (CLIP-seq) and total RNA-seq showed that Hfq interacts with different regions of the P. aeruginosa transcriptome under planktonic versus biofilm conditions. In the present approach, P. aeruginosa Hfq preferentially interacted with repeats of the AAN triplet motif at mRNA 5’ UTRs and sRNAs, and U-rich sequences at rho-independent terminators. Further transcriptome analysis suggested that sRNAs association with Hfq is primarily a function of their expression levels, strongly supporting that the pool of Hfq-associated RNAs is equilibrated by RNA concentration-driven cycling on and off Hfq. Overall, our combinatorial CLIP-seq and total RNA-seq approach highlights conditional sRNA associations with Hfq as a novel aspect of post-transcriptional regulation in P. aeruginosa. To disentangle differences in gene expression from those in protein binding, we performed RNA-seq for an ad hoc normalization of CLIP-seq