In vivo RNA interactome profiling reveals 3’UTR-processed small RNA targeting a central regulatory hub

Small noncoding RNAs (sRNAs) are crucial regulators of gene expression in bacteria. Acting in concert with major RNA chaperones such as Hfq or ProQ, sRNAs directly base-pair with multiple target mRNAs, together forming a large and complex RNA-RNA interaction network. To systematically investigate the RNA-RNA interactome in living cells, we have developed a streamlined in vivo approach LiRIP-seq (LiRIP-seq, ligation RIP-seq). This generic approach is highly robust, illustrating the dynamic sRNA interactomes in Salmonella enterica across multiple stages of growth. Strikingly, we have identified the OmpD porin mRNA as a central regulatory hub that is targeted by more than a dozen sRNAs. These include a novel sRNA FadZ that is processed from the conserved 3’ UTR of fadBA mRNA by RNase E. Our results show that both ompD and its regulator FadZ are activated by the same transcription factor upstream, constituting a type I incoherent feed-forward loop in the fatty acid metabolism pathway. Altogether, we have established a novel approach to profile RNA-RNA interactomes in live cells, providing insights into the complexity of post-transcriptional regulatory hubs in RNA interaction networks.