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 base-pair with multiple target mRNAs and form large RNA-RNA interaction networks. To systematically investigate the RNA-RNA interactome in living cells, we have developed a streamlined in vivo approach iRIL-seq (intracellular RIL-seq). This generic approach is highly robust, illustrating the dynamic sRNA interactomes in Salmonella enterica across multiple stages of growth. We have identified the OmpD porin mRNA as a central regulatory hub that is targeted by a dozen sRNAs, including FadZ cleaved from the conserved 3'UTR of fadBA mRNA. Both ompD and FadZ are activated by CRP, constituting a type I incoherent feed-forward loop in the fatty acid metabolism pathway. Altogether, we have established an approach to profile RNA-RNA interactomes in live cells, highlighting the complexity of RNA regulatory hubs and RNA networks.