Based on our results, we conclude that small RNAs are much more widespread than previously imagined and that these versatile molecules may play important roles in the fine-tuning of cell responses to changing environments.

SummarySmall RNAs (sRNAs) associated with the RNA chaperon protein Hfq are key posttranscriptional regulators of gene expression in bacteria. Deciphering the sRNA-target interactome is an essential step toward understanding the roles of sRNAs in the cellular networks. We developed a broadly applicable methodology termed RIL-seq (RNA interaction by ligation and sequencing), which integrates experimental and computational tools for in vivo transcriptome-wide identification of interactions involving Hfq-associated sRNAs. By applying this methodology to Escherichia coli we discovered an extensive network of interactions involving RNA pairs showing sequence complementarity. We expand the ensemble of targets for known sRNAs, uncover additional Hfq-bound sRNAs encoded in various genomic regions along with their trans encoded targets, and provide insights into binding and possible cycling of RNAs on Hfq. Comparison of the sRNA interactome under various conditions has revealed changes in the sRNA repertoire as well as substantial re-wiring of the network between conditions.

The OxyS regulatory RNA integrates the adaptive response to hydrogen peroxide with other cellular stress responses and protects against DNA damage. Among the OxyS targets is the rpoS-encoded σ s subunit of RNA polymerase. σ s is a central regulator of genes induced by osmotic stress, starvation and entry into stationary phase. We examined the mechanism whereby OxyS represses rpoS expression and found that the OxyS RNA inhibits translation of the rpoS message. This repression is dependent on the hfqencoded RNA-binding protein (also denoted host factor I, HF-I). Co-immunoprecipitation and gel mobility shift experiments revealed that the OxyS RNA binds Hfq, suggesting that OxyS represses rpoS translation by altering Hfq activity.

More than 60 small RNAs (sRNA) have been identified in E. coli. The functions of the majority of these sRNAs are still unclear. For the few sRNAs characterized, expression and functional studies indicate that they act under stress conditions. Here, we describe a novel E. coli chromosome locus that is part of the SOS response to DNA damage. This locus encodes two sRNAs, IstR-1 and IstR-2, and a toxic peptide, TisB, encoded by tisAB mRNA. Transcription of tisAB and istR-2 is SOS regulated, whereas IstR-1 is present throughout growth. IstR-1 inhibits toxicity by base-pairing to a short region in the tisAB mRNA. This antisense interaction entails RNase III-dependent cleavage, thereby inactivating the mRNA for translation. In the absence of the SOS response, IstR-1 is present in high excess over its target. However, SOS induction leads to depletion of the IstR-1 pool, concomitant with accumulation of tisAB mRNA. Under such conditions, TisB exerts its toxic effect, slowing down growth. We propose that the inhibitory sRNA prevents inadvertent TisB synthesis during normal growth and, possibly, also limits SOS-induced toxicity. Our study adds the SOS regulon to the growing list of global regulatory circuits controlled by sRNA genes.

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