Hfq CLASH uncovers sRNA-target interaction networks linked to nutrient availability adaptation [CLASH]

By shaping gene expression profiles, small RNAs (sRNAs) enable bacteria to efficiently adapt to changes in their environment. To better understand how Escherichia coli acclimatizes to nutrient availability, we performed UV cross-linking, ligation and sequencing of hybrids (CLASH) to uncover Hfq-associated RNA-RNA interactions at specific growth stages. We demonstrate that Hfq CLASH robustly captures bona fide RNA-RNA interactions identified hundreds of novel sRNA base-pairing interactions, including many sRNA-sRNA interactions and involving 3’UTR-derived sRNAs. We rediscovered known and identified novel sRNA seed sequences. The sRNA-mRNA interactions identified by CLASH have strong base-pairing potential and are highly enriched for complementary sequence motifs, even those supported by only a few reads. Yet, steady state levels of most mRNA targets were not significantly affected upon over-expression of the sRNA regulator. Our results reinforce the idea that the reproducibility of the interaction, not base-pairing potential, is a stronger predictor for a regulatory outcome. Cells expressing HIS6-TEV-3xFLAG tagged Hfq were grown in LB medium from early exponential phase to stationary phase. At various optical densities, cells were UV cross-linked in the Vari-X-linker and harvested by filtration. CLASH was subsequently performed on each sample using the tagged Hfq as bait to enrich for sRNA-terget interactions. The resulting associated RNAs were sequenced on Illumina platforms. We included two biological replicates of the density series. In addition, for each density, we generated rRNA-depleted total RNA sequencing data.