The conserved regulatory RNA RsaE down-regulates the arginine degradation pathway in Staphylococcus aureus [Hybrid-Trap-seq]. The conserved regulatory RNA RsaE down-regulates the arginine degradation pathway in Staphylococcus aureus [Hybrid-Trap-seq]

Bacterial regulatory RNAs (sRNA) generally act by base-pairing with target mRNAs. While identification of sRNA targets is the essential step in sRNA characterization, it remains a stumbling block in most studies. To study sRNA-regulated networks in the major human pathogen Staphylococcus aureus, we used a RNA-RNA interactome screening method for identifying sRNA targets based on synthetic sRNAs that are used in vitro as bait to trap their corresponding targets. The key to target discovery lies in the differential analysis of RNA-seq data from captures with different sRNAs and from ΔrsaE and RsaE over-expressing strains. This strategy was applied to study RsaE, a regulatory RNA highly conserved amongst Firmicutes and revealed that RsaE targets the arginase rocF mRNA via direct interactions involving G-rich motifs. Two duplicated C-rich motifs of RsaE can independently downregulate rocF expression. However, the relative activity of these motifs is substrate dependent. Metabolite quantifications in wild-type and ΔrsaE cultures indicate that the absence of RsaE affects amino acid metabolism. Collectively, the data support the model that RsaE acts as a global regulator downregulating functions associated to metabolic adaptation. Overall design: The protocol called Hybrid-Trap-seq used in this study allows identifying RNA targets interacting in vitro with a sRNA. In brief, a synthetic sRNA was produced, biotinylated, and fixed to streptavidin-associated magnetic beads. The resulting complex was incubated with total RNA extracts, washed, and RNAs bound to sRNAs were eluted. Recovered RNAs were quantified by RNA-seq using Illumina Genome Analyzer IIx generating single-end 40-nt reads. Hypothesizing that unspecific RNAs recovered via this protocol would be the same with different sRNAs used as baits; therefore, background noise could be subtracted by a differential analysis of Hybrid-Trap-seq data obtained with several different baits. In this study, we run Hybrid-Trap-seq with Staphylococcus aureus RsaE regulatory RNA and we used 3 other sRNAs (RsaA, RsaH and RNAIII) dataset as control. The processed data file (putative_RsaE-targets_revised_20180425.csv) compiles read counts for S. aureus CDSs and bona-fide sRNAs (described by Liu et al 2018, doi: 10.3389/fmicb.2018.00228) for the 4 RNA-seq samples as well as DEseq output (RsaE sample versus the 3 other samples used as control).