SRA

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. In addition, the transcriptomic effects of sRNA deletion and sRNA accumulation were analyzed by differential expression analyzes of RNA-seq data. 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 ?rsaE 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: Two transcriptome comparisons were analyzed: i) Staphylococcus aureus ?rsaE mutant compared to HG003 wild-type strain, and ii) ?rsaE containing rsaE controlled by an inducible promoter (PtetO-rsaE) under induced relative to non-induced conditions. Total RNA were extracted, mRNA enriched using the MICROBExpress kit (Ambion), then RNA-seq libraries were generated with the TruSeq SBS Kit v3 (Illumina). The sequencing was performed with a HiSeq using 100 x 2 (Paired-End) bases run. Transcriptomic changes were determined by running a differential expression analysis of RNA-seq data using DESeq2.