Genome-wide detection of novel regulatory RNAs in E. coli

The intergenic regions in bacterial genomes can contain regulatory leader sequences and small RNAs (sRNAs), which both serve to modulate gene expression. Computational analyses have predicted the presence of hundreds of these noncoding regulatory RNAs in Escherichia coli; however, only about 80 have been experimentally validated. By applying a deep-sequencing approach, we detected and quantified the vast majority of the previously validated regulatory elements and identified 10 new sRNAs and nine new regulatory leader sequences in the intergenic regions of E. coli. Half of the newly discovered sRNAs displayed enhanced stability in the presence of the RNA-binding protein Hfq, which is vital to the function of many of the known E. coli sRNAs. Whereas previous methods have often relied on phylogenetic conservation to identify regulatory leader sequences, only five of the newly discovered E. coli leader sequences were present in the genomes of other enteric species. For those newly identified regulatory elements having orthologs in Salmonella, evolutionary analyses showed that these regions encoded new noncoding elements rather than small, unannotated protein-coding transcripts. In addition to discovering new noncoding regulatory elements, we validated 53 sRNAs that were previously predicted but never detected and showed that the presence, within intergenic regions, of σ(70) promoters and sequences with compensatory mutations that maintain stable RNA secondary structures across related species is a good predictor of novel sRNAs.