Abstract
A number of computational or experimental tools have been developed to identify targets of small RNA (sRNA) regulation. Here, we modified one of these methods, based on in vivo proximity ligation of sRNAs bound to their targets, referred to as rGRIL-seq, that can be used to capture sRNA regulators of a gene of interest. Intracellular expression of bacteriophage T4 RNA ligase leads to a covalent linking of sRNAs base-paired with mRNAs, and the chimeras are captured using oligonucleotides complementary to the mRNA, followed by sequencing. This allows the identification of known as well as novel sRNAs. We applied rGRIL-seq toward finding sRNA regulators of expression of the stress response sigma factor RpoS in Escherichia coli, Pseudomonas aeruginosa, and Vibrio cholerae In E. coli, we confirmed the regulatory role of known sRNAs and discovered a new negative regulator, asYbiE. When applied to P. aeruginosa and V. cholerae, we identified two novel sRNAs (s03661 and s0223) in P. aeruginosa and two known sRNAs (TfoR and Vcr043) in V. cholerae as direct regulators of rpoS The use of rGRIL-seq for defining multiple posttranscriptional regulatory inputs into individual mRNAs represents a step toward a more comprehensive understanding of the workings of bacterial regulatory networks.IMPORTANCE With the recognition of the importance of posttranscriptional regulation mediated by bacterial small RNAs (sRNAs), their contribution to global gene expression regulatory networks needs to be addressed in a truly comprehensive manner. While a single sRNA targets multiple RNAs, an mRNA can be regulated by multiple sRNAs that can be either transcribed individually or derived by processing of mRNAs. In this paper, we developed a tool (referred to as rGRIL-seq) to identify sRNAs that regulate mRNAs regardless of their origin. We demonstrated the utility of this approach by identifying positive and negative sRNA regulators of the rpoS mRNA in three bacterial species. We not only described known sRNAs of E. coli or P. aeruginosa that control rpoS but also identified several new rpoS regulators in V. cholerae Therefore, rGRIL-seq can be used to identify species-specific sRNAs targeting a conserved mRNA, and they likely play an important role in bacterial adaptation to specific environmental niches.