Toward a Comprehensive Analysis of Posttranscriptional Regulatory Networks: a New Tool for the Identification of Small RNA Regulators of Specific mRNAs

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.