Functional dissection of sRNA translational regulators by nonhomologous random recombination and in vivo selection

Small nontranslated RNAs (sRNAs) regulate a variety of biological processes. DsrA and OxyS are two E. coli sRNAs that regulate the translation of rpoS, which encodes a protein sigma factor. Due to their structural complexity, the functional dissection of sRNAs solely by designing and assaying mutants can be challenging. Here, we present a complementary approach to the study of functional RNAs, in which highly diversified RNA libraries are generated by nonhomologous random recombination (NRR) and processed efficiently by in vivo selections that link RNA activities to cell survival. When applied to DsrA and OxyS, this approach rapidly identified essential and nonessential regions of both sRNAs. Resulting hypotheses about DsrA and OxyS structure-function relationships were tested and further refined experimentally. Our findings demonstrate an efficient, unbiased approach to the functional dissection of nucleic acids.