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Small RNAs (sRNAs) posttranscriptionally regulate mRNA targets, typically under conditions of environmental stress. Although hundreds of sRNAs have been discovered in diverse bacterial genomes, most sRNAs remain uncharacterized, even in model organisms. Identification of mRNA targets directly regulated by sRNAs is rate-limiting for sRNA functional characterization. To address this, we developed a computational pipeline that we named SPOT for s RNA target p rediction o rganizing t ool. SPOT incorporates existing computational tools to search for sRNA binding sites, allows filtering based on experimental data, and organizes the results into a standardized report. SPOT sensitivity (number of correctly predicted targets/number of total known targets) was equal to or exceeded any individual method when used on 12 characterized sRNAs. Using SPOT, we generated a set of target predictions for the sRNA RydC, which was previously shown to positively regulate cfa mRNA, encoding cyclopropane fatty acid synthase. SPOT identified cfa along with additional putative mRNA targets, which we then tested experimentally. Our results demonstrated that in addition to cfa mRNA, RydC also regulates trpE and pheA mRNAs, which encode aromatic amino acid biosynthesis enzymes. Our results suggest that SPOT can facilitate elucidation of sRNA target regulons to expand our understanding of the many regulatory roles played by bacterial sRNAs. IMPORTANCE Small RNAs (sRNAs) regulate gene expression in diverse bacteria by interacting with mRNAs to change their structure, stability, or translation. Hundreds of sRNAs have been identified in bacteria, but characterization of their regulatory functions is limited by difficulty with sensitive and accurate identification of mRNA targets. Thus, new robust methods of bacterial sRNA target identification are in demand. Here, we describe our s mall RNA target p rediction o rganizing t ool (SPOT), which streamlines the process of sRNA target prediction by providing a single pipeline that combines available computational prediction tools with customizable results filtering based on experimental data. SPOT allows the user to rapidly produce a prioritized list of predicted sRNA-target mRNA interactions that serves as a basis for further experimental characterization. This tool will facilitate elucidation of sRNA regulons in bacteria, allowing new discoveries regarding the roles of sRNAs in bacterial stress responses and metabolic regulation.

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sRNA Target Prediction Organizing Tool (SPOT) Integrates Computational and Experimental Data To Facilitate Functional Characterization of Bacterial Small RNAs