A Method to Efficiently Select mRNA Targets and Identify sRNA Regulators

FARE Award Winner

Authors

• J Chen
• S Gottesman

Abstract

Small regulatory RNAs are present in all three kingdoms of life. Bacterial small regulatory RNAs (sRNAs) are key stress-response regulators that can shield bacteria against adverse conditions through fine-tuning a broad repertoire of stress-response genes. Mechanistically, bacterial sRNA regulation is achieved through controlling the translation and/or stability of target mRNAs by an antisense mechanism. Here we present an effective and efficient combinatorial method to select mRNA targets and identify sRNA regulators. The candidate mRNA targets were selected based on physical association with Hfq protein plus transcript level change in the presence versus absence of Hfq. By analyzing genomic data, we predicted 94 candidate mRNA targets. We validated the selection method by experimentally identifying sRNA regulators for 2 selected candidate mRNA targets (mutS and yhcN) using a reporter-based, sRNA overexpression library method. We report here that Spot42 sRNA strongly represses yhcN, and ArcZ sRNA represses mutS gene expression, both by a post-transcriptional mechanism. More importantly, we showed that in both cases ablation of physiological levels of the sRNA is sufficient to disturb target mRNA expression, indicating a bona fide regulation related to the bacterial physiology. This was further corroborated by studying sRNA regulation of MutS function where we showed sRNA-mediated dampening of mutS expression led to elevated stress-induced bacterial mutagenesis. Taken together, this study builds a pipeline for efficiently identifying sRNA regulators involved in specific biological events of interest by providing guidelines for selecting candidate mRNA targets and experimentally identifying and validating sRNA regulators in action.

Scientific Focus Area: Microbiology and Infectious Diseases

This page was last updated on Friday, March 26, 2021