Small non-coding RNAs (sRNAs) have been found by the hundreds in bacterial genomes. They control gene expression by interacting with target mRNAs and altering their translation and/or stability. Despite intensive study, it is not understood how a single sRNA distinguishes between multiple mRNA targets or how multiple sRNAs function simultaneously to control a particular mRNA target. We discovered that five homologous sRNAs (Qrr sRNAs) lie at the heart of vibrio quorum-sensing systems to control 20 mRNA targets. Multiple sRNAs controlling multiple mRNAs is unprecedented in sRNA systems. These features of the vibrio system positioned us to attack questions concerning mRNA target selection and specificity by dissecting pairing regimes, target preferences, and modes of regulation. We combined experiments with mathematical modeling to demonstrate that, remarkably, the Qrr sRNAs use four mechanisms to control their particular targets: catalytic degradation, coupled degradation, sequestration, and activation-induced degradation. The Qrr sRNAs form different base-pairing interactions with each mRNA target; the particular pairing strategy determines which regulatory mechanism ensues. Indeed, we could convert a particular regulatory mechanism at will into a different one by altering the base-pairing position or binding strength. The different interaction mechanisms result in distinct dynamical behaviors of the mRNA targets, which we showed is critical for properly timed quorum-sensing responses.
To explore the roles individual Qrr nucleotides play in mRNA target regulation, we developed an analysis method we call RSort-Seq that combines saturating mutagenesis, FACS, high-throughput sequencing, and mutual information theory. We developed companion biochemical assays to assign a specific molecular mechanism by which each important base exerts its effect. This strategy yielded a comprehensive understanding of the precise inter-molecular and intra-molecular interactions required for Qrr stability, Hfq interaction, stem-loop formation, and base-pairing to single or multiple mRNA targets. Importantly, the RSort-Seq analysis provided strikingly different results from those predicted by commonly used RNA folding algorithms. Our approach is applicable to any RNA-RNA interaction, including other bacterial sRNAs and regulatory RNAs in higher organisms.