Comprehensive architecture of the bacterial RNA interactome

RNA-based regulation pervades bacteria, but obtaining a transcriptome-wide map of native RNA contacts has been technically challenging. Here TRIC-seq (Total RNA Interaction Capture) is introduced as an in situ, genetics-free proximity-ligation approach that preserves cellular context and resolves both intramolecular (structure) and intermolecular (regulatory) RNA contacts at high resolution and specificity. In Escherichia coli , TRIC-seq captures thousands of unique interactions, recovering known small RNA (sRNA) regulons and revealing non-canonical contacts among rRNAs, tRNAs and mRNAs, including a widespread interaction between the 3′ extension of 16S rRNA and 5’UTRs of stress-response mRNAs. Unsupervised analysis exposes a highly modular interactome organized by sRNA hubs. Beyond E. coli , TRIC-seq maps interactomes in diverse bacteria, enabling de novo discovery of novel sRNAs along with their targets. Finally, TRIC-seq uncovers a large, functionally coherent cohort of stress-related mRNAs that co-aggregate and are depleted for ribosome contacts.