Comprehensive architecture of the bacterial RNA Interactome

RNA-based regulation is ubiquitous in all microbes yet generating a global map of RNA–RNA interactions has been a formidable challenge. Here, TRIC-seq is introduced as an unbiased approach to map transcriptome-wide RNA–RNA interactions in several bacterial species. Applying TRIC-seq to Escherichia coli captured thousands of unique interactions, unveiling not only the targets of regulatory small RNAs (sRNAs) but also novel non-canonical regulatory interactions involving ribosomal RNAs, transfer RNAs, and messenger RNAs, including a widespread interaction between the 3’ extension of the 16S rRNA and the 5'UTRs of stress-response mRNAs. Unsupervised clustering of the interactome revealed a highly modular architecture, clustered by sRNA interactions. TRIC-seq is presented as a genetics-free approach that can be applied to any bacteria to de novo reveal regulatory RNAs along with their regulons at high specificity and resolution. TRIC-seq revealed a vast, functionally coherent cluster of mRNAs that aggregates and is excluded from ribosomes, providing compelling evidence for bacterial RNA condensates that share key properties with eukaryotic stress granules. TRIC-seq’s robust mapping provides a powerful platform to uncover RNA structures and generating foundational data for the next generation of biological models. TRIC-seq of wild type microbes grown to stationary phase - E. coli, S. stutzeri, M. xanthus and S. aureus. TRIC-seq captures global RNA-RNA interactions in bacteria using a in-situ proximity ligation approach in bacteria