High-throughput RNA sequencing and allied techniques for profiling RNA in bacterial cells have uncovered an extraordinarily rich transcriptome, replete with hundreds of short regulatory RNAs. Recent work has also demonstrated that both mRNAs and tRNAs can generate short and stable sub-fragments that have regulatory activity. These (often) non-coding RNAs function in gene regulation and base-pair with target RNAs through short regions of complementarity. Because they contain few conserved sequences or structural motifs, identifying and predicting their function has been a major challenge.
In this talk I will overview some of the new methodologies that we have implemented for capturing regulatory RNA functions transcriptome-wide. Using UV-crosslinking and RNA-sequencing we have profiled RNA interactions with key regulatory RNA-binding proteins at nucleotide resolution providing insights into binding sites and identifying new regulatory RNAs. A major technical advance came with our ability to use this technique to capture regulatory RNA-mRNA interactions by RNA proximity-dependant ligation (termed CLASH). This methodology ligates regulatory RNA and target together in vivo allowing sequencing of interacting RNA pairs. Using this approach, we have profiled the regulatory RNA “interactome” and uncovered the function of hundreds of regulatory RNAs, many for the first time.
Gram positive bacteria pose a unique problem for understanding regulatory RNA function as they largely lack the regulatory RNA machinery found in model Gram negatives. In recent work we have applied CLASH to Staphylococcus aureus by using the endoribonuclease RNase III as a ‘bait’ protein. The S. aureus RNA interactome has allowed us to identify RNAs required for antibiotic tolerance and toxin production including an mRNA 3’UTR required for vancomycin tolerance.
These studies have identified interaction partners for many of the hundreds of regulatory RNAs identified to date and shed light on their functions in bacterial pathogens.