Transcriptome-wide analysis of protein–RNA and RNA–RNA interactions in pathogenic bacteria

Regulatory RNAs and RNA-binding proteins (RBPs) play critical roles in virulence gene expression in pathogenic bacteria. A wealth of regulatory RNAs have been identified in bacterial pathogens using RNA-seq and recent technical advances are uncovering their mRNA targets. UV-crosslinking is a powerful tool for identifying protein binding sites throughout the transcriptome providing base-pair resolution of sites in vivo. With minor modifications to the protocol, RNA–RNA interactions can also be captured by proximity-dependent ligation of RNA pairs on the protein. Here, we described a high-stringency UV-crosslinking method for recovery of both protein–RNA interactions (CRAC) and RNA–RNA interactions occurring on the bait protein (CLASH). These analyses provide complementary data that provide insights into RBP, and regulatory RNA function.