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.