Global RNA recognition patterns of post-transcriptional regulators Hfq and CsrA revealed by UV crosslinking in vivo

The molecular roles of many RNA-binding proteins in bacterial post-transcriptional gene regulation are not well understood. Approaches combining in vivo UV crosslinking with RNA deep sequencing (CLIP-seq) have begun to revolutionize the transcriptome-wide mapping of eukaryotic RNA-binding protein target sites. We have applied CLIP-seq to chart the target landscape of two major bacterial post-transcriptional regulators, Hfq and CsrA, in the model pathogen Salmonella Typhimurium. By detecting binding sites at single-nucleotide resolution, we identify RNA preferences and structural constraints of Hfq and CsrA during their interactions with hundreds of cellular transcripts. This reveals 3'-located Rho-independent terminators as a universal motif involved in Hfq–RNA interactions. Additionally, Hfq preferentially binds 5' to sRNA-target sites in mRNAs, and 3' to seed sequences in sRNAs, reflecting a simple logic in how Hfq facilitates sRNA–mRNA interactions. Importantly, global knowledge of Hfq sites significantly improves sRNA-target predictions. CsrA binds AUGGA sequences in apical loops and targets many Salmonella virulence mRNAs. Overall, our generic CLIP-seq approach will bring new insights into post-transcriptional gene regulation by RNA-binding proteins in diverse bacterial species. Overall design: To detect the binding sites for Salmonella Hfq and CsrA we collected three biological replicates of Salmonella strain SL1344 where either the hfq or csrA gene was fused to a 3xFLAG tag. Half of each replicate culture was irradiated with UV light (254 nm, 800 mJ/cm2) (+CL), while the other half was left untreated (-CL). Bacteria were lysed and the FLAG-tagged protein was immunoprecipitated using a monoclonal anti-FLAG antibody. The samples were treated with benzonase nuclease, calf intestine phophorylase, and polynucleotide kinase in the presence of radioactive gamma-ATP. Samples were separated with SDS-PAGE followed by transfer to nitrocellulose membranes. Radioactively labelled RNA-protein complexes were eluted from membranes and treated with Proteinase K. Purified RNA was used as input for library preparation using the NEB Next Small RNA Library kit.