title: Global RNA recognition patterns of post-transcriptional regulators Hfq and CsrA revealed by UV crosslinking in vivo.
abstract: 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.
authors.affiliation: Institute for Molecular Infection Biology, University of Würzburg, Würzburg, Germany.
authors.forename: Erik
authors.initials: E
authors.lastname: Holmqvist
authors.affiliation: Bioinformatics Group, Department of Computer Science, Albert Ludwig University Freiburg, Freiburg, Germany.
authors.forename: Patrick R
authors.initials: PR
authors.lastname: Wright
authors.affiliation: Institute for Molecular Infection Biology, University of Würzburg, Würzburg, Germany.
authors.forename: Lei
authors.initials: L
authors.lastname: Li
authors.affiliation: Institute for Molecular Infection Biology, University of Würzburg, Würzburg, Germany.
authors.forename: Thorsten
authors.initials: T
authors.lastname: Bischler
authors.affiliation: Institute for Molecular Infection Biology, University of Würzburg, Würzburg, Germany.
authors.forename: Lars
authors.initials: L
authors.lastname: Barquist
authors.affiliation: Max Planck Genome Centre Cologne, Max Planck Institute for Plant Breeding Research, Cologne, Germany.
authors.forename: Richard
authors.initials: R
authors.lastname: Reinhardt
authors.affiliation: Bioinformatics Group, Department of Computer Science, Albert Ludwig University Freiburg, Freiburg, Germany BIOSS Centre for Biological Signaling Studies, University of Freiburg, Freiburg, Germany backofen@informatik.uni-freiburg.de joerg.vogel@uni-wuerzburg.de.
authors.forename: Rolf
authors.initials: R
authors.lastname: Backofen
authors.affiliation: Institute for Molecular Infection Biology, University of Würzburg, Würzburg, Germany backofen@informatik.uni-freiburg.de joerg.vogel@uni-wuerzburg.de.
authors.forename: Jörg
authors.identifier: 0000-0003-2220-1404
authors.initials: J
authors.lastname: Vogel
mesh_terms: D001665:Binding Sites; D035001:Host Factor 1 Protein / Q000378:metabolism*; D011485:Protein Binding; D012333:RNA, Messenger / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D012486:Salmonella typhimurium / Q000201:enzymology*; D014466:Ultraviolet Rays
chemical_list: D035001:Host Factor 1 Protein; D012333:RNA, Messenger; D016601:RNA-Binding Proteins
keywords: CLIP; CsrA; Hfq; non‐coding RNA; peak calling; post‐transcriptional control; small RNA; terminator; translation