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EMBO J. 2016 May 2;35(9):991-1011. doi: 10.15252/embj.201593360. Epub 2016 Apr 4.

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

Author information

1
Institute for Molecular Infection Biology, University of Würzburg, Würzburg, Germany.
2
Bioinformatics Group, Department of Computer Science, Albert Ludwig University Freiburg, Freiburg, Germany.
3
Max Planck Genome Centre Cologne, Max Planck Institute for Plant Breeding Research, Cologne, Germany.
4
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.
5
Institute for Molecular Infection Biology, University of Würzburg, Würzburg, Germany backofen@informatik.uni-freiburg.de joerg.vogel@uni-wuerzburg.de.

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.

KEYWORDS:

CLIP; CsrA; Hfq; non‐coding RNA; peak calling; post‐transcriptional control; small RNA; terminator; translation

PMID:
27044921
PMCID:
PMC5207318
DOI:
10.15252/embj.201593360
[Indexed for MEDLINE]
Free PMC Article

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