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High-Resolution, High-Throughput Analysis of Hfq-Binding Sites Using UV Crosslinking and Analysis of cDNA (CRAC)

  • Brandon Sy
  • Julia Wong
  • Sander Granneman
  • David Tollervey
  • David Gally
  • Jai J. Tree
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1737)

Abstract

Small regulatory nonprotein-coding RNAs (sRNAs) have emerged as ubiquitous and abundant regulators of gene expression in a diverse cross section of bacteria. They play key roles in most aspects of bacterial physiology, including central metabolism, nutrient acquisition, virulence, biofilm formation, and outer membrane composition. RNA sequencing technologies have accelerated the identification of bacterial regulatory RNAs and are now being employed to understand their functions. Many regulatory RNAs require protein partners for activity, or modulate the activity of interacting proteins. Understanding how and where proteins interact with the transcriptome is essential to elucidate the functions of the many sRNAs. Here, we describe the implementation in bacteria of a UV-crosslinking technique termed CRAC that allows stringent, transcriptome-wide recovery of bacterial RNA–protein interaction sites in vivo and at base-pair resolution. We have used CRAC to map protein–RNA interaction sites for the RNA chaperone Hfq and ribonuclease RNase E in pathogenic E. coli, and toxins from toxin–antitoxin systems in Mycobacterium smegmatis, demonstrating the broad applicability of this technique.

Keywords

Protein–RNA interaction Small RNA Noncoding RNA RBP RNA-binding protein 

References

  1. 1.
    Gama-Castro S, Salgado H, Santos-Zavaleta A et al (2016) RegulonDB version 9.0: high-level integration of gene regulation, coexpression, motif clustering and beyond. Nucleic Acids Res 44:D133–D143CrossRefPubMedGoogle Scholar
  2. 2.
    Ishihama A (2010) Prokaryotic genome regulation: multifactor promoters, multitarget regulators and hierarchic networks. FEMS Microbiol Rev 34:628–645CrossRefPubMedGoogle Scholar
  3. 3.
    Waters LS, Storz G (2009) Regulatory RNAs in bacteria. Cell 136:615–628CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Melamed S, Peer A, Faigenbaum-Romm R et al (2016) Global mapping of small RNA-target interactions in bacteria. Mol Cell 63:884–897CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Bouvier M, Sharma CM, Mika F et al (2008) Small RNA binding to 5′ mRNA coding region inhibits translational initiation. Mol Cell 32:827–837CrossRefPubMedGoogle Scholar
  6. 6.
    Storz G, Vogel J, Wassarman KM (2011) Regulation by small RNAs in bacteria: expanding frontiers. Mol Cell 43:880–891CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Smirnov A, Förstner KU, Holmqvist E et al (2016) Grad-seq guides the discovery of ProQ as a major small RNA-binding protein. Proc Natl Acad Sci U S A 113:11591–11596CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Smirnov A, Wang C, Drewry LL et al (2017) Molecular mechanism of mRNA repression in trans by a ProQ-dependent small RNA. EMBO J 36:1029–1045CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Bandyra KJ, Said N, Pfeiffer V et al (2012) The seed region of a small RNA drives the controlled destruction of the target mRNA by the endoribonuclease RNase E. Mol Cell 47:943–953CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Soper T, Mandin P, Majdalani N et al (2010) Positive regulation by small RNAs and the role of Hfq. Proc Natl Acad Sci 107:9602–9607CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Papenfort K, Sun Y, Miyakoshi M et al (2013) Small RNA-mediated activation of sugar phosphatase mRNA regulates glucose homeostasis. Cell 153:426–437CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Tree JJ, Granneman S, McAteer SP et al (2014) Identification of bacteriophage-encoded anti-sRNAs in pathogenic Escherichia coli. Mol Cell 55:199–213CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Miyakoshi M, Chao Y, Vogel J (2015) Cross talk between ABC transporter mRNAs via a target mRNA-derived sponge of the GcvB small RNA. EMBO J 34:1478–1492CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Bossi L, Schwartz A, Guillemardet B et al (2012) A role for Rho-dependent polarity in gene regulation by a noncoding small RNA. Genes Dev 26:1864–1873CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Rabhi M, Espéli O, Schwartz A et al (2011) The Sm-like RNA chaperone Hfq mediates transcription antitermination at Rho-dependent terminators. EMBO J 30:2805–2816CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Sedlyarova N, Shamovsky I, Bharati BK et al (2016) sRNA-mediated control of transcription termination in E. coli. Cell 167:111–121.e13CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Winther K, Tree JJ, Tollervey D et al (2016) VapCs of Mycobacterium tuberculosis cleave RNAs essential for translation. Nucleic Acids Res 44:9860–9871CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    van Nues R, Schweikert G, de Leau E et al (2017) Kinetic CRAC uncovers a role for Nab3 in determining gene expression profiles during stress. Nat Commun 8:12CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Granneman S, Kudla G, Petfalski E et al (2009) Identification of protein binding sites on U3 snoRNA and pre-rRNA by UV cross-linking and high-throughput analysis of cDNAs. Proc Natl Acad Sci U S A 106:9613–9618CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Holmqvist E, Wright PR, Li L et al (2016) Global RNA recognition patterns of post-transcriptional regulators Hfq and CsrA revealed by UV crosslinking in vivo. EMBO J 35:e201593360CrossRefGoogle Scholar
  21. 21.
    Chao Y, Li L, Girodat D et al (2017) In vivo cleavage map illuminates the central role of RNase E in coding and noncoding RNA pathways. Mol Cell 65:39–51CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Waters SA, McAteer SP, Kudla G et al (2017) Small RNA interactome of pathogenic E. coli revealed through crosslinking of RNase E. EMBO J 36:374–387CrossRefPubMedGoogle Scholar
  23. 23.
    Lalaouna D, Carrier MC, Semsey S et al (2015) A 3′ external transcribed spacer in a tRNA transcript acts as a sponge for small RNAs to prevent transcriptional noise. Mol Cell 58:393–405CrossRefPubMedGoogle Scholar
  24. 24.
    Han K, Tjaden B, Lory S (2016) GRIL-seq provides a method for identifying direct targets of bacterial small regulatory RNA by in vivo proximity ligation. Nat Microbiol 16239:1–10Google Scholar
  25. 25.
    Dahan S, Knutton S, Shaw RK et al (2004) Transcriptome of enterohemorrhagic Escherichia coli O157 adhering to eukaryotic plasma membranes. Infect Immun 72:5452–5459CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Van Nostrand EL, Pratt GA, Shishkin AA et al (2016) Robust transcriptome-wide discovery of RNA-binding protein binding sites with enhanced CLIP (eCLIP). Nat Methods 13:508–514CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Webb S, Hector RD, Kudla G et al (2014) PAR-CLIP data indicate that Nrd1-Nab3-dependent transcription termination regulates expression of hundreds of protein coding genes in yeast. Genome Biol 15:R8CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Dodt M, Roehr J, Ahmed R et al (2012) FLEXBAR—flexible barcode and adapter processing for next-generation sequencing platforms. Biology 1:895–905CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Uhl M, Houwaart T, Corrado G et al (2016) Computational analysis of CLIP-seq data. Methods 118–119:60–72Google Scholar
  30. 30.
    Langenberger D, Bermudez-Santana C, Hertel J et al (2009) Evidence for human microRNA-offset RNAs in small RNA sequencing data. Bioinformatics 25:2298–2301CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  • Brandon Sy
    • 1
  • Julia Wong
    • 1
  • Sander Granneman
    • 2
  • David Tollervey
    • 3
  • David Gally
    • 4
  • Jai J. Tree
    • 1
  1. 1.School of Biotechnology and Biomolecular Sciences, University of New South Wales SydneySydneyAustralia
  2. 2.Institute of Structural and Molecular Biology, Centre for Synthetic and Systems Biology (SynthSys), University of EdinburghEdinbughUK
  3. 3.Wellcome Trust Centre for Cell Biology, University of EdinburghEdinburghUK
  4. 4.Division of Infection and ImmunityThe Roslin Institute, University of EdinburghEdinburghUK

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