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

Methods Mol Biol. 2018;1737:251-272. doi: 10.1007/978-1-4939-7634-8_15.

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: Noncoding RNA; Protein–RNA interaction; RBP; RNA-binding protein; Small RNA.

MeSH terms

  • Binding Sites
  • Cross-Linking Reagents / metabolism*
  • DNA, Complementary / analysis*
  • DNA, Complementary / genetics
  • Escherichia coli / genetics
  • Escherichia coli / metabolism*
  • Escherichia coli Proteins / genetics
  • Escherichia coli Proteins / metabolism*
  • Gene Expression Regulation, Bacterial
  • Gene Library
  • High-Throughput Nucleotide Sequencing / methods*
  • Host Factor 1 Protein / genetics
  • Host Factor 1 Protein / metabolism*
  • Mycobacterium smegmatis / genetics
  • Mycobacterium smegmatis / metabolism*
  • Protein Binding
  • RNA, Bacterial / genetics
  • RNA, Bacterial / metabolism
  • RNA, Small Untranslated / genetics
  • RNA, Small Untranslated / metabolism*
  • RNA-Binding Proteins / genetics
  • RNA-Binding Proteins / metabolism
  • Toxin-Antitoxin Systems
  • Transcriptome
  • Ultraviolet Rays

Substances

  • Cross-Linking Reagents
  • DNA, Complementary
  • Escherichia coli Proteins
  • Hfq protein, E coli
  • Host Factor 1 Protein
  • RNA, Bacterial
  • RNA, Small Untranslated
  • RNA-Binding Proteins