Transcriptome-Wide Analysis of Protein-RNA and RNA-RNA Interactions in Pathogenic Bacteria

Methods Enzymol. 2018;612:467-488. doi: 10.1016/bs.mie.2018.08.009. Epub 2018 Sep 22.

Abstract

Regulatory RNAs and RNA-binding proteins (RBPs) play critical roles in virulence gene expression in pathogenic bacteria. A wealth of regulatory RNAs have been identified in bacterial pathogens using RNA-seq and recent technical advances are uncovering their mRNA targets. UV-crosslinking is a powerful tool for identifying protein binding sites throughout the transcriptome providing base-pair resolution of sites in vivo. With minor modifications to the protocol, RNA-RNA interactions can also be captured by proximity-dependent ligation of RNA pairs on the protein. Here, we described a high-stringency UV-crosslinking method for recovery of both protein-RNA interactions (CRAC) and RNA-RNA interactions occurring on the bait protein (CLASH). These analyses provide complementary data that provide insights into RBP, and regulatory RNA function.

Keywords: Hfq; ProQ; RNase; Regulatory noncoding RNA; Ribonuclease; Small RNA; sRNA interactome.

MeSH terms

  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Binding Sites
  • Gene Expression Profiling / methods*
  • Gene Expression Regulation, Bacterial / genetics
  • Gene Expression Regulation, Bacterial / physiology
  • Protein Binding
  • RNA, Bacterial / genetics
  • RNA, Bacterial / metabolism*
  • RNA-Binding Proteins / genetics
  • RNA-Binding Proteins / metabolism*
  • Transcriptome / genetics*

Substances

  • Bacterial Proteins
  • RNA, Bacterial
  • RNA-Binding Proteins