PAR-CLIP for Discovering Target Sites of RNA-Binding Proteins

Methods Mol Biol. 2018:1720:55-75. doi: 10.1007/978-1-4939-7540-2_5.

Abstract

RNA-binding proteins (RBPs) establish posttranscriptional gene regulation (PTGR) by coordinating the maturation, editing, transport, stability, and translation of cellular RNAs. A variety of experimental approaches have been developed to characterize the RNAs associated with RBPs in vitro as well as in vivo. Our laboratory developed Photoactivatable-Ribonucleoside-Enhanced Cross-Linking and Immunoprecipitation (PAR-CLIP), which in combination with next-generation sequencing enables the identification of RNA targets of RBPs at a nucleotide-level resolution. Here we present an updated and condensed step-by-step PAR-CLIP protocol followed by the description of our RNA-seq data analysis pipeline.

Keywords: Next-generation sequencing; PAR-CLIP Suite; RNA recognition element; Ribonucleoprotein.

MeSH terms

  • Animals
  • Binding Sites
  • Computational Biology / methods
  • Cross-Linking Reagents
  • High-Throughput Nucleotide Sequencing / methods*
  • Humans
  • Immunoprecipitation / methods*
  • Nucleotides / chemistry
  • Protein Binding
  • RNA / chemistry
  • RNA / isolation & purification
  • RNA / metabolism*
  • RNA-Binding Proteins / chemistry
  • RNA-Binding Proteins / metabolism*
  • Ribonucleosides / chemistry
  • Sequence Analysis, RNA / methods*
  • Staining and Labeling / methods
  • Ultraviolet Rays

Substances

  • Cross-Linking Reagents
  • Nucleotides
  • RNA-Binding Proteins
  • Ribonucleosides
  • RNA