Optimization of PAR-CLIP for transcriptome-wide identification of binding sites of RNA-binding proteins

Methods. 2017 Apr 15:118-119:24-40. doi: 10.1016/j.ymeth.2016.10.007. Epub 2016 Oct 17.

Abstract

Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP) in combination with next-generation sequencing is a powerful method for identifying endogenous targets of RNA-binding proteins (RBPs). Depending on the characteristics of each RBP, key steps in the PAR-CLIP procedure must be optimized. Here we present a comprehensive step-by-step PAR-CLIP protocol with detailed explanations of the critical steps. Furthermore, we report the application of a new PAR-CLIP data analysis pipeline to three distinct RBPs targeting different annotation categories of cellular RNAs.

Keywords: Next-generation sequencing; Photocrosslinking; Ribonucleoprotein analysis; UV RNA-protein crosslinking.

Publication types

  • Review
  • Research Support, Non-U.S. Gov't
  • Research Support, N.I.H., Extramural

MeSH terms

  • Antibodies / chemistry
  • Base Sequence
  • Binding Sites
  • Cell Line, Tumor
  • Fragile X Mental Retardation Protein / genetics
  • Fragile X Mental Retardation Protein / metabolism
  • Gene Library
  • HEK293 Cells
  • High-Throughput Nucleotide Sequencing / methods*
  • Humans
  • Immunoprecipitation / methods*
  • Nucleic Acid Conformation
  • Phosphoproteins / genetics
  • Phosphoproteins / metabolism
  • Phosphorus Radioisotopes
  • Protein Binding
  • RNA / chemistry*
  • RNA / genetics
  • RNA / metabolism
  • RNA-Binding Protein FUS / genetics
  • RNA-Binding Protein FUS / metabolism
  • RNA-Binding Proteins / genetics*
  • RNA-Binding Proteins / metabolism
  • Ribonucleases / chemistry
  • Sequence Alignment
  • Sequence Analysis, RNA / methods*
  • Thiouridine / chemistry
  • Thiouridine / metabolism*
  • Ultraviolet Rays

Substances

  • Antibodies
  • FMR1 protein, human
  • FUS protein, human
  • La protein, human
  • Phosphoproteins
  • Phosphorus Radioisotopes
  • RNA-Binding Protein FUS
  • RNA-Binding Proteins
  • Fragile X Mental Retardation Protein
  • Thiouridine
  • RNA
  • Ribonucleases