iCLIP: protein-RNA interactions at nucleotide resolution

Ina Huppertz, Jan Attig, Andrea D'Ambrogio, Laura E Easton, Christopher R Sibley, Yoichiro Sugimoto, Mojca Tajnik, Julian König, Jernej Ule

Research output: Contribution to journalArticlepeer-review

Abstract

RNA-binding proteins (RBPs) are key players in the post-transcriptional regulation of gene expression. Precise knowledge about their binding sites is therefore critical to unravel their molecular function and to understand their role in development and disease. Individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) identifies protein-RNA crosslink sites on a genome-wide scale. The high resolution and specificity of this method are achieved by an intramolecular cDNA circularization step that enables analysis of cDNAs that truncated at the protein-RNA crosslink sites. Here, we describe the improved iCLIP protocol and discuss critical optimization and control experiments that are required when applying the method to new RBPs.

Original languageEnglish
Pages (from-to)274-287
Number of pages14
JournalMethods
Volume65
Issue number3
DOIs
Publication statusPublished - Feb 2014

Keywords

  • binding sites
  • DNA
  • Gene Expression Regulation
  • gene library
  • HeLa Cells
  • High-Throughput Nucleotide Sequencing
  • humans
  • Immunoprecipitation/methods
  • protein binding
  • RNA/chemistry
  • RNA-Binding Proteins/chemistry
  • ultraviolet rays
  • circular/chemistry

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