CLIP: A method for identifying protein-RNA interaction sites in living cells

Jernej Ule, Kirk Jensen, Aldo Mele, Robert B. Darnell

Research output: Contribution to journalArticle

404 Citations (Scopus)

Abstract

Nucleic-acid binding proteins constitute nearly one-fourth of all functionally annotated human genes. Genome-wide analysis of protein-nucleic acid contacts has not yet been performed for most of these proteins, restricting attempts to establish a comprehensive understanding of protein function. UV cross-linking is a method typically used to determine the position of direct interactions between proteins and nucleic acids. We have developed the cross-linking and immunoprecipitation assay, which exploits the covalent protein-nucleic acid cross-linking to stringently purify a specific protein-RNA complex using immunoprecipitation followed by SDS-PAGE separation. In this way, the vast majority of non-specific contaminating RNA, which can bind to co-immunoprecipitated proteins or beads, can be removed. Here, we present an improved protocol that performs RNA linker ligation before the SDS-PAGE step, and describe its application to the specific purification and amplification of RNA ligands of Nova in neurons.

LanguageEnglish
Pages376-386
Number of pages11
JournalMethods
Volume37
Issue number4
DOIs
Publication statusPublished - 1 Dec 2005

Keywords

  • CLIP
  • Immunoprecipitation
  • Nova
  • Protein-RNA binding
  • UV cross-linking

ASJC Scopus subject areas

  • Molecular Biology
  • Biochemistry, Genetics and Molecular Biology(all)

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