RNA-binding protein immunopurification-microarray (RIP-Chip) analysis to profile localized RNAs

Methods Mol Biol. 2011:714:369-85. doi: 10.1007/978-1-61779-005-8_23.

Abstract

Post-transcriptional gene regulation is largely mediated by RNA-binding proteins (RBPs) that modulate mRNA expression at multiple levels, from RNA processing to translation, localization, and degradation. Thereby, the genome-wide identification of mRNAs regulated by RBPs is crucial to uncover post--transcriptional gene regulatory networks. In this chapter, we provide a detailed protocol for one of the techniques that has been developed to systematically examine RNA targets for RBPs. This technique involves the purification of endogenously formed RBP-mRNA complexes with specific antibodies from cellular extracts, followed by the identification of associated RNAs using DNA microarrays. Such RNA-binding protein immunopurification-microarray profiling, also called RIP-Chip, has also been applied to identify mRNAs that are transported to distinct subcellular compartments by RNP-motor complexes. The application and further development of this method could provide global insights into the subcellular architecture of the RBP-RNA network, and how it is restructured upon changing environmental conditions, during development, and possibly in disease.

MeSH terms

  • Animals
  • Antibodies / immunology
  • Antibodies / metabolism
  • Antibody Specificity
  • Cell Extracts
  • Color
  • Edetic Acid / chemistry
  • Fluorescent Dyes / metabolism
  • Humans
  • Microspheres
  • Nerve Tissue Proteins / metabolism
  • Nucleic Acid Hybridization
  • Oligonucleotide Array Sequence Analysis / methods*
  • RNA / analysis
  • RNA / genetics
  • RNA / isolation & purification
  • RNA / metabolism*
  • RNA Transport
  • RNA, Antisense / genetics
  • RNA, Antisense / isolation & purification
  • RNA, Antisense / metabolism
  • RNA-Binding Proteins / immunology
  • RNA-Binding Proteins / isolation & purification*
  • RNA-Binding Proteins / metabolism*
  • Ribonucleoproteins / isolation & purification
  • Ribonucleoproteins / metabolism
  • Sepharose / chemistry
  • Staphylococcal Protein A / metabolism
  • Time Factors

Substances

  • Antibodies
  • Cell Extracts
  • Fluorescent Dyes
  • G-substrate
  • Nerve Tissue Proteins
  • PUM1 protein, human
  • PUM2 protein, human
  • RNA, Antisense
  • RNA-Binding Proteins
  • Ribonucleoproteins
  • Staphylococcal Protein A
  • RNA
  • Sepharose
  • Edetic Acid