Systematic Detection of Poly(A)+ RNA-Interacting Proteins and Their Differential Binding

Methods Mol Biol. 2018:1649:405-417. doi: 10.1007/978-1-4939-7213-5_27.

Abstract

RNA-binding proteins are dynamic posttranscriptional regulators of gene expression. Identification of mRNA-binding proteins in a given experimental setting is thus of great importance. We describe a procedure to enrich for direct poly(A)+ RNA protein binders by 4-thiouridine-enhanced UV cross-linking and oligo(dT) purification. Subsequent nuclease-mediated release of RNA-binding proteins (RBPs) from mRNA allows for detection of eluted proteins by mass spectrometry. In addition, we provide a comparative approach to detect differences in RBP binding activity upon a biological stimulus.

Keywords: 4-Thiouridine; Mass spectrometry; Oligo(dT) affinity purification; Photoactivatable ribonucleoside; Protein–RNA interactions; RNA-binding proteins; UV cross-linking.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Fractionation
  • Cell Line
  • Cross-Linking Reagents / chemistry
  • Humans
  • Molecular Biology / methods*
  • Poly A / metabolism*
  • Protein Binding
  • Quality Control
  • RNA-Binding Proteins / metabolism*
  • Ultraviolet Rays

Substances

  • Cross-Linking Reagents
  • RNA-Binding Proteins
  • Poly A