Systematic detection of poly(A)(+) RNA-interacting proteins and their differential binding

Autor/innen

  • M. Milek
  • M. Landthaler

Journal

  • Methods in Molecular Biology

Quellenangabe

  • Methods Mol Biol 1649: 405-417

Zusammenfassung

  • 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.


DOI

doi:10.1007/978-1-4939-7213-5_27