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RNA Detection pp 405-417 | Cite as

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

  • Miha Milek
  • Markus LandthalerEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1649)

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.

Key words

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

Notes

Acknowledgment

We thank Koshi Imami and Matthias Selbach (Max Delbrück Center for Molecular Medicine, Berlin) for their expertise in mass spectrometry. This work was supported by an International European Fellowship (Maria Sklodowska Actions FP7-PEOPLE-2011-IEF) and DFG grant LA 2941/5-1.

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Copyright information

© Springer Science+Business Media LLC 2018

Authors and Affiliations

  1. 1.RNA Biology and Posttranscriptional Regulation, Max Delbrück Center for Molecular Medicine BerlinBerlin Institute for Molecular Systems BiologyBerlinGermany

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