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

Item Type:Article
Title:Systematic detection of poly(A)(+) RNA-interacting proteins and their differential binding
Creators Name:Milek, M. and Landthaler, M.
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:Protein-RNA Interactions, RNA-Binding Proteins, Photoactivatable Ribonucleoside, 4-Thiouridine, UV Cross-Linking, Oligo(dT) Affinity Purification, Mass Spectrometry
Source:Methods in Molecular Biology
Series Name:Methods in Molecular Biology
Title of Book:RNA Detection
ISSN:1064-3745
ISBN:978-1-4939-7212-8
Publisher:Springer / Humana Press (U.S.A.)
Volume:1649
Page Range:405-417
Date:2018
Official Publication:https://doi.org/10.1007/978-1-4939-7213-5_27
PubMed:View item in PubMed

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