RNA Detection

Chapter 27

Systematic Detection of Poly(A)

+

RNA-Interacting Proteins

and Their Differential Binding

Miha Milek and Markus Landthaler

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 wordsProtein–RNA interactions, RNA-binding proteins, Photoactivatable ribonucleoside, 4-

Thiouridine, UV cross-linking, Oligo(dT) affinity purification, Mass spectrometry

1 Introduction

To systematically identify proteins binding to mRNA, a quantitative

approach termed mRNA interactome capture was recently pub-

lished [1, 2]. This method exploits in vivo UV cross-linking, oligo

(dT) affinity purification, and mass spectrometry to identify the

proteins directly bound to poly(A)+RNA. Since its initial publica-

tion, interactome capture has been successfully applied to other

cultured mammalian cells [3–5],Drosophilaembryos [6, 7],Cae-

norhabditis elegans[8],Plasmodium falciparum[9],Arabidopsis

thaliana[10, 11], and yeast [4, 12]. A common feature of this

approach is to stabilize the protein–RNA interactions by either

conventional (UV 254 nm) or photoactivatable ribonucleoside

(PAR)-enhanced (UV 365 nm) cross-linking. As in cross-linking

and immunoprecipitation (CLIP)-related techniques [13–16],

covalent bonds are formed at the site of direct contact between a

nucleotide in the RNA and an amino acid in the binding protein

molecule [17]. This linkage allows for stringent oligo(dT) affinity

purification under denaturing conditions and highly selective

recovery of protein–mRNA complexes.

Imre Gaspar (ed.),RNA Detection: Methods and Protocols, Methods in Molecular Biology,
vol. 1649, DOI 10.1007/978-1-4939-7213-5_27,©Springer Science+Business Media LLC 2018

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