Purification of cross-linked RNA-protein complexes by phenol-toluol extraction

Erika C. Urdaneta, Carlos H. Vieira-Vieira, Timon Hick, Hans-Herrmann Wessels, Davide Figini, Rebecca Moschall, Jan Medenbach, Uwe Ohler, Sander Granneman, Matthias Selbach and Benedikt M. Beckmann ()
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Erika C. Urdaneta: Humboldt University
Carlos H. Vieira-Vieira: Max-Delbrück-Center for Molecular Medicine
Timon Hick: Humboldt University
Hans-Herrmann Wessels: Berlin Institute for Medical Systems Biology, Max-Delbrück-Center for Molecular Medicine
Davide Figini: Humboldt University
Rebecca Moschall: Biochemistry I, University of Regensburg
Jan Medenbach: Biochemistry I, University of Regensburg
Uwe Ohler: Berlin Institute for Medical Systems Biology, Max-Delbrück-Center for Molecular Medicine
Sander Granneman: University of Edinburgh, Max Born Crescent
Matthias Selbach: Max-Delbrück-Center for Molecular Medicine
Benedikt M. Beckmann: Humboldt University

Nature Communications, 2019, vol. 10, issue 1, 1-17

Abstract: Abstract Recent methodological advances allowed the identification of an increasing number of RNA-binding proteins (RBPs) and their RNA-binding sites. Most of those methods rely, however, on capturing proteins associated to polyadenylated RNAs which neglects RBPs bound to non-adenylate RNA classes (tRNA, rRNA, pre-mRNA) as well as the vast majority of species that lack poly-A tails in their mRNAs (including all archea and bacteria). We have developed the Phenol Toluol extraction (PTex) protocol that does not rely on a specific RNA sequence or motif for isolation of cross-linked ribonucleoproteins (RNPs), but rather purifies them based entirely on their physicochemical properties. PTex captures RBPs that bind to RNA as short as 30 nt, RNPs directly from animal tissue and can be used to simplify complex workflows such as PAR-CLIP. Finally, we provide a global RNA-bound proteome of human HEK293 cells and the bacterium Salmonella Typhimurium.

Date: 2019
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DOI: 10.1038/s41467-019-08942-3

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