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Nat Commun. 2019 Mar 1;10(1):990. doi: 10.1038/s41467-019-08942-3.

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

Author information

1
IRI Life Sciences, Humboldt University, Philippstr. 13, 10115, Berlin, Germany.
2
Max-Delbrück-Center for Molecular Medicine, Robert-Rössle-Str. 10, 13125, Berlin, Germany.
3
Berlin Institute for Medical Systems Biology, Max-Delbrück-Center for Molecular Medicine, Robert-Rössle-Str. 10, 13125, Berlin, Germany.
4
Department of Biology, Humboldt University, Philippstr. 13, 10115, Berlin, Germany.
5
Biochemistry I, University of Regensburg, Universitätsstrasse 31, 93053, Regensburg, Germany.
6
Centre for Systems and Synthetic Biology (SynthSys), University of Edinburgh, Max Born Crescent, Edinburgh, EH9 3BF, UK.
7
IRI Life Sciences, Humboldt University, Philippstr. 13, 10115, Berlin, Germany. benedikt.beckmann@iri-lifesciences.de.

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.

PMID:
30824702
PMCID:
PMC6397201
DOI:
10.1038/s41467-019-08942-3
[Indexed for MEDLINE]
Free PMC Article

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