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

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Item Type:

Article

Title:

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

Creators Name:

Urdaneta, E.C. and Vieira-Vieira, C.H. and Hick, T. and Wessels, H.H. and Figini, D. and Moschall, R. and Medenbach, J. and Ohler, U. and Granneman, S. and Selbach, M. and Beckmann, B.M.

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.

Keywords:

Base Sequence, Brain, HEK293 Cells, Inbred C57BL Mice, Messenger RNA, Molecular Biology, Multiprotein Complexes, Phenol, Proteome, Proteomics, Ribonucleoproteins, RNA, RNA-Binding Proteins, Salmonella typhimurium, Sensitivity and Specificity, Toluene, Animals, Mice

Source:

Nature Communications

ISSN:

2041-1723

Publisher:

Nature Publishing Group

Volume:

Number:

Page Range:

990

Date:

1 March 2019