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

Erika C Urdaneta,Benedikt M Beckmann,Sander Granneman,Rebecca Moschall,Davide Figini,Carlos H Vieira-Vieira,Uwe Ohler,Timon Hick,Matthias Selbach,Jan Medenbach,Hans-Herrmann Wessels

doi:10.1038/s41467-019-08942-3

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.

Highlights

Recent methodological advances allowed the identification of an increasing number of RNAbinding proteins (RBPs) and their RNA-binding sites
As RNA interactome capture relies on the purification of crosslinked RNPs based on hybridisation of oligo-dT beads to oligo-A sequences typically found in eukaryotic messenger RNAs, RBPs that exclusively associate with non-adenylate RNA species such as e.g. recruited to RNA polymerase I (rRNA), tRNAs, snRNAs, histone mRNAs, or numerous lncRNAs cannot be identified
We tested for additional well-established RBPs (Fig. 1f), namely polypyrimidine tract binding protein 1 (PTBP1), fused in sarcoma (FUS), and the more recently identified RNAbinding enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and enolase (Eno1);[3] all of which are enriched by Phenol Toluol extraction (PTex) in a UV-irradiation-dependent manner whereas the highly abundant DNA-binding histone H3 is depleted

Summary

Introduction

Recent methodological advances allowed the identification of an increasing number of RNAbinding proteins (RBPs) and their RNA-binding sites Most of those methods rely, on capturing proteins associated to polyadenylated RNAs which neglects RBPs bound to nonadenylate 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). Exploiting the stability of cross-linked RNPs, new methods have been developed to identify and analyse RNPs: (i) RNA interactome capture in which poly-A RNA and its bound proteins are first selected by oligo-dT beads and co-purified proteins subsequently identified by mass spectrometry (MS). This led to the discovery of hundreds of hitherto unknown RBPs7,8. The pH during extraction allows to control if DNA and RNA (neutral pH) or only RNA (acidic pH) accumulate in the aqueous phase (acidic conditions shown in Fig. 1b, right panel)

Methods

Results

Conclusion