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

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.

Fast and unbiased purification of RNA-protein complexes after UV cross-linking

“…• Maybe the most important caveat of UV cross-linking is its very low efficiency. According to own results only up to 5% of a given RBP can be cross-linked to RNA [5,7,12,28]. The efficiency for individual RNA molecules was reported to be higher [29] (note that a single transcript is usually bound by many proteins [3,4]).…”

“…Efficiency is even lower when working with tissue or turbid liquid cultures due to the poor penetration of UV light in these media. To overcome this obstacle to some extend, an array of UV bulbs [12,30] can be used, as e.g. done in a Vari-X-Link device [31].…”

“…Here, we present an approach that separates RNPs solely by physicochemical features that are specific for RNA-protein complexes rather than for individual RNA sequences or for protein epitopes. In our method called Phenol Toluol extraction (PTex) [12]), we are using organic liquid-liquid extractions to enrich UV cross-linked RNPs directly from biological sources such as human cell culture, bacteria or animal tissue.…”

mSphere of Influence: Modifying an Old Method To Study RNA-Protein Interactions

“…It is worth mentioning that since coming across this paper, alternative variants of this method have been published (3–5). It turns out that three of these groups (1, 3, 4) were independently working on the same idea and coordinated efforts to simultaneously submit to bioRxiv.…”

“…It turns out that three of these groups (1, 3, 4) were independently working on the same idea and coordinated efforts to simultaneously submit to bioRxiv.…”