Recent methodological advances allowed the identification of an increasing number of RNAbinding proteins (RBPs) and their RNA-binding sites. RNA interactome capture is, however, limited to proteins interacting with polyadenylated RNAs while RBPs associating with nonadenylate RNA classes cannot be purified. Moreover, the vast majority of species that lack poly-A tails in their mRNAs (including all archea and bacteria) are not amenable to RNA interactome capture studies. To overcome these limitations, we have developed a novel protocol, Phenol Toluol extraction (PTex), 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 poly-A RNA, but also proteins associating with non-adenylate RNA species (rRNA, tRNA) as short as 30nt. PTex can be used to simplify complex work ows such as PAR-CLIP and reliably recovers RBPs from tissues and bacteria thus significantly expanding the experimental toolbox to species that could previously not be assessed experimentally.
Urdaneta EC, Vieira-Vieira CH, Hick T, Wessels HH, Figini D, Moschall R, Medenbach J, Ohler U, Granneman S, Selbach M, Beckmann BM. Purification of cross-linked RNA-protein complexes by phenol-toluol extraction. Nat Commun. 2019 10(1):990 PubMed: 30824702 ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier <PXD009571>. https://www.ebi.ac.uk/pride/archive/projects/PXD009571