Purification of Cross-linked RNA-Protein Complexes by Phenol-Toluol Extraction

Abstract

Recent methodological advances allowed the identification of an increasing number of RNAbinding 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 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). 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 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 first global RNA-bound proteome of human HEK293 cells and Salmonella Typhimurium as a bacterial species.

System-wide analyses of the fission yeast poly(A)+ RNA interactome reveal insights into organisation and function of RNA-protein complexes

“…We assume this to be the case because removal of the poly(A) tail from the 3' end during degradation prevents capture of the crosslinked complex during oligo-d(T) pull-down. In accordance with this assumption, recovery of crosslinked exosome core subunits from HEK293 cells was reported to be much more efficient when crosslinked RNA-protein complexes were enriched by chemical extraction rather than by poly(A)+ RNA selection (Urdaneta et al, 2019).…”

“…Comparison to alternative crosslinking methods, such as formaldehyde crosslinking, would help to remove crosslinking bias. Likewise, the data should be complemented by non-poly(A) RIC data, for which several methods have recently been developed (Asencio et al, 2018;Shchepachev et al, 2019;Trendel et al, 2019;Urdaneta et al, 2019)this would help to clean up the substoichiometric category of nonpoly(A) RNA binders and pave the way for a comprehensive classification of RBDs based on in vivo RNA-binding activity. As another caveat, RIC measures enrichment of proteins, not protein domains.…”

EuRBPDB: a comprehensive resource for annotation, functional and oncological investigation of eukaryotic RNA binding proteins (RBPs)

“…Therefore, clearly it is insufficient to identify RBPs merely based on RBD searching. On the other hand, RBPome methods are likewise incapable of detecting all RBPs because of the limitation of total RBP purification strategy and relative low sensitivity of MS technology (14,15,19). Thus, presently a comprehensive way to acquire a more complete RBP repertoire is to combine the computational RBP searching with RBPome profiling.…”

“…For example, RBPDB is a database focusing on the collection of experimentally validated RBPs and RNA binding domains (RBDs), and it contained only 1,171 RBPs from human, mouse, fly and worm (5). ATtRACT is a manually curated database that collects compiled information for only 370 well-characterized RBPs from 39 species (6 (16)(17)(18)(19), click chemistry-based RNA interactome capture (13), and orthogonal organic phase separation (OOPS) of RBPs (14,15,19). These methods crosslink the RBPs with RNA using UV, then apply different strategies to extract total RBPs from cells or tissues.…”

“…These RBPome technologies have been applied to many eukaryotes, including human (15,16,19), mouse (12) and fly (18), and identified a large number of novel canonical and non-canonical RBPs. It should be noted that as a experimental method, none of RBPome technologies is capable of capturing the complete category of RBPs, due to the limitation of total RBP purification strategy and MS technology (12)(13)(14)(15)(16)(17)(18)(19).…”