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

Abstract

Post-transcriptional regulation of gene expression in cells is facilitated by formation of RNA-protein complexes (RNPs). While many methods to study eukaryotic (m)RNPs rely on purification of polyadenylated RNA, other important regulatory RNA classes or bacterial mRNA could not be investigated at the same depth. To overcome this limitation, we developed Phenol Toluol extraction (PTex), a novel and unbiased method for the purification of UV cross-linked RNPs in living cells. PTex is a fast (2-3 hrs) and simple protocol. The purification principle is solely based on physicochemical properties of cross-linked RNPs, enabling us to interrogate RNA-protein interactions system-wide and beyond poly(A) RNA from a variety of species and source material. Here, we are presenting an introduction of the underlying separation principles and give a detailed discussion of the individual steps as well as incorporation of PTex in high-throughput pipelines.

In vivo discovery of RNA proximal proteins via proximity-dependent biotinylation

“…UV has very low crosslinking efficiency, necessitating large numbers of cells (~100-800 million) be used as input [14,16], which may not be feasible for slow-growing model systems such as primary cell cultures. Moreover, UVcrosslinking induces RNA modifications [17] that can alter the binding affinity of RNA to certain RBPs [18] and impair downstream protein analysis [19]. An alternative approach, tagging of endogenous RNA, requires genetic manipulation and may interfere with endogenous RNA function [20].…”