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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 poly-adenylated RNA, other important regulatory RNA classes or bacterial mRNA could not be investigated at the same depth. To overcome this limitation, we developed PTex (Phenol Toluol extraction), a novel and unbiased method for the purification of UV cross-linked RNPs in living cells. PTex is a fast (2-3 hrs) low-tech protocol; its purification principle is solely based on physicochemical properties of cross-linked RNPs. This enables us now for the first time 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 utilisation of PTex in high-throughput pipelines.

Protein-RNA interactions regulate all aspects of RNA metabolism and are crucial to the function of catalytic ribonucleoproteins. Until recently, the available technologies to capture RNA-bound proteins have been biased toward poly(A) RNA-binding proteins (RBPs) or involve molecular labeling, limiting their application. With the advent of organic-aqueous phase separation-based methods, we now have technologies that efficiently enrich the complete suite of RBPs and enable quantification of RBP dynamics. These flexible approaches to study RBPs and their bound RNA open up new research avenues for systems-level interrogation of protein-RNA interactions.