RNA sequencing of HCT116 samples processed with TREX. Homo sapiens

Targeted RNase H-mediated Extraction of crosslinked(X) RBPs (TREX) is an RNA-centric method developed to identify RNA-protein interactions at the region specific level in living cells. Briefly, the method combines UV-C crosslinking with organic phase separation, followed by isolation of RNA binding proteins (RBPs) and annealing of DNA oligonucleotides fully complementary to the RNA sequence of interest, the latter resulting in DNA-RNA hybrids at the regions of interest. The addition of RNAseH digests the RNA-DNA hybrids and releases the interacting proteins, which will be consequently isolated with another organic phase separation followed by identification through mass spectrometry.We have leveraged this method to profile the interactome of several RNA molecules of interest (e.g. U1, 45S). We managed to validate known interactors but also identified new ones, thus confirming the potential of TREX in discovering novel RNA interactors in living cells.