Defining the RNA Interactome by Total RNA-Associated Protein Purification

UV crosslinking can be used to identify precise RNA targets for individual proteins, transcriptome-wide. We sought to develop a technique to generate reciprocal data, identifying precise sites of RNA-binding proteome-wide. The resulting technique, total RNA-associated protein purification (TRAPP), relies on SILAC labelling to quantify RNA-associated protein recovery in the presence and absence of irradiation. We utilised TRAPP to study alterations in RNA-protein interactions upon exposure to weak acid stress in yeast. In addition, as UV irradiation induces short distance crosslinks between proteins and nucleic acids, the identity of crosslinked amino acids reveals the exact protein-RNA interacting interface. Precise sites of crosslinking at the level of individual amino acids (iTRAPP) were identified following phospho-peptide enrichment combined with a bioinformatic pipeline (Xi).