RNA-Protein Crosslink Mapping Using TEV Protease

Characterization of novel RNA-protein interactions often demands physical mapping of the RNA binding sites in the protein. This can sometimes be accomplished using radioactively labeled RNA in covalent RNA-protein crosslinking experiments. The position of the radioactive label crosslinked to the protein can then be determined by fragmentation of the protein using a battery of sequence-specific proteolytic enzymes or chemical reagents. However, there are typically many cleavage sites in the natural protein sequence, and for large proteins, particularly when there are multiple sites of RNA-protein interaction, it may be difficult or impossible to determine the sites of crosslink formation unambiguously using this traditional physical mapping approach. We have developed an alternative method for physical mapping of RNA-protein crosslinks based on random insertion into the protein of a short peptide tag that includes the target sequence ENLYFQG (Glu-Asn-Leu-Tyr-Phe-Gln-Gly) for the highly specific TEV protease from tobacco etch virus. Covalent RNA-protein crosslinks can then be physically mapped by TEV protease digestion, fractionation of the proteolytic digestion products by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and visualization of the labeled protein fragments by phosphorimaging.