HyperTRIBE uncovers MEX3A RNA-binding activity in colorectal cancer (CRC) cells

To address this gap, we applied the high-throughput HyperTRIBE (Targets of RNA Identified By Editing) methodology in the SW480 cell line in order to map the MEX3A RNA interactome in CRC. An hyperactive version of the catalytic domain (cd) of the fruit fly RNA-editing enzyme Adenosine Deaminase Acting on RNA (ADAR) was fused either to the human MEX3A coding sequence (MEX3A-hyperADARcd) or its RNA-binding mutant version (MEX3ADKH-hyperADARcd) as a control.

Once expressed, the MEX3A-hyperADARcd fusion protein creates irreversible adenosine-to-inosine (A-to-I) RNA editing events that are identified as adenosine-to-guanosine (A-to-G) mismatches near MEX3A-binding sites by RNA-sequencing. Editing specificity is guaranteed by the lack of RNA-binding of ADARcd. To prevent analytical bias by ADAR continuous activity, the MEX3A-hyperADARcd and MEX3ADKH-hyperADARcd sequences were placed under the control of a doxycycline (DOX)-inducible promoter for transient expression, together with a TurboRFP reporter for sorting of positive cells expressing the constructs.