Fluorescent shift assay for APOBEC-mediated RNA editing.

Abstract

Cytidine (C) to Uridine (U) RNA editing is a post-transcriptional modification that is involved in diverse biological processes. The APOBEC deaminase family acts in various cellular processes mostly through inducing C-to-U mutation in single-stranded RNA (or DNA). However, comparing the activity of different RNA editing enzymes to one another is difficult due to the limited number of systems that can provide direct and efficient readout. In this report, a system in which RNA editing directly prompts a change in the subcellular localization of a modified eGFP structure is described in detail. This approach allows us to compare relative fluorescence intensity based on the RNA editing level. When observed through a fluorescence detection system, like a scanning confocal microscope, the cellular nucleus can be readily identified using a DNA-binding stain, such as DAPI or Hoechst, so that the accurate calculation of the ratio of nuclear to cytosolic eGFP intensity can be applied for an individual cell. This method provides a useful and flexible tool to examine and quantify RNA editing activity within cells, and it is not only limited to APOBEC proteins, but can also be applied more generally to other RNA editing enzymatic assays.