Live-Cell Quantification of APOBEC1-Mediated RNA Editing: A Comparison of RNA Editing Assays
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APOBEC1 is a member of the AID/APOBECs, a group of deaminases responsible for the editing of C>U in both DNA and RNA. APOBEC1 is physiologically involved in C>U RNA editing: while hundreds of targets have been discovered in mice, in humans the only well-characterized target of APOBEC1 is the apolipoprotein B (ApoB) transcript. APOBEC1 edits a CAA codon into a stop codon, which causes the translation of a truncated form of ApoB. A number of assays have been developed to investigate this process. Early assays, poisoned primer extension and Sanger sequencing, have focused on accuracy and sensitivity but rely on extraction of the RNA from tissues and cells. More recently, the need to visualize the RNA editing process directly in live cells have led to the development of fluorescence-based tools. These assays detect RNA editing through reporters whose editing causes a change in cellular localization or a change in fluorescent properties. Here we review the available assays to quantify RNA editing, and we present the protocol for cytofluorimetric analysis using a double-fluorescent reporter.
Key wordsC>U RNA editing APOBEC1 Apolipoprotein B Sequencing Subcellular localization Flow cytometer Methods
This work was supported by the Regione Toscana; Italian Ministry of Health [PE-2013-02357669]; and the Associazione Italiana per la Ricerca sul Cancro [IG-17701].
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