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RNA Editing pp 69-81 | Cite as

Live-Cell Quantification of APOBEC1-Mediated RNA Editing: A Comparison of RNA Editing Assays

  • Martina Chieca
  • Serena Torrini
  • Silvestro G. ConticelloEmail author
Protocol
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Part of the Methods in Molecular Biology book series (MIMB, volume 2181)

Abstract

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 words

C>U RNA editing APOBEC1 Apolipoprotein B Sequencing Subcellular localization Flow cytometer Methods 

Notes

Acknowledgment

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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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2021

Authors and Affiliations

  • Martina Chieca
    • 1
    • 2
  • Serena Torrini
    • 1
    • 2
  • Silvestro G. Conticello
    • 1
    • 3
    Email author
  1. 1.Core Research Laboratory, ISPRO—Institute for Cancer Research, Prevention and Clinical NetworkFirenzeItaly
  2. 2.Department of Medical BiotechnologiesUniversità di SienaSienaItaly
  3. 3.Institute of Clinical Physiology, CNRPisaItaly

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