A GFP-based assay for rapid screening of compounds affecting ARE-dependent mRNA turnover

Nucleic Acids Research
Don BenjaminChristoph Moroni

Abstract

A reporter transcript containing the green fluorescent protein (GFP) gene upstream of the destabilizing 3'-untranslated region (3'-UTR) of the murine IL-3 gene was inserted in mouse PB-3c-15 mast cells. The GFP-IL-3 transcript was inherently unstable due to the presence of an adenosine-uridine (AU)-rich element (ARE) in the 3'-UTR and was subject to rapid decay giving a low baseline of GFP fluorescence. Transcript stabilization with ionomycin resulted in an increase of fluorescence that is quantitated by FACS analysis of responding cells. Using this system we have identified okadaic acid as a novel stabilizing compound, and investigated the upstream signaling pathways leading to stabilization. This reporter system has the advantage of speed and simplicity over standard methods currently in use and in addition to serving as a research tool it can be easily automated to increase throughput for drug discovery.

References

Jan 1, 1990·Proceedings of the National Academy of Sciences of the United States of America·A Wodnar-Filipowicz, C Moroni
Apr 16, 1999·Science·G LaroiaR J Schneider
Apr 8, 2000·Current Opinion in Genetics & Development·P Mitchell, D Tollervey
Mar 20, 2001·Gene·J Guhaniyogi, G Brewer
Nov 22, 2001·Proceedings of the National Academy of Sciences of the United States of America·B L BennettD W Anderson
Aug 29, 2002·The EMBO Journal·Georg StoecklinChristoph Moroni
Jan 10, 2003·Nucleic Acids Research·Tala BakheetKhalid S A Khabar

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Citations

Aug 7, 2004·Bioinformatics·Vincent J CareyRobert Gentleman
Jan 12, 2007·Molecular BioSystems·Don BenjaminChristoph Moroni
Aug 23, 2011·Methods : a Companion to Methods in Enzymology·Jennifer A CorcoranCraig McCormick
Oct 18, 2011·BioEssays : News and Reviews in Molecular, Cellular and Developmental Biology·Simon TrowitzschImre Berger
Jul 5, 2005·Developmental Biology·Valérie HilgersJulien Dubrulle

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