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Nucleic Acids Research 2005 33(6):e54; doi:10.1093/nar/gni052
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Published online 30 March 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions{at}oupjournals.org

Methods Online

A GFP-based reporter system to monitor nonsense-mediated mRNA decay

Alexandra Paillusson, Nadine Hirschi, Claudio Vallan1, Claus M. Azzalin2 and Oliver Mühlemann*

Institute of Cell Biology, University of Bern CH-3012 Bern, Switzerland 1Institute of Pathology, University of Bern CH-3012 Bern, Switzerland 2Swiss Institute for Experimental Cancer Research (ISREC) Epalinges sur Lausanne, Switzerland

*To whom correspondence should be addressed. Tel: +41 31 631 4627; Fax: +41 31 631 4616; Email: oliver.muehlemann{at}izb.unibe.ch

Received August 24, 2004. Revised October 27, 2004. Accepted March 1, 2005.

Aberrant mRNAs whose open reading frame (ORF) is truncated by the presence of a premature translation-termination codon (PTC) are recognized and degraded in eukaryotic cells by a process called nonsense-mediated mRNA decay (NMD). Here, we report the development of a reporter system that allows monitoring of NMD in mammalian cells by measuring the fluorescence of green fluorescent protein (GFP). The NMD reporter gene consists of a T-cell receptor-ß minigene construct, in which the GFP-ORF was inserted such that the stop codon of GFP is recognized as PTC. The reporter mRNA is therefore subjected to NMD, resulting in a low steady-state mRNA level, an accordingly low protein level and hence a very low green fluorescence in normal, NMD-competent cells that express this reporter gene. We show that the inactivation of NMD by RNAi-mediated knockdown of the essential NMD factor hUpf1 or hSmg6 increases the NMD reporter mRNA level, resulting in a proportional increase of the green fluorescence that can be detected by flow cytometry, spectrofluorometry and fluorescence microscopy. With these properties, our GFP-based NMD reporter system could be used for large-scale screenings to identify NMD-inhibiting drugs or NMD-deficient mutant cells.


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