A GFP-based reporter system to monitor nonsense-mediated mRNA decay.
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-beta 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.
Paillusson
Alexandra
A
Institute of Cell Biology, University of Bern CH-3012 Bern, Switzerland.
Hirschi
Nadine
N
Vallan
Claudio
C
Azzalin
Claus M
CM
Mühlemann
Oliver
O
D002352:Carrier Proteins / Q000235:genetics; D018389:Codon, Nonsense; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000032:analysis; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D008856:Microscopy, Fluorescence; D020365:RNA Helicases; D034622:RNA Interference; D020871:RNA Stability; D012333:RNA, Messenger / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D015534:Trans-Activators / Q000235:genetics
D002352:Carrier Proteins; D018389:Codon, Nonsense; D005456:Fluorescent Dyes; D012333:RNA, Messenger; D015534:Trans-Activators; D049452:Green Fluorescent Proteins; D020365:RNA Helicases; C101828:UPF1 protein, human