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

Nucleic Acids Res. 2005 Mar 30;33(6):e54. doi: 10.1093/nar/gni052.


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.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Carrier Proteins / genetics
  • Codon, Nonsense*
  • Flow Cytometry
  • Fluorescent Dyes* / analysis
  • Genes, Reporter*
  • Green Fluorescent Proteins / analysis
  • Green Fluorescent Proteins / genetics*
  • HeLa Cells
  • Humans
  • Microscopy, Fluorescence
  • RNA Helicases
  • RNA Interference
  • RNA Stability*
  • RNA, Messenger / metabolism*
  • Spectrometry, Fluorescence
  • Trans-Activators / genetics


  • Carrier Proteins
  • Codon, Nonsense
  • Fluorescent Dyes
  • RNA, Messenger
  • Trans-Activators
  • Green Fluorescent Proteins
  • RNA Helicases
  • UPF1 protein, human