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

Nonsense-mediated mRNA decay (NMD) is a surveillance pathway that mediates rapid degradation of transcripts bearing premature translation termination codons (PTCs) and thereby limits the expression of unproductively processed mRNAs and the synthesis of C-terminally truncated peptides. Both its impor...

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Bibliographic Details
Published in:Biochemical and biophysical research communications 2006-10, Vol.349 (1), p.186-191
Main Authors: Boelz, Stephanie, Neu-Yilik, Gabriele, Gehring, Niels H., Hentze, Matthias W., Kulozik, Andreas E.
Format: Article
Language:English
Subjects:
Androstadienes - pharmacology
Chemical biology
Dose-Response Relationship, Drug
Enzyme Inhibitors - pharmacology
Gene Expression Regulation
Genes, Reporter
Genetic Techniques
HeLa Cells
Humans
Luciferase
Luminescence
Nonsense-mediated mRNA decay
Phosphatidylinositol 3-Kinases - antagonists & inhibitors
Protein Structure, Tertiary
Renilla
Reporter assay
RNA Interference
RNA, Messenger - chemistry
RNA, Messenger - metabolism
Trans-Activators - metabolism
Transfection
UPF1 phosphorylation
Wortmannin
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Summary:Nonsense-mediated mRNA decay (NMD) is a surveillance pathway that mediates rapid degradation of transcripts bearing premature translation termination codons (PTCs) and thereby limits the expression of unproductively processed mRNAs and the synthesis of C-terminally truncated peptides. Both its importance as a means to control gene expression and in the context of genetic and acquired human diseases call for an exploration of the mammalian NMD pathway using chemical biology approaches. Here, we describe a novel cell-based chemiluminescence reporter system that recapitulates the hallmark features of mammalian NMD. The assay is characterized by its high sensitivity, robustness, and its potential for automated handling. Limiting NMD efficiency by RNAi-mediated depletion of the essential NMD factor UPF1 markedly and specifically increased the NMD reporter mRNA level and resulted in a proportional increase in protein expression reflected by Renilla luminescence. The PI 3-kinase inhibitor wortmannin has previously been found to up-modulate PTC-containing transcripts by inhibiting the UPF1 kinase SMG1. Wortmannin treatment enhanced NMD reporter expression in our system in a dose-dependent way, illustrating its utility for small molecule screening.
ISSN:0006-291X
1090-2104
DOI:10.1016/j.bbrc.2006.08.017