Published online 23 January 2004
Nucleic Acids Research, 2004, Vol. 32, No. 2 488-494
© 2004 Oxford University Press
Premature termination codons enhance mRNA decapping in human cells
Institut Jacques Monod du CNRS, Universités Paris 6–7, Tour 43, 2 Place Jussieu, 75251 Paris Cedex 05, France
*To whom correspondence should be addressed. Tel: + 33 1 44275707; Fax: +33 1 44275716; Email: grange{at}ccr.jussieu.fr
Present address:
P. Couttet, Department of Human Genetics, The Sanger Institute, The Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK
Nonsense-mediated mRNA decay (NMD) is a eukaryotic surveillance process that promotes selective degradation of imperfect messages containing premature translation termination codons (PTCs). In yeast, PTCs trigger both deadenylylation-independent mRNA decapping, thereby allowing their rapid degradation by a 5' to 3' exonuclease, and to a smaller extent accelerated deadenylylation. It is not clear to what extent this decay pathway is conserved in higher eukaryotes. We used a transcriptional pulse strategy relying on a tetracycline-regulated promoter to study the decay of a PTC- containing ß-globin mRNA in human cells. We show that a PTC destabilizes the mRNA and decreases its half-life from >16 h to 3 h. The deadenylylation rate is increased, but not sufficiently to account for the decreased half-life on its own. Using a circularization RT–PCR (cRT–PCR) strategy, we could detect decapped degradation intermediates and measure simultaneously their poly(A) tail length. This allowed us to show that a PTC enhances the rate of mRNA decapping and that decapped products have been deadenylylated to a certain extent. Thus the major feature of the NMD pathway, enhanced decapping, is conserved from yeast to man even though the kinetic details might differ between various mRNAs and/or species.
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