title: Premature termination codons enhance mRNA decapping in human cells.
abstract: 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 beta-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.
authors.affiliation: Institut Jacques Monod du CNRS, Universités Paris 6-7, Tour 43, 2 Place Jussieu, 75251 Paris Cedex 05, France.
authors.forename: P
authors.initials: P
authors.lastname: Couttet
authors.forename: T
authors.initials: T
authors.lastname: Grange
mesh_terms: D018389:Codon, Nonsense / Q000235:genetics*; D005818:Genetic Engineering; D005914:Globins / Q000235:genetics*; D006207:Half-Life; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D012315:RNA Caps / Q000235:genetics* / Q000378:metabolism*; D020871:RNA Stability / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D018389:Codon, Nonsense; D012315:RNA Caps; D005914:Globins