Premature termination codons enhance mRNA decapping in human cells

Nucleic Acids Res. 2004 Jan 23;32(2):488-94. doi: 10.1093/nar/gkh218. Print 2004.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Codon, Nonsense / genetics*
  • Genetic Engineering
  • Globins / genetics*
  • Half-Life
  • HeLa Cells
  • Humans
  • Kinetics
  • RNA Caps / genetics*
  • RNA Caps / metabolism*
  • RNA Stability / genetics*
  • Reverse Transcriptase Polymerase Chain Reaction

Substances

  • Codon, Nonsense
  • RNA Caps
  • Globins