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

Premature termination codons enhance mRNA decapping in human cells

P Couttet  1 , T Grange
Affiliations

Premature termination codons enhance mRNA decapping in human cells

P Couttet et al. Nucleic Acids Res. .

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.

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Figures

Figure 1
Figure 1
Tetracycline-regulated chimeric β-globin genes used for transfection. The mature mRNA is represented by a box that includes the ORF (hatched box). The Tet-regulated minimal CMV promoter (tetO CMV) and polyadenylylation site [poly(A)] are represented by lines. The locations of introns 1 and 2 (int.1, int.2) are indicated above the box; the introns themselves are not represented. The initiation (AUG) and the normal and mutant termination codons (UAA and UAG, respectively) are represented above the ORF.
Figure 2
Figure 2
A premature termination codon enhances β-globin mRNA decay without affecting its deadenylylation rate. (A) Northern blot analysis of the decay of both WT and mutant PTC-containing β-globin mRNAs following a pulse of transcription. tTA-expressing HeLa cells (38) grown in the presence of doxycycline (Dox) were transiently transfected with constructs containing the Tet-regulated CMV promoter driving expression of either the WT or PTC39 β-globin genes. One day after transfection (lanes 2 and 8), transcription was induced by growing the cells for 3 h in Dox-depleted medium (lanes 3 and 9) and then stopped by addition of Dox. mRNA was extracted 1–6 h later as indicated (lanes 4–6 and 10–13). The amount of total RNA loaded on the gel is twice as much for the PTC39-transfected cells (lanes 7–13) to compensate for the lower absolute level of β-globin mRNA produced. Poly(A)– mRNA was obtained after oligo(dT)/RNase H treatment of the induced RNA preparations (lanes 1 and 7). The estimated size of the poly(A) tail is represented on the left-hand side (0 and 200 As). This estimation was performed using as size references the poly(A)– β-globin mRNA, rRNA and the mRNAs used as internal controls of transfection efficiency and RNA loading [luciferase and poly(A)-binding protein mRNAs]. (B) Semi-log plot showing decay of WT and PTC39 β-globin mRNAs. Data from three independent transfection experiments were quantified using a phosphorimager (Molecular Dynamics) and are expressed relative to the β-globin mRNA level measured at the end of the transcription pulse. The mean ± SD is represented as well as the exponential regression curve that allows us to estimate mRNA half-lives (WT, continuous line; PTC39, broken line). (C) Comparison of the deadenylylation rates of WT and PTC39 mRNAs. The average distributions of the poly(A) tail length measured on four independent gels are represented.
Figure 3
Figure 3
A premature termination codon enhances β-globin mRNA decapping. (A) Strategy used for detecting specific decapped mRNAs and measuring their poly(A) tail length. mRNA molecules are represented as dotted lines and DNA molecules as thick lines. The cap and poly(A) tail (AAAAA) are indicated by a diamond and a rectangle, respectively. The circle indicates the 5′-phosphate group of the 5′ most base of the original mRNA. (B) Arrangement of the primers used. (C) cRT–PCR analysis of the decapped β-globin mRNA degradation products. The pulse of transcription was performed as described in Figure 1 except that equal amounts of total RNA were used for analysis of each point. The size of the poly(A) tail was deduced by comparison with a size standard and subtraction of the predicted length of the poly (A)– PCR product.
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References

    1. Maquat L.E. (1995) When cells stop making sense: effects of nonsense codons on RNA metabolism in vertebrate cells. RNA, 1, 453–465. - PMC - PubMed
    1. Hentze M.W. and Kulozik,A.E. (1999) A perfect message: RNA surveillance and nonsense-mediated decay. Cell, 96, 307–310. - PubMed
    1. Hilleren P. and Parker,R. (1999) mRNA surveillance in eukaryotes: kinetic proofreading of proper translation termination as assessed by mRNP domain organization? RNA, 5, 711–719. - PMC - PubMed
    1. Wilusz C.J., Wang,W. and Peltz,S.W. (2001) Curbing the non-sense: the activation and regulation of mRNA surveillance. Genes Dev., 15, 2781–2785. - PubMed
    1. Oliveira C.C. and McCarthy,J.E. (1995) The relationship between eukaryotic translation and mRNA stability. A short upstream open reading frame strongly inhibits translational initiation and greatly accelerates mRNA degradation in the yeast Saccharomyces cerevisiae. J. Biol. Chem., 270, 8936–8943. - PubMed

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