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Nonsense-mediated messenger RNA decay is initiated by endonucleolytic cleavage in Drosophila

Nature volume 429, pages 575–578 (2004)Cite this article

Abstract

In eukaryotic cells, messenger RNAs harbouring premature termination codons (PTCs) are rapidly degraded by a conserved post-transcriptional mechanism referred to as nonsense-mediated mRNA decay (NMD)1,2, which prevents the synthesis of truncated proteins that could be deleterious for the cell1,2. Studies in yeast and mammals indicate that degradation by means of this pathway can occur from both the 5′ end of the message (involving decapping and 5′-to-3′ exonucleolytic digestion by XRN1) or the 3′ end (through accelerated deadenylation and exosome-mediated 3′-to-5′ decay)3,4,5,6,7,8,9. Here we show that, contrary to expectation, degradation of PTC-containing messages in Drosophila is initiated by endonucleolytic cleavage(s) in the vicinity of the nonsense codon. The resulting 5′ fragment is rapidly degraded by exonucleolytic digestion by the exosome, whereas the 3′ fragment is degraded by XRN1. This decay route is shown for several PTC-containing reporters, as well as an endogenous mRNA that is naturally regulated by NMD. We conclude that, despite conservation in the NMD machinery, PTC-containing transcripts are degraded in Drosophila by a mechanism that differs considerably from those described in yeast and mammals3,6,7.

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Figure 1: Accumulation of a 5′-truncated intermediate in XRN1-depleted cells.
Figure 2: The size of the decay intermediates is correlated with the position of the PTC.
Figure 3: The 5′ intermediate is capped, whereas the 3′ intermediate is polyadenylated.
Figure 4: Endonucleolytic cleavage(s) of nonsense transcripts occurs in the vicinity of the PTC.

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Acknowledgements

We thank J. Rehwinkel for sharing information on the microarray analysis, K. Czaplinski for helpful discussions, and E. D. Andrulis, J. T. Lis, R. Lührmann and S. F. Newbury for antibodies. This study was supported by the European Molecular Biology Organisation (EMBO) and the Human Frontier Science Program Organization (HFSPO).

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  1. European Molecular Biology Laboratory, Meyerhofstrasse 1, D-69117, Heidelberg, Germany

    David Gatfield & Elisa Izaurralde

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  1. David Gatfield
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  2. Elisa Izaurralde
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Correspondence to Elisa Izaurralde.

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The authors declare that they have no competing financial interests.

Supplementary information

Supplementary Figure 1

a, Efficiency of the XRN1 depletion. b, Comparison of decay rates of inducible CAT transcripts after inhibition of transcription by actinomycin D in untreated cells or cells depleted of XRN1 or UPF1.

Supplementary Figure 2

a, Depletion of individual components of the exosome or of SKI2 in cells expressing the CAT-PTC reporter leads to the accumulation of a 5' intermediate. b, Efficiency of the RRP4 depletion. c, The 3' degradation intermediate derived from the adh-64 reporter is polyadenylated.

Supplementary Figure 3

Mapping of the ends of the 5' and 3' intermediates for adh-64 and ODA mRNAs.

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Gatfield, D., Izaurralde, E. Nonsense-mediated messenger RNA decay is initiated by endonucleolytic cleavage in Drosophila. Nature 429, 575–578 (2004). https://doi.org/10.1038/nature02559

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