Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

SMG6 promotes endonucleolytic cleavage of nonsense mRNA in human cells

Nature Structural & Molecular Biology volume 16, pages 49–55 (2009)Cite this article

Abstract

From yeast to humans, mRNAs harboring premature termination codons (PTCs) are recognized and degraded by nonsense-mediated mRNA decay (NMD). However, degradation mechanisms of NMD have been suggested to differ between species. In Drosophila melanogaster, NMD is initiated by endonucleolysis near the PTC, whereas in yeast and human cells the current view posits that NMD occurs by exonucleolysis from one or both RNA termini. Here we report that degradation of human nonsense mRNAs can be initiated by PTC-proximal endonucleolytic cleavage. We identify the metazoan-specific NMD factor SMG6 as the responsible endonuclease by demonstrating that mutation of conserved residues in its nuclease domain—the C-terminal PIN motif—abolishes endonucleolysis in vivo and in vitro. Our data lead to a revised mechanistic model for degradation of nonsense mRNA in human cells and suggest that endonucleolytic cleavage is a conserved feature in metazoan NMD.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Distinct 3′ and 5′ fragments are produced from nonsense mRNA upon depletion of exonucleases.
Figure 2: Sizes of the 3′ end decay intermediates correlate with the position of the PTC.
Figure 3: SMG6 induces endocleavage of nonsense mRNA through its PIN domain.
Figure 4: The PIN domain of SMG6 can cleave a circular RNA.
Figure 5: Model for NMD in mammals.

Similar content being viewed by others

References

  1. Doma, M.K. & Parker, R. RNA quality control in eukaryotes. Cell 131, 660–668 (2007).

    Article  CAS  Google Scholar 

  2. Amrani, N., Sachs, M.S. & Jacobson, A. Early nonsense: mRNA decay solves a translational problem. Nat. Rev. Mol. Cell Biol. 7, 415–425 (2006).

    Article  CAS  Google Scholar 

  3. Behm-Ansmant, I. et al. mRNA quality control: an ancient machinery recognizes and degrades mRNAs with nonsense codons. FEBS Lett. 581, 2845–2853 (2007).

    Article  CAS  Google Scholar 

  4. Isken, O. & Maquat, L.E. Quality control of eukaryotic mRNA: safeguarding cells from abnormal mRNA function. Genes Dev. 21, 1833–1856 (2007).

    Article  CAS  Google Scholar 

  5. Muhlemann, O., Eberle, A.B., Stalder, L. & Zamudio Orozco, R. Recognition and elimination of nonsense mRNA. Biochim. Biophys. Acta 1779, 538–549 (2008).

    Article  Google Scholar 

  6. Lewis, B.P., Green, R.E. & Brenner, S.E. Evidence for the widespread coupling of alternative splicing and nonsense-mediated mRNA decay in humans. Proc. Natl. Acad. Sci. USA 100, 189–192 (2003).

    Article  CAS  Google Scholar 

  7. Rehwinkel, J., Raes, J. & Izaurralde, E. Nonsense-mediated mRNA decay: target genes and functional diversification of effectors. Trends Biochem. Sci. 31, 639–646 (2006).

    Article  CAS  Google Scholar 

  8. Stalder, L. & Muhlemann, O. The meaning of nonsense. Trends Cell Biol. 18, 315–321 (2008).

    Article  CAS  Google Scholar 

  9. He, F. et al. Genome-wide analysis of mRNAs regulated by the nonsense-mediated and 5′ to 3′ mRNA decay pathways in yeast. Mol. Cell 12, 1439–1452 (2003).

    Article  CAS  Google Scholar 

  10. Mendell, J.T., Sharifi, N.A., Meyers, J.L., Martinez-Murillo, F. & Dietz, H.C. Nonsense surveillance regulates expression of diverse classes of mammalian transcripts and mutes genomic noise. Nat. Genet. 36, 1073–1078 (2004).

    Article  CAS  Google Scholar 

  11. Rehwinkel, J., Letunic, I., Raes, J., Bork, P. & Izaurralde, E. Nonsense-mediated mRNA decay factors act in concert to regulate common mRNA targets. RNA 11, 1530–1544 (2005).

    Article  CAS  Google Scholar 

  12. Wittmann, J., Hol, E.M. & Jack, H.M. hUPF2 silencing identifies physiologic substrates of mammalian nonsense-mediated mRNA decay. Mol. Cell. Biol. 26, 1272–1287 (2006).

    Article  CAS  Google Scholar 

  13. Johansson, M.J., He, F., Spatrick, P., Li, C. & Jacobson, A. Association of yeast Upf1p with direct substrates of the NMD pathway. Proc. Natl. Acad. Sci. USA 104, 20872–20877 (2007).

    Article  CAS  Google Scholar 

  14. Holbrook, J.A., Neu-Yilik, G., Hentze, M.W. & Kulozik, A.E. Nonsense-mediated decay approaches the clinic. Nat. Genet. 36, 801–808 (2004).

    Article  CAS  Google Scholar 

  15. Amrani, N. et al. A faux 3′-UTR promotes aberrant termination and triggers nonsense-mediated mRNA decay. Nature 432, 112–118 (2004).

    Article  CAS  Google Scholar 

  16. Behm-Ansmant, I., Gatfield, D., Rehwinkel, J., Hilgers, V. & Izaurralde, E. A conserved role for cytoplasmic poly(A)-binding protein 1 (PABPC1) in nonsense-mediated mRNA decay. EMBO J. 26, 1591–1601 (2007).

    Article  CAS  Google Scholar 

  17. Eberle, A.B., Stalder, L., Mathys, H., Zamudio Orozco, R. & Muhlemann, O. Posttranscriptional gene regulation by spatial rearrangement of the 3′ untranslated region. PLoS Biol. 6, e92 (2008).

    Article  Google Scholar 

  18. Ivanov, P.V., Gehring, N.H., Kunz, J.B., Hentze, M.W. & Kulozik, A.E. Interactions between UPF1, eRFs, PABP and the exon junction complex suggest an integrated model for mammalian NMD pathways. EMBO J. 27, 736–747 (2008).

    Article  CAS  Google Scholar 

  19. Longman, D., Plasterk, R.H., Johnstone, I.L. & Caceres, J.F. Mechanistic insights and identification of two novel factors in the C. elegans NMD pathway. Genes Dev. 21, 1075–1085 (2007).

    Article  CAS  Google Scholar 

  20. Silva, A.L., Ribeiro, P., Inacio, A., Liebhaber, S.A. & Romao, L. Proximity of the poly(A)-binding protein to a premature termination codon inhibits mammalian nonsense-mediated mRNA decay. RNA 14, 563–576 (2008).

    Article  CAS  Google Scholar 

  21. Singh, G., Rebbapragada, I. & Lykke-Andersen, J. A competition between stimulators and antagonists of Upf complex recruitment governs human nonsense-mediated mRNA decay. PLoS Biol. 6, e111 (2008).

    Article  Google Scholar 

  22. Thermann, R. et al. Binary specification of nonsense codons by splicing and cytoplasmic translation. EMBO J. 17, 3484–3494 (1998).

    Article  CAS  Google Scholar 

  23. Cao, D. & Parker, R. Computational modeling and experimental analysis of nonsense-mediated decay in yeast. Cell 113, 533–545 (2003).

    Article  CAS  Google Scholar 

  24. Hagan, K.W., Ruiz-Echevarria, M.J., Quan, Y. & Peltz, S.W. Characterization of cis-acting sequences and decay intermediates involved in nonsense-mediated mRNA turnover. Mol. Cell. Biol. 15, 809–823 (1995).

    Article  CAS  Google Scholar 

  25. Muhlrad, D. & Parker, R. Premature translational termination triggers mRNA decapping. Nature 370, 578–581 (1994).

    Article  CAS  Google Scholar 

  26. Chen, C.Y. & Shyu, A.B. Rapid deadenylation triggered by a nonsense codon precedes decay of the RNA body in a mammalian cytoplasmic nonsense-mediated decay pathway. Mol. Cell. Biol. 23, 4805–4813 (2003).

    Article  CAS  Google Scholar 

  27. Couttet, P. & Grange, T. Premature termination codons enhance mRNA decapping in human cells. Nucleic Acids Res. 32, 488–494 (2004).

    Article  CAS  Google Scholar 

  28. Lejeune, F., Li, X. & Maquat, L.E. Nonsense-mediated mRNA decay in mammalian cells involves decapping, deadenylating, and exonucleolytic activities. Mol. Cell 12, 675–687 (2003).

    Article  CAS  Google Scholar 

  29. Yamashita, A. et al. Concerted action of poly(A) nucleases and decapping enzyme in mammalian mRNA turnover. Nat. Struct. Mol. Biol. 12, 1054–1063 (2005).

    Article  CAS  Google Scholar 

  30. Gatfield, D. & Izaurralde, E. Nonsense-mediated messenger RNA decay is initiated by endonucleolytic cleavage in Drosophila. Nature 429, 575–578 (2004).

    Article  CAS  Google Scholar 

  31. Mohn, F., Buhler, M. & Muhlemann, O. Nonsense-associated alternative splicing of T-cell receptor β genes: no evidence for frame dependence. RNA 11, 147–156 (2005).

    Article  CAS  Google Scholar 

  32. Stoecklin, G., Stoeckle, P., Lu, M., Muehlemann, O. & Moroni, C. Cellular mutants define a common mRNA degradation pathway targeting cytokine AU-rich elements. RNA 7, 1578–1588 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Kammler, S., Lykke-Andersen, S. & Jensen, T.H. The RNA exosome component hRrp6 is a target for 5-fluorouracil in human cells. Mol. Cancer Res. 6, 990–995 (2008).

    Article  CAS  Google Scholar 

  34. Clissold, P.M. & Ponting, C.P. PIN domains in nonsense-mediated mRNA decay and RNAi. Curr. Biol. 10, R888–R890 (2000).

    Article  CAS  Google Scholar 

  35. Glavan, F., Behm-Ansmant, I., Izaurralde, E. & Conti, E. Structures of the PIN domains of SMG6 and SMG5 reveal a nuclease within the mRNA surveillance complex. EMBO J. 25, 5117–5125 (2006).

    Article  CAS  Google Scholar 

  36. Huntzinger, E., Kashima, I., Fauser, M., Sauliere, J. & Izaurralde, E. SMG6 is the catalytic endonuclease that cleaves mRNAs containing nonsense codons in metazoan. RNA published online doi:10.1261/rna.1386208 (30 October 2008).

  37. Lebreton, A., Tomecki, R., Dziembowski, A. & Séraphin, B. Endonucleolytic RNA cleavage by a eukaryotic exosome. Nature advance online publication, doi:10.1038/nature07480 (7 December 2008).

  38. Schaeffer, D. et al. The exosome contains domains with specific endoribonuclease, exoribonuclease and cytoplasmic mRNA decay activities. Nat. Struct. Mol. Biol. advance online publication, doi:10.1038/nsmb.1528 (7 December 2008).

  39. Kashima, I. et al. Binding of a novel SMG-1-Upf1-eRF1-eRF3 complex (SURF) to the exon junction complex triggers Upf1 phosphorylation and nonsense-mediated mRNA decay. Genes Dev. 20, 355–367 (2006).

    Article  CAS  Google Scholar 

  40. Anders, K.R., Grimson, A. & Anderson, P. SMG-5, required for C. elegans nonsense-mediated mRNA decay, associates with SMG-2 and protein phosphatase 2A. EMBO J. 22, 641–650 (2003).

    Article  CAS  Google Scholar 

  41. Chiu, S.Y., Serin, G., Ohara, O. & Maquat, L.E. Characterization of human Smg5/7a: a protein with similarities to Caenorhabditis elegans SMG5 and SMG7 that functions in the dephosphorylation of Upf1. RNA 9, 77–87 (2003).

    Article  CAS  Google Scholar 

  42. Fukuhara, N. et al. SMG7 is a 14–3-3-like adaptor in the nonsense-mediated mRNA decay pathway. Mol. Cell 17, 537–547 (2005).

    Article  CAS  Google Scholar 

  43. Ohnishi, T. et al. Phosphorylation of hUPF1 induces formation of mRNA surveillance complexes containing hSMG-5 and hSMG-7. Mol. Cell 12, 1187–1200 (2003).

    Article  CAS  Google Scholar 

  44. Buhler, M., Paillusson, A. & Muhlemann, O. Efficient downregulation of immunoglobulin μ mRNA with premature translation-termination codons requires the 5′-half of the VDJ exon. Nucleic Acids Res. 32, 3304–3315 (2004).

    Article  Google Scholar 

  45. Damgaard, C.K. et al. A 5′ splice site enhances the recruitment of basal transcription initiation factors in vivo. Mol. Cell 29, 271–278 (2008).

    Article  CAS  Google Scholar 

  46. Brummelkamp, T.R., Bernards, R. & Agami, R. A system for stable expression of short interfering RNAs in mammalian cells. Science 296, 550–553 (2002).

    Article  CAS  Google Scholar 

  47. Lykke-Andersen, S., Pinol-Roma, S. & Kjems, J. Alternative splicing of the ADAR1 transcript in a region that functions either as a 5′-UTR or an ORF. RNA 13, 1732–1744 (2007).

    Article  CAS  Google Scholar 

  48. Mendell, J.T., ap Rhys, C.M. & Dietz, H.C. Separable roles for rent1/hUpf1 in altered splicing and decay of nonsense transcripts. Science 298, 419–422 (2002).

    Article  CAS  Google Scholar 

  49. Paillusson, A., Hirschi, N., Vallan, C., Azzalin, C.M. & Muhlemann, O. A GFP-based reporter system to monitor nonsense-mediated mRNA decay. Nucleic Acids Res. 33, e54 (2005).

    Article  Google Scholar 

  50. Lewis, D.L., Hagstrom, J.E., Loomis, A.G., Wolff, J.A. & Herweijer, H. Efficient delivery of siRNA for inhibition of gene expression in postnatal mice. Nat. Genet. 32, 107–108 (2002).

    Article  CAS  Google Scholar 

  51. Buhler, M., Steiner, S., Mohn, F., Paillusson, A. & Muhlemann, O. EJC-independent degradation of nonsense immunoglobulin-μ mRNA depends on 3′ UTR length. Nat. Struct. Mol. Biol. 13, 462–464 (2006).

    Article  Google Scholar 

Download references

Acknowledgements

We thank J. Lykke-Andersen (University of Colorado, USA), G. Pruijn (Radboud University Nijmegen, The Netherlands), J. Lingner (Ecole Polytechnique Federale de Lausanne, Switzerland), M.-D. Ruepp and D. Schümperli (both University of Bern, Switzerland) for reagents; J. Lykke-Andersen and M. Rosbash for comments on the manuscript; and D. Riishøj and K. Jürgensen for excellent technical assistance. B. Seraphin and A. Dziembowski are acknowledged for communication of unpublished results. The work was supported by the Danish National Research Foundation and the Danish Natural Science Research Council (S.L.-A. and T.H.J.), and by the Swiss National Science Foundation, the Max Cloëtta Foundation and a Starting Grant of the European Research Council (A.B.E. and O.M.).

Author information

Author notes

  1. Andrea B Eberle and Søren Lykke-Andersen: These authors contributed equally to this work.

Authors and Affiliations

  1. Institute of Cell Biology, University of Bern, Baltzerstrasse 4, Bern, CH-3012, Switzerland

    Andrea B Eberle & Oliver Mühlemann

  2. Department of Molecular Biology, Centre for mRNP Biogenesis and Metabolism, Aarhus University, C.F. Møllers Alle, Building 1130, Aarhus, 8000, Denmark

    Søren Lykke-Andersen & Torben Heick Jensen

Authors
  1. Andrea B Eberle
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Søren Lykke-Andersen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Oliver Mühlemann
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Torben Heick Jensen
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Oliver Mühlemann or Torben Heick Jensen.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4 and Supplementary Methods (PDF 3171 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Eberle, A., Lykke-Andersen, S., Mühlemann, O. et al. SMG6 promotes endonucleolytic cleavage of nonsense mRNA in human cells. Nat Struct Mol Biol 16, 49–55 (2009). https://doi.org/10.1038/nsmb.1530

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.1530

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing