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:

Structure of the active form of Dcp1–Dcp2 decapping enzyme bound to m7GDP and its Edc3 activator

Nature Structural & Molecular Biology volume 23, pages 982–986 (2016)Cite this article

Subjects

Abstract

Elimination of the 5′ cap of eukaryotic mRNAs, known as decapping, is considered to be a crucial, irreversible and highly regulated step required for the rapid degradation of mRNA by Xrn1, the major cytoplasmic 5′-3′ exonuclease. Decapping is accomplished by the recruitment of a protein complex formed by the Dcp2 catalytic subunit and its Dcp1 cofactor. However, this complex has a low intrinsic enzymatic activity and requires several accessory proteins such as the Lsm1–7 complex, Pat1, Edc1–Edc2 and/or Edc3 to be fully active. Here we present the crystal structure of the active form of the yeast Kluyveromyces lactis Dcp1–Dcp2 enzyme bound to its product (m7GDP) and its potent activator Edc3. This structure of the Dcp1–Dcp2 complex bound to a cap analog further explains previously published data on substrate binding and provides hints as to the mechanism of Edc3-mediated Dcp2 activation.

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: Structure of the K. lactis Dcp1–Dcp2–Edc3-m7GDP complex.
Figure 2: m7GDP binding mode.
Figure 3: The mRNA channel.

Similar content being viewed by others

Accession codes

Primary accessions

Protein Data Bank

Referenced accessions

Protein Data Bank

References

  1. Garneau, N.L., Wilusz, J. & Wilusz, C.J. The highways and byways of mRNA decay. Nat. Rev. Mol. Cell Biol. 8, 113–126 (2007).

    Article  CAS  PubMed  Google Scholar 

  2. Chen, C.Y. & Shyu, A.B. Mechanisms of deadenylation-dependent decay. Wiley Interdiscip. Rev. RNA 2, 167–183 (2011).

    Article  CAS  PubMed  Google Scholar 

  3. Li, Y. & Kiledjian, M. Regulation of mRNA decapping. Wiley Interdiscip. Rev. RNA 1, 253–265 (2010).

    Article  PubMed  Google Scholar 

  4. Steiger, M., Carr-Schmid, A., Schwartz, D.C., Kiledjian, M. & Parker, R. Analysis of recombinant yeast decapping enzyme. RNA 9, 231–238 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Nissan, T., Rajyaguru, P., She, M., Song, H. & Parker, R. Decapping activators in Saccharomyces cerevisiae act by multiple mechanisms. Mol. Cell 39, 773–783 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Dunckley, T. & Parker, R. The DCP2 protein is required for mRNA decapping in Saccharomyces cerevisiae and contains a functional MutT motif. EMBO J. 18, 5411–5422 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. van Dijk, E. et al. Human Dcp2: a catalytically active mRNA decapping enzyme located in specific cytoplasmic structures. EMBO J. 21, 6915–6924 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. She, M. et al. Crystal structure and functional analysis of Dcp2p from Schizosaccharomyces pombe. Nat. Struct. Mol. Biol. 13, 63–70 (2006).

    Article  CAS  PubMed  Google Scholar 

  9. Piccirillo, C., Khanna, R. & Kiledjian, M. Functional characterization of the mammalian mRNA decapping enzyme hDcp2. RNA 9, 1138–1147 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Arribas-Layton, M., Wu, D., Lykke-Andersen, J. & Song, H. Structural and functional control of the eukaryotic mRNA decapping machinery. Biochim. Biophys. Acta 1829, 580–589 (2013).

    Article  CAS  PubMed  Google Scholar 

  11. Harigaya, Y., Jones, B.N., Muhlrad, D., Gross, J.D. & Parker, R. Identification and analysis of the interaction between Edc3 and Dcp2 in Saccharomyces cerevisiae. Mol. Cell. Biol. 30, 1446–1456 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. He, F. & Jacobson, A. Control of mRNA decapping by positive and negative regulatory elements in the Dcp2 C-terminal domain. RNA 21, 1633–1647 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Kshirsagar, M. & Parker, R. Identification of Edc3p as an enhancer of mRNA decapping in Saccharomyces cerevisiae. Genetics 166, 729–739 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Sharif, H. et al. Structural analysis of the yeast Dhh1-Pat1 complex reveals how Dhh1 engages Pat1, Edc3 and RNA in mutually exclusive interactions. Nucleic Acids Res. 41, 8377–8390 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Fromm, S.A. et al. The structural basis of Edc3- and Scd6-mediated activation of the Dcp1:Dcp2 mRNA decapping complex. EMBO J. 31, 279–290 (2012).

    Article  CAS  PubMed  Google Scholar 

  16. Decourty, L. et al. Linking functionally related genes by sensitive and quantitative characterization of genetic interaction profiles. Proc. Natl. Acad. Sci. USA 105, 5821–5826 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Deshmukh, M.V. et al. mRNA decapping is promoted by an RNA-binding channel in Dcp2. Mol. Cell 29, 324–336 (2008).

    Article  CAS  PubMed  Google Scholar 

  18. She, M. et al. Structural basis of dcp2 recognition and activation by dcp1. Mol. Cell 29, 337–349 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Floor, S.N., Jones, B.N., Hernandez, G.A. & Gross, J.D. A split active site couples cap recognition by Dcp2 to activation. Nat. Struct. Mol. Biol. 17, 1096–1101 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Aglietti, R.A., Floor, S.N., McClendon, C.L., Jacobson, M.P. & Gross, J.D. Active site conformational dynamics are coupled to catalysis in the mRNA decapping enzyme Dcp2. Structure 21, 1571–1580 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Valkov, E. et al. Structure of the Dcp2-Dcp1 mRNA-decapping complex in the activated conformation. Nat. Struct. Mol. Biol. 23, 574–579 (2016).

    Article  CAS  PubMed  Google Scholar 

  22. Floor, S.N., Borja, M.S. & Gross, J.D. Interdomain dynamics and coactivation of the mRNA decapping enzyme Dcp2 are mediated by a gatekeeper tryptophan. Proc. Natl. Acad. Sci. USA 109, 2872–2877 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. She, M. et al. Crystal structure of Dcp1p and its functional implications in mRNA decapping. Nat. Struct. Mol. Biol. 11, 249–256 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Vasilyev, N. & Serganov, A. Structures of RNA complexes with the Escherichia coli RNA pyrophosphohydrolase RppH unveil the basis for specific 5′-end-dependent mRNA decay. J. Biol. Chem. 290, 9487–9499 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Gaudon, C., Chambon, P. & Losson, R. Role of the essential yeast protein PSU1 in transcriptional enhancement by the ligand-dependent activation function AF-2 of nuclear receptors. EMBO J. 18, 2229–2240 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Borja, M.S., Piotukh, K., Freund, C. & Gross, J.D. Dcp1 links coactivators of mRNA decapping to Dcp2 by proline recognition. RNA 17, 278–290 (2010).

    Article  PubMed  Google Scholar 

  27. Wurm, J.P., Overbeck, J. & Sprangers, R. The S. pombe mRNA decapping complex recruits cofactors and an Edc1-like activator through a single dynamic surface. RNA 22, 1360–1372 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Ling, S.H. et al. Crystal structure of human Edc3 and its functional implications. Mol. Cell. Biol. 28, 5965–5976 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Cohen, L.S. et al. Dcp2 Decaps m2,2,7GpppN-capped RNAs, and its activity is sequence and context dependent. Mol. Cell. Biol. 25, 8779–8791 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Studier, F.W. Protein production by auto-induction in high density shaking cultures. Protein Expr. Purif. 41, 207–234 (2005).

    Article  CAS  PubMed  Google Scholar 

  31. Kabsch, W. Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants. J. Appl. Crystallogr. 26, 795–800 (1993).

    Article  CAS  Google Scholar 

  32. Evans, P.R. & Murshudov, G.N. How good are my data and what is the resolution? Acta Crystallogr. D Biol. Crystallogr. 69, 1204–1214 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Schneider, T.R. & Sheldrick, G.M. Substructure solution with SHELXD. Acta Crystallogr. D Biol. Crystallogr. 58, 1772–1779 (2002).

    Article  PubMed  Google Scholar 

  34. Vonrhein, C., Blanc, E., Roversi, P. & Bricogne, G. Automated structure solution with autoSHARP. Methods Mol. Biol. 364, 215–230 (2007).

    CAS  PubMed  Google Scholar 

  35. Vagin, A. & Teplyakov, A. MOLREP: an automated program for molecular replacement. J. Appl. Crystallogr. 30, 1022–1025 (1997).

    Article  CAS  Google Scholar 

  36. Winn, M.D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D Biol. Crystallogr. 67, 235–242 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. McCoy, A.J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Emsley, P., Lohkamp, B., Scott, W.G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. BUSTER version 2.10.2. (Global Phasing Ltd., 2016).

Download references

Acknowledgements

We are indebted to K. Breunig (Martin-Luther University Halle–Wittenberg, Germany) for the kind gift of the K. lactis yeast strain. We acknowledge SOLEIL and ESRF for provision of synchrotron radiation facilities. This work was supported by Ecole Polytechnique (M.G.), the Centre National pour la Recherche Scientifique (B.S. and M.G.) including specific support by the ATIP–AVENIR program (to M.G.), the Agence Nationale pour la Recherche (grant ANR-11-BSV8-009 to B.S. and M.G., study ANR-10-LABX-0030-INRT, performed under the program Investissements d'Avenir ANR-10-IDEX-0002-02 (to B.S.), the Ligue Contre le Cancer (Equipe Labellisée 2014) (to B.S.) and the CERBM–IGBMC and INSERM (to B.S.). C.C. is supported by a PhD fellowship from the French Ministère de l'Enseignement Supérieur et de la Recherche (MESR) and ENS Cachan.

Author information

Authors and Affiliations

  1. Laboratoire de Biochimie, Ecole Polytechnique, CNRS, Université Paris-Saclay, Palaiseau, France

    Clément Charenton, Régis Back & Marc Graille

  2. Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Illkirch, France

    Valerio Taverniti, Claudine Gaudon-Plesse & Bertrand Séraphin

  3. Centre National de Recherche Scientifique (CNRS) UMR 7104, Illkirch, France

    Valerio Taverniti, Claudine Gaudon-Plesse & Bertrand Séraphin

  4. Institut National de Santé et de Recherche Médicale (INSERM) U964, Illkirch, France

    Valerio Taverniti, Claudine Gaudon-Plesse & Bertrand Séraphin

  5. Université de Strasbourg, Illkirch, France

    Valerio Taverniti, Claudine Gaudon-Plesse & Bertrand Séraphin

Authors
  1. Clément Charenton
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Valerio Taverniti
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Claudine Gaudon-Plesse
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Régis Back
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Bertrand Séraphin
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Marc Graille
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

C.C. performed the biochemical and crystallographic experiments. V.T. performed enzymatic assays. C.G.-P. and R.B. performed cloning, and C.G.-P. performed preliminary binding assays. C.C., B.S. and M.G. designed research. C.C., B.S. and M.G. analyzed the data and wrote the paper.

Corresponding authors

Correspondence to Bertrand Séraphin or Marc Graille.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Identification of the ScDcp2 minimal region interacting with both ScDcp1 and the ScEdc3 LSm domain.

Pull-down experiment of untagged ScEdc3 LSm with various ScDcp1-Dcp2-His6 fragments. Input and eluted (His pull-down) samples were analyzed on 15% SDS-PAGE and Coomassie Blue staining.

Supplementary Figure 2 Experimental and anomalous electron density maps.

A. Experimental electron density map (contour: 1σ) obtained by Se-MAD. The m7GDP molecule bound to Dcp2 active site is shown in red sticks. Color code is the same as for Fig.1A.

B. Anomalous difference electron density map (contour: 4σ) calculated from the dataset collected at the energy corresponding to the peak of Se absorption spectra, showing the location of Se atoms from selenomethionines. The methionine side chains as built in the final model are shown as sticks, validating side chain assignment in our structure.

Supplementary Figure 3 m7GDP-binding site.

A. The molecule bound to KlDcp1-Dcp2-Edc3 active site in the crystals is m7GDP. Several crystals were harvested and dissolved in water. Upon addition of nucleoside diphosphate kinase (NDPK) and ATP-ɣP32 to dissolved crystals, the formation of m7GTP confirms the presence of m7GDP in the crystals (Lane 3). As negative and positive controls, NDPK and ATP-ɣP32 were incubated with water (Lane 1) or m7GDP (Lane 2). The content of the reaction was analyzed by TLC and the nature of the m7GTP product was confirmed by migration in a different TLC buffer (data not shown).

B. Alignment of Dcp2 sequences. For the sake of clarity, only Dcp2 regions corresponding to NRD and Nudix are shown. Strictly conserved residues are in white on a black background. Partially conserved residues are boxed. Residues involved in m7GDP binding are indicated by black stars below the alignment. Secondary structure elements as observed in our structure of KlDcp1-Dcp2-Edc3 and in SpDcp1-Dcp2 compact form (PDB code: 2QKM, chain B) are indicated. This panel was generated using the ESPript server (Robert, X. & Gouet, P. Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Res 42, W320, (2014)).

C. Representation of the sequence conservation score at the surface of KlDcp1-Dcp2-Edc3 complex. The m7GDP is shown as red sticks. Coloring is from gray (low conservation) to cyan (highly conserved).

D. Stereo view representation of m7GDP binding mode.

Supplementary Figure 4 Superimposition of Dcp2 and bound ligands with E. coli RppH and associated RNA.

Superimposition of ligands bound to KlDcp1-Dcp2-Edc3, SpDcp1-Dcp2 and E. coli RppH. m7GDP bound to KlDcp1-Dcp2-Edc3 (grey), ATP bound to the compact form of SpDcp1-Dcp2 (yellow sticks ; She, M. et al. Structural basis of dcp2 recognition and activation by dcp1. Mol Cell 29, 337, (2008)) and RNA fragment bound to RppH (magenta ; Vasilyev, N. & Serganov, A. Structures of RNA complexes with the Escherichia coli RNA pyrophosphohydrolase RppH unveil the basis for specific 5'-end-dependent mRNA decay. J Biol Chem 290, 9487, (2015)) are shown as sticks. This figure was generated by superimposing Nudix domains from our structure of KlDcp1-Dcp2-Edc3-m7GDP complex and from E. coli RppH onto the compact form of the SpDcp1-Dcp2 complex. SpDcp1-Dcp2 and E. coli RppH are not shown. For the sake of clarity, neither SpDcp1-Dcp2 nor E. coli RppH are shown as ribbons. SpY220 (KlF223 or ScY222), which stacks with adenine ring in SpDcp1-Dcp2 compact form, is shown as cyan sticks.

Supplementary Figure 5 KlDcp2-Edc3 interface.

A. Sequence alignment of the Sp, Sc and KlDcp2 region involved in Edc3 binding. Strictly conserved residues are in white on a black background. Partially conserved residues are boxed. Residues involved in Edc3 binding are indicated by black stars below the alignment.

B. Sequence alignment of Sp, Sc and KlEdc3 LSm domain. Strictly conserved residues are in white on a black background. Partially conserved residues are boxed. Residues involved in Dcp2 binding are indicated by black stars below the alignment. Panels A and B were generated using the ESPript server.

C. Detailed representation of KlDcp2-Edc3 interface. Some side chain residues from the interface are shown as sticks.

D. Superimposition of SpDcp2-Edc3 complex determined by NMR (SpDcp2 and SpEdc3 LSm are in yellow and grey, respectively) onto KlDcp2-Edc3 as observed in our structure. Some side chain residues from both Dcp2 proteins are shown as sticks.

E. Comparison of Edc3 LSm residues involved in Dcp2 binding. The superimposition shown in panel E is viewed from a different angle as panel D using the same color code. Some side chains from SpEdc3 and KlEdc3 involved in Dcp2 binding are shown as sticks.

Supplementary Figure 6 KlEdc3 LSm stimulates KlDcp1–Dcp2 enzymatic activity and RNA binding.

A. Fluorescence quenching analyses of RNA binding to KlDcp1-Dcp2 or KlDcp1-Dcp2-Edc3. FAM-labeled RNA (10 nM) was incubated with increasing amount of purified recombinant complexes. The graph represents the difference in fluorescence (ΔF) between the free fluorescent RNA and the reaction mix as a function of complex concentration. The curves obtained after fitting of the experimental data with equation (A) from the materials and methods section, are shown as a solid line. Error bars were calculated from triplicate experiments. Kd values determined for KlDcp1-Dcp2 or KlDcp1-Dcp2-Edc3 complexes are 3.8 µM ± 0.4 and 1.9 µM ± 0.1, respectively.

B. Specific activation of KlDcp1-Dcp2 by KlEdc3 LSm domain. 32P cap-labeled RNA was incubated with equimolar amounts of KlDcp1-Dcp2, KlDcp1-Dcp2-Edc3 or KlDcp1-Dcp2 supplemented with BSA as a non-specific carrier. Left: Formation of m7GDP as a result of decapping was monitored after 0, 10, 30 and 90 minutes by TLC analysis and autoradiography. Right: Results of three biochemical reactions were quantified and the amount of m7GDP formation as a function of time and standard deviations from triplicate experiments were plotted.

Supplementary Figure 7 Model of Edc1 bound to the KlDcp1–Dcp2–Edc3 ternary complex.

Model of Edc1 binding to Dcp1 in the KlDcp1-Dcp2-Edc3-m7GDP complex. This representation has been generated by superimposing the Dcp1 EVH1 domains from SpDcp1-Dcp2-Edc1 (Valkov, E. et al. Structure of the Dcp2-Dcp1 mRNA-decapping complex in the activated conformation. Nature Structural & Molecular Biology 23, 574-579, (2016)) and KlDcp1-Dcp2-Edc3 structures. The SpEdc1 peptide is shown in magenta with the YAG conserved motif shown as sticks. For the sake of clarity, SpDcp1-Dcp2 complex as bound to SpEdc1 has been omitted in this representation.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7 and Supplementary Table 1 (PDF 1198 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Charenton, C., Taverniti, V., Gaudon-Plesse, C. et al. Structure of the active form of Dcp1–Dcp2 decapping enzyme bound to m7GDP and its Edc3 activator. Nat Struct Mol Biol 23, 982–986 (2016). https://doi.org/10.1038/nsmb.3300

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nsmb.3300

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