Article | Published:

Structural basis of mRNA-cap recognition by Dcp1–Dcp2

Nature Structural & Molecular Biology volume 23, pages 987994 (2016) | Download Citation

Abstract

Removal of the 5′ cap on mRNA by the decapping enzyme Dcp2 is a critical step in 5′-to-3′ mRNA decay. Understanding the structural basis of Dcp2 activity has been a challenge because Dcp2 is dynamic and has weak affinity for the cap substrate. Here we present a 2.6-Å-resolution crystal structure of a heterotrimer of fission yeast Dcp2, its essential activator Dcp1, and the human NMD cofactor PNRC2, in complex with a tight-binding cap analog. Cap binding is accompanied by a conformational change in Dcp2, thereby forming a composite nucleotide-binding site comprising conserved residues in the catalytic and regulatory domains. Kinetic analysis of PNRC2 revealed that a conserved short linear motif enhances both substrate affinity and the catalytic step of decapping. These findings explain why Dcp2 requires a conformational change for efficient catalysis and reveals that coactivators promote RNA binding and the catalytic step of decapping, possibly through different conformational states.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Accessions

References

  1. 1.

    From birth to death: the complex lives of eukaryotic mRNAs. Science 309, 1514–1518 (2005).

  2. 2.

    , , & Cap and cap-binding proteins in the control of gene expression. Wiley Interdiscip. Rev. RNA 2, 277–298 (2011).

  3. 3.

    RNA degradation in Saccharomyces cerevisae. Genetics 191, 671–702 (2012).

  4. 4.

    , , & Structural and functional control of the eukaryotic mRNA decapping machinery. Biochim. Biophys. Acta 1829, 580–589 (2013).

  5. 5.

    & Regulation of mRNA decapping. Wiley Interdiscip. Rev. RNA 1, 253–265 (2010).

  6. 6.

    et al. Structures and mechanisms of Nudix hydrolases. Arch. Biochem. Biophys. 433, 129–143 (2005).

  7. 7.

    , , & XRN 5′ → 3′ exoribonucleases: structure, mechanisms and functions. Biochim. Biophys. Acta 1829, 590–603 (2013).

  8. 8.

    , , , & Decapping of long noncoding RNAs regulates inducible genes. Mol. Cell 45, 279–291 (2012).

  9. 9.

    , , & Mammalian microRNAs predominantly act to decrease target mRNA levels. Nature 466, 835–840 (2010).

  10. 10.

    & Towards a molecular understanding of microRNA-mediated gene silencing. Nat. Rev. Genet. 16, 421–433 (2015).

  11. 11.

    & NMD: a multifaceted response to premature translational termination. Nat. Rev. Mol. Cell Biol. 13, 700–712 (2012).

  12. 12.

    & Organizing principles of mammalian nonsense-mediated mRNA decay. Annu. Rev. Genet. 47, 139–165 (2013).

  13. 13.

    , & Human proline-rich nuclear receptor coregulatory protein 2 mediates an interaction between mRNA surveillance machinery and decapping complex. Mol. Cell 33, 75–86 (2009).

  14. 14.

    et al. Staufen1-mediated mRNA decay functions in adipogenesis. Mol. Cell 46, 495–506 (2012).

  15. 15.

    & Staufen-mediated mRNA decay. Wiley Interdiscip. Rev. RNA 4, 423–435 (2013).

  16. 16.

    et al. mRNA decapping factors and the exonuclease Xrn2 function in widespread premature termination of RNA polymerase II transcription. Mol. Cell 46, 311–324 (2012).

  17. 17.

    et al. Codon optimality is a major determinant of mRNA stability. Cell 160, 1111–1124 (2015).

  18. 18.

    & Quality control of assembly-defective U1 snRNAs by decapping and 5′-to-3′ exonucleolytic digestion. Proc. Natl. Acad. Sci. USA 111, E3277–E3286 (2014).

  19. 19.

    , , , & Dcp2 decapping protein modulates mRNA stability of the critical interferon regulatory factor (IRF) IRF-7. Mol. Cell. Biol. 32, 1164–1172 (2012).

  20. 20.

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

  21. 21.

    , & Two related proteins, Edc1p and Edc2p, stimulate mRNA decapping in Saccharomyces cerevisiae. Genetics 157, 27–37 (2001).

  22. 22.

    , , & Dcp1 links coactivators of mRNA decapping to Dcp2 by proline recognition. RNA 17, 278–290 (2011).

  23. 23.

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

  24. 24.

    et al. In vitro reconstitution of a cellular phase-transition process that involves the mRNA decapping machinery. Angew. Chem. Int. Ed. Engl. 53, 7354–7359 (2014).

  25. 25.

    , , , & Identification and analysis of the interaction between Edc3 and Dcp2 in Saccharomyces cerevisiae. Mol. Cell. Biol. 30, 1446–1456 (2010).

  26. 26.

    , , , & The activation of the decapping enzyme DCP2 by DCP1 occurs on the EDC4 scaffold and involves a conserved loop in DCP1. Nucleic Acids Res. 42, 5217–5233 (2014).

  27. 27.

    et al. Structural basis of the PNRC2-mediated link between mRNA surveillance and decapping. Structure 20, 2025–2037 (2012).

  28. 28.

    , & The decapping activator Lsm1p-7p-Pat1p complex has the intrinsic ability to distinguish between oligoadenylated and polyadenylated RNAs. RNA 13, 998–1016 (2007).

  29. 29.

    & Activation of decapping involves binding of the mRNA and facilitation of the post-binding steps by the Lsm1-7-Pat1 complex. RNA 15, 1837–1848 (2009).

  30. 30.

    & General translational repression by activators of mRNA decapping. Cell 122, 875–886 (2005).

  31. 31.

    , & The DEAD-box protein Dhh1 promotes decapping by slowing ribosome movement. PLoS Biol. 10, e1001342 (2012).

  32. 32.

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

  33. 33.

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

  34. 34.

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

  35. 35.

    , & Functional characterization of the mammalian mRNA decapping enzyme hDcp2. RNA 9, 1138–1147 (2003).

  36. 36.

    , & 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).

  37. 37.

    , , & A split active site couples cap recognition by Dcp2 to activation. Nat. Struct. Mol. Biol. 17, 1096–1101 (2010).

  38. 38.

    & The role of disordered protein regions in the assembly of decapping complexes and RNP granules. Genes Dev. 27, 2628–2641 (2013).

  39. 39.

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

  40. 40.

    et al. Two-headed tetraphosphate cap analogs are inhibitors of the Dcp1/2 RNA decapping complex. RNA 22, 518–529 (2016).

  41. 41.

    et al. Phosphate-modified analogues of m7GTP and m7Gppppm7G: synthesis and biochemical properties. Bioorg. Med. Chem. 23, 5369–5381 (2015).

  42. 42.

    , , & Cocrystal structure of the messenger RNA 5′ cap-binding protein (eIF4E) bound to 7-methyl-GDP. Cell 89, 951–961 (1997).

  43. 43.

    , , & Large-scale induced fit recognition of an m7GpppG cap analogue by the human nuclear cap-binding complex. EMBO J. 21, 5548–5557 (2002).

  44. 44.

    et al. The crystal structure of diadenosine tetraphosphate hydrolase from Caenorhabditis elegans in free and binary complex forms. Structure 10, 589–600 (2002).

  45. 45.

    et al. Crystal structure of human MTH1 and the 8-oxo-dGMP product complex. FEBS Lett. 585, 2617–2621 (2011).

  46. 46.

    , , & A kinetic assay to monitor RNA decapping under single-turnover conditions. Methods Enzymol. 448, 23–40 (2008).

  47. 47.

    & Mechanisms of DNA-binding specificity and functional gene regulation by transcription factors. Curr. Opin. Struct. Biol. 38, 68–74 (2016).

  48. 48.

    From “simple” DNA-protein interactions to the macromolecular machines of gene expression. Annu. Rev. Biophys. Biomol. Struct. 36, 79–105 (2007).

  49. 49.

    et al. Structure and flexibility adaptation in nonspecific and specific protein-DNA complexes. Science 305, 386–389 (2004).

  50. 50.

    & Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997).

  51. 51.

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

  52. 52.

    et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

  53. 53.

    , , & Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

  54. 54.

    , , & PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Crystallogr. 26, 283–291 (1993).

  55. 55.

    et al. PDB2PQR: expanding and upgrading automated preparation of biomolecular structures for molecular simulations. Nucleic Acids Res. 35, W522–W525 (2007).

  56. 56.

    , , , & Electrostatics of nanosystems: application to microtubules and the ribosome. Proc. Natl. Acad. Sci. USA 98, 10037–10041 (2001).

Download references

Acknowledgements

The authors thank X. Liu, J. Binning, and C. Waddling at UCSF for valuable help and advice on crystallography experiments, and J. Holton and G. Meigs at Lawrence Berkeley National Laboratory, Advanced Light Source beamline 8.3.1, for help with X-ray data collection. We also thank J. Kowalska for helpful discussions on the design and synthesis of the two-headed cap analog. This work was supported by the US National Institutes of Health (R01 GM078360 to J.D.G. and NRSA fellowship F32 GM105313 to J.S.M.) and the National Science Centre, Poland (grant no. UMO-2012/05/E/ST5/03893 to J.J. and fellowship no. UMO-2014/12/T/NZ1/00528 to M.Z.). The Advanced Light Source is supported by the US Department of Energy under contract no. DE-AC02-05CH11231.

Author information

Affiliations

  1. Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, California, USA.

    • Jeffrey S Mugridge
    • , Marcin Ziemniak
    •  & John D Gross
  2. Division of Biophysics, Institute of Experimental Physics, Faculty of Physics, University of Warsaw, Warsaw, Poland.

    • Marcin Ziemniak
  3. Centre of New Technologies, University of Warsaw, Warsaw, Poland.

    • Jacek Jemielity

Authors

  1. Search for Jeffrey S Mugridge in:

  2. Search for Marcin Ziemniak in:

  3. Search for Jacek Jemielity in:

  4. Search for John D Gross in:

Contributions

J.S.M. designed and purified all protein constructs, carried out crystallization experiments, collected and refined crystallographic data, carried out decapping kinetics experiments, wrote the manuscript, and prepared the figures. M.Z. and J.J. designed and synthesized the two-headed cap analog. J.D.G. supervised the project and experimental design and guided manuscript preparation and editing. All authors read and commented on the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to John D Gross.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–7 and Supplementary Table 1

Excel files

  1. 1.

    Supplementary Data Set 1

    Kinetic data associated with Figure 4 and Supplementary Figure 1a,b.

Videos

  1. 1.

    Conformational change in Dcp1–Dcp2 accompanied by cap-analog binding.

    This video shows a morph between the apo Dcp1–Dcp2 conformation (PDB 5KQ1) and the two-headed cap analog-bound Dcp1–Dcp2 conformation (PDB 5KQ4).

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nsmb.3301

Further reading