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.

Structure and function of the bacterial decapping enzyme NudC

Nature Chemical Biology volume 12, pages 730–734 (2016)Cite this article

Subjects

Abstract

RNA capping and decapping are thought to be distinctive features of eukaryotes. The redox cofactor NAD was recently discovered to be attached to small regulatory RNAs in bacteria in a cap-like manner, and Nudix hydrolase NudC was found to act as a NAD-decapping enzyme in vitro and in vivo. Here, crystal structures of Escherichia coli NudC in complex with substrate NAD and with cleavage product NMN reveal the catalytic residues lining the binding pocket and principles underlying molecular recognition of substrate and product. Biochemical mutation analysis identifies the conserved Nudix motif as the catalytic center of the enzyme, which needs to be homodimeric, as the catalytic pocket is composed of amino acids from both monomers. NudC is single-strand specific and has a purine preference for the 5′-terminal nucleotide. The enzyme strongly prefers NAD-linked RNA (NAD–RNA) over NAD and binds to a diverse set of cellular RNAs in an unspecific manner.

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

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it

$39.95

Learn more

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

Figure 1: Crystal structure E. coli NudC in complex with bound NAD.
Figure 2: Crystal structure of E. coli NudC in complex with bound NMN.
Figure 3: Mutagenesis of NudC.
Figure 4: RNA mutagenesis.
Figure 5: Comparison of the active site conformation in decapping enzymes.

Accession codes

Primary accessions

GenBank/EMBL/DDBJ

Protein Data Bank

Referenced accessions

Protein Data Bank

References

  1. Topisirovic, I., Svitkin, Y.V., Sonenberg, N. & Shatkin, A.J. Cap and cap-binding proteins in the control of gene expression. Wiley Interdiscip. Rev. RNA 2, 277–298 (2011).

    Article  CAS  Google Scholar 

  2. 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  Google Scholar 

  3. Chen, Y.G., Kowtoniuk, W.E., Agarwal, I., Shen, Y. & Liu, D.R. LC/MS analysis of cellular RNA reveals NAD-linked RNA. Nat. Chem. Biol. 5, 879–881 (2009).

    Article  CAS  Google Scholar 

  4. Cahová, H., Winz, M.L., Höfer, K., Nübel, G. & Jäschke, A. NAD captureSeq indicates NAD as a bacterial cap for a subset of regulatory RNAs. Nature 519, 374–377 (2015).

    Article  Google Scholar 

  5. Jäschke, A., Höfer, K., Nübel, G. & Frindert, J. Cap-like structures in bacterial RNA and epitranscriptomic modification. Curr. Opin. Microbiol. 30, 44–49 (2016).

    Article  Google Scholar 

  6. Luciano, D.J. & Belasco, J.G. NAD in RNA: unconventional headgear. Trends Biochem. Sci. 40, 245–247 (2015).

    Article  CAS  Google Scholar 

  7. Marbaniang, C.N. & Vogel, J. Emerging roles of RNA modifications in bacteria. Curr. Opin. Microbiol. 30, 50–57 (2016).

    Article  CAS  Google Scholar 

  8. Mackie, G.A. Ribonuclease E is a 5′-end-dependent endonuclease. Nature 395, 720–723 (1998).

    Article  CAS  Google Scholar 

  9. Deana, A., Celesnik, H. & Belasco, J.G. The bacterial enzyme RppH triggers messenger RNA degradation by 5′ pyrophosphate removal. Nature 451, 355–358 (2008).

    Article  CAS  Google Scholar 

  10. McLennan, A.G. The Nudix hydrolase superfamily. Cell. Mol. Life Sci. 63, 123–143 (2006).

    Article  CAS  Google Scholar 

  11. McLennan, A.G. Substrate ambiguity among the Nudix hydrolases: biologically significant, evolutionary remnant, or both? Cell. Mol. Life Sci. 70, 373–385 (2013).

    Article  CAS  Google Scholar 

  12. Song, M.G., Bail, S. & Kiledjian, M. Multiple Nudix family proteins possess mRNA decapping activity. RNA 19, 390–399 (2013).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  14. Frick, D.N. & Bessman, M.J. Cloning, purification, and properties of a novel NADH pyrophosphatase. Evidence for a nucleotide pyrophosphatase catalytic domain in MutT-like enzymes. J. Biol. Chem. 270, 1529–1534 (1995).

    Article  CAS  Google Scholar 

  15. 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  Google Scholar 

  16. Olejnik, K. et al. Mutational analysis of the AtNUDT7 Nudix hydrolase from Arabidopsis thaliana reveals residues required for protein quaternary structure formation and activity. Acta Biochim. Pol. 56, 291–300 (2009).

    Article  CAS  Google Scholar 

  17. O'Handley, S.F., Frick, D.N., Dunn, C.A. & Bessman, M.J. Orf186 represents a new member of the Nudix hydrolases, active on adenosine(5′)triphospho(5′)adenosine, ADP-ribose, and NADH. J. Biol. Chem. 273, 3192–3197 (1998).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  20. 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  Google Scholar 

  21. Bessman, M.J., Frick, D.N. & O'Handley, S.F. The MutT proteins or “Nudix” hydrolases, a family of versatile, widely distributed, “housecleaning” enzymes. J. Biol. Chem. 271, 25059–25062 (1996).

    Article  CAS  Google Scholar 

  22. Lee, C.R., Kim, M., Park, Y.H., Kim, Y.R. & Seok, Y.J. RppH-dependent pyrophosphohydrolysis of mRNAs is regulated by direct interaction with DapF in Escherichia coli. Nucleic Acids Res. 42, 12746–12757 (2014).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  25. 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  Google Scholar 

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

    Article  CAS  Google Scholar 

  27. Laskowski, R.A., Macarthur, M.W., Moss, D.S. & Thornton, J.M. PROCHECK: a program to check the stereochemical quality of protein structures. J. Appl. Crystallogr. 26, 283–291 (1993).

    Article  CAS  Google Scholar 

  28. Chen, V.B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21 (2010).

    Article  CAS  Google Scholar 

  29. Halbritter, F., Vaidya, H.J. & Tomlinson, S.R. GeneProf: analysis of high-throughput sequencing experiments. Nat. Methods 9, 7–8 (2012).

    Article  CAS  Google Scholar 

  30. Kersey, P.J. et al. Ensembl Genomes 2016: more genomes, more complexity. Nucleic Acids Res. 44, D1, D574–D580 (2016).

    Article  Google Scholar 

  31. Langmead, B., Trapnell, C., Pop, M. & Salzberg, S.L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009).

    Article  Google Scholar 

  32. Höfer, K., Abele, F., Schlotthauer, J. & Jäschke, A. Synthesis of 5′-NAD-capped RNA. Bioconjug. Chem. 27, 874–877 (2016).

    Article  Google Scholar 

Download references

Acknowledgements

We are grateful to L. Obenauer (Heidelberg University) for assistance with NGS data analysis, L. Kiss for experimental assistance and to W. Shi at beamline X29A at the Brookhaven National Laboratory for support in diffraction data collection. A.J. is supported by the German Research Foundation (DFG SPP 1784, grant JA 794/10-1) and by the Baden-Württemberg Stiftung (BWST-NCRNA-045). The structural research was supported by funds from US National Institutes of Health (NIH) grant GM104962 to D.J.P. and the Thousand Young Talent Program of China and the Chinese Academy of Sciences to J.D.

Author information

Author notes

  1. Katharina Höfer and Sisi Li: These authors contributed equally to this work.

Authors and Affiliations

  1. Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Heidelberg, Germany

    Katharina Höfer, Florian Abele, Jens Frindert, Jasmin Schlotthauer, Julia Grawenhoff & Andres Jäschke

  2. Structural Biology Program, Memorial Sloan Kettering Cancer Center, New York, New York, USA

    Sisi Li & Dinshaw J Patel

  3. Shanghai Center for Plant Stress Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China

    Jiamu Du

Authors
  1. Katharina Höfer
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sisi Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Florian Abele
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jens Frindert
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jasmin Schlotthauer
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Julia Grawenhoff
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Jiamu Du
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Dinshaw J Patel
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Andres Jäschke
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

K.H., S.L., D.J.P., and A.J. designed research; S.L. and J.D. performed the crystallographic investigation; K.H., F.A., J.F., J.S., and J.G. carried out the functional studies on NudC; all authors analyzed data; K.H., S.L., J.F., J.D., D.J.P., and A.J. wrote the paper.

Corresponding authors

Correspondence to Dinshaw J Patel or Andres Jäschke.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Figures 1–8 and Supplementary Tables 1 and 2. (PDF 3030 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Höfer, K., Li, S., Abele, F. et al. Structure and function of the bacterial decapping enzyme NudC. Nat Chem Biol 12, 730–734 (2016). https://doi.org/10.1038/nchembio.2132

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nchembio.2132

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