Nothing Special   »   [go: up one dir, main page]
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.

  • Letter
  • Published:

Structural insights into mechanisms of the small RNA methyltransferase HEN1

Nature volume 461, pages 823–827 (2009)Cite this article

Abstract

RNA silencing is a conserved regulatory mechanism in fungi, plants and animals that regulates gene expression and defence against viruses and transgenes1. Small silencing RNAs of ∼20–30 nucleotides and their associated effector proteins, the Argonaute family proteins, are the central components in RNA silencing2. A subset of small RNAs, such as microRNAs and small interfering RNAs (siRNAs) in plants, Piwi-interacting RNAs in animals and siRNAs in Drosophila, requires an additional crucial step for their maturation; that is, 2′-O-methylation on the 3′ terminal nucleotide3,4,5,6. A conserved S-adenosyl-l-methionine-dependent RNA methyltransferase, HUA ENHANCER 1 (HEN1), and its homologues are responsible for this specific modification3,4,5,7,8. Here we report the 3.1 Å crystal structure of full-length HEN1 from Arabidopsis in complex with a 22-nucleotide small RNA duplex and cofactor product S-adenosyl-l-homocysteine. Highly cooperative recognition of the small RNA substrate by multiple RNA binding domains and the methyltransferase domain in HEN1 measures the length of the RNA duplex and determines the substrate specificity. Metal ion coordination by both 2′ and 3′ hydroxyls on the 3′-terminal nucleotide and four invariant residues in the active site of the methyltransferase domain suggests a novel Mg2+-dependent 2′-O-methylation mechanism.

Access through your institution
Buy or subscribe

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

Access options

Access through your institution

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

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

Figure 1: Structures of HEN1 in complex with a small RNA duplex and AdoHcy.
Figure 2: Small RNA substrate recognition by dsRBDs and LCD.
Figure 3: Small RNA substrate recognition by the MTase domain.
Figure 4: Proposed model for the specific recognition of small RNA substrates by HEN1 and the Mg 2+ -dependent 2′- O -methyltransferase mechanism.

Similar content being viewed by others

Accession codes

Primary accessions

Protein Data Bank

References

  1. Ghildiyal, M. & Zamore, P. D. Small silencing RNAs: an expanding universe. Nature Rev. Genet. 10, 94–108 (2009)

    Article  CAS  Google Scholar 

  2. Farazi, T. A., Juranek, S. A. & Tuschl, T. The growing catalog of small RNAs and their association with distinct Argonaute/Piwi family members. Development 135, 1201–1214 (2008)

    Article  CAS  Google Scholar 

  3. Yu, B. et al. Methylation as a crucial step in plant microRNA biogenesis. Science 307, 932–935 (2005)

    Article  ADS  CAS  Google Scholar 

  4. Horwich, M. D. et al. The Drosophila RNA methyltransferase, DmHen1, modifies germline piRNAs and single-stranded siRNAs in RISC. Curr. Biol. 17, 1265–1272 (2007)

    Article  CAS  Google Scholar 

  5. Saito, K. et al. Pimet, the Drosophila homolog of HEN1, mediates 2'-O-methylation of Piwi-interacting RNAs at their 3′ ends. Genes Dev. 21, 1603–1608 (2007)

    Article  CAS  Google Scholar 

  6. Kirino, Y. & Mourelatos, Z. Mouse Piwi-interacting RNAs are 2'-O-methylated at their 3′ termini. Nature Struct. Mol. Biol. 14, 347–348 (2007)

    Article  CAS  Google Scholar 

  7. Kirino, Y. & Mourelatos, Z. The mouse homolog of HEN1 is a potential methylase for Piwi-interacting RNAs. RNA 13, 1397–1401 (2007)

    Article  CAS  Google Scholar 

  8. Kurth, H. M. & Mochizuki, K. 2'-O-methylation stabilizes Piwi-associated small RNAs and ensures DNA elimination in Tetrahymena . RNA 15, 675–685 (2009)

    Article  CAS  Google Scholar 

  9. Chen, X., Liu, J., Cheng, Y. & Jia, D. HEN1 functions pleiotropically in Arabidopsis development and acts in C function in the flower. Development 129, 1085–1094 (2002)

    Article  CAS  Google Scholar 

  10. Park, W., Li, J., Song, R., Messing, J. & Chen, X. CARPEL FACTORY, a Dicer homolog, and HEN1, a novel protein, act in microRNA metabolism in Arabidopsis thaliana . Curr. Biol. 12, 1484–1495 (2002)

    Article  CAS  Google Scholar 

  11. Yang, Z., Ebright, Y. W., Yu, B. & Chen, X. HEN1 recognizes 21–24 nt small RNA duplexes and deposits a methyl group onto the 2' OH of the 3′ terminal nucleotide. Nucleic Acids Res. 34, 667–675 (2006)

    Article  CAS  Google Scholar 

  12. Li, J., Yang, Z., Yu, B., Liu, J. & Chen, X. Methylation protects miRNAs and siRNAs from a 3′-end uridylation activity in Arabidopsis . Curr. Biol. 15, 1501–1507 (2005)

    Article  CAS  Google Scholar 

  13. Ramachandran, V. & Chen, X. Degradation of microRNAs by a family of exoribonucleases in Arabidopsis . Science 321, 1490–1492 (2008)

    Article  ADS  CAS  Google Scholar 

  14. Chen, X. A marked end. Nature Struct. Mol. Biol. 14, 259–260 (2007)

    Article  CAS  Google Scholar 

  15. Tkaczuk, K., Obarska, A. & Bujnicki, J. Molecular phylogenetics and comparative modeling of HEN1, a methyltransferase involved in plant microRNA biogenesis. BMC Evol. Biol. 6, 6 (2006)

    Article  Google Scholar 

  16. Kang, C. B., Dhe-Paganon, S. & Yoon, H. S. FKBP family proteins: immunophilins with versatile biological functions. Neurosignals 16, 318–325 (2008)

    Article  CAS  Google Scholar 

  17. Tian, B., Bevilacqua, P. C., Diegelman-Parente, A. & Mathews, M. B. The double-stranded-RNA-binding motif: interference and much more. Nature Rev. Mol. Cell Biol. 5, 1013–1023 (2004)

    Article  CAS  Google Scholar 

  18. Curry, S. & Conte, M. R. A terminal affair: 3′-end recognition by the human La protein. Trends Biochem. Sci. 31, 303–305 (2006)

    Article  CAS  Google Scholar 

  19. Maraia, R. J. & Bayfield, M. A. The La protein-RNA complex surfaces. Mol. Cell 21, 149–152 (2006)

    Article  CAS  Google Scholar 

  20. Teplova, M. et al. Structural basis for recognition and sequestration of UUUOH 3′ temini of nascent RNA polymerase III transcripts by La, a rheumatic disease autoantigen. Mol. Cell 21, 75–85 (2006)

    Article  CAS  Google Scholar 

  21. Vargason, J. M., Szittya, G., Burgyán, J. & Hall, T. M. T. Size selective recognition of siRNA by an RNA silencing suppressor. Cell 115, 799–811 (2003)

    Article  CAS  Google Scholar 

  22. Ye, K., Malinina, L. & Patel, D. J. Recognition of small interfering RNA by a viral suppressor of RNA silencing. Nature 426, 874–878 (2003)

    Article  ADS  CAS  Google Scholar 

  23. Yu, B., Chapman, E. J., Yang, Z., Carrington, J. C. & Chen, X. Transgenically expressed viral RNA silencing suppressors interfere with microRNA methylation in Arabidopsis . FEBS Lett. 580, 3117–3120 (2006)

    Article  CAS  Google Scholar 

  24. Schubert, H. L., Blumenthal, R. M. & Cheng, X. Many paths to methyltransfer: a chronicle of convergence. Trends Biochem. Sci. 28, 329–335 (2003)

    Article  CAS  Google Scholar 

  25. Ryter, J. M. & Schultz, S. C. Molecular basis of double-stranded RNA-protein interactions: structure of a dsRNA-binding domain complexed with dsRNA. EMBO J. 17, 7505–7513 (1998)

    Article  CAS  Google Scholar 

  26. MacRae, I. J. et al. Structural basis for double-stranded RNA processing by Dicer. Science 311, 195–198 (2006)

    Article  ADS  CAS  Google Scholar 

  27. Lingel, A., Simon, B., Izaurralde, E. & Sattler, M. Nucleic acid 3′-end recognition by the Argonaute2 PAZ domain. Nature Struct. Mol. Biol. 11, 576–577 (2004)

    Article  CAS  Google Scholar 

  28. Ma, J.-B., Ye, K. & Patel, D. J. Structural basis for overhang-specific small interfering RNA recognition by the PAZ domain. Nature 429, 318–322 (2004)

    Article  ADS  CAS  Google Scholar 

  29. Yang, Z. et al. Approaches for studying microRNA and small interfering RNA methylation in vitro and in vivo . Methods Enzymol. 427, 139–154 (2007)

    Article  CAS  Google Scholar 

  30. Stump, W. T. & Hall, K. B. Crosslinking of an iodo-uridine-RNA hairpin to a single site on the human U1A N-terminal RNA binding domain. RNA 1, 55–63 (1995)

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Doublié, S. & Carter, C. W. Jr. Preparation of selenomethionyl proteins for phase determination. Methods Enzymol. 276, 523–530 (1997)

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  33. Hendrickson, W. A. Determination of macromolecular structures from anomalous diffraction of synthrotron radiation. Science 254, 51–58 (1991)

    Article  ADS  CAS  Google Scholar 

  34. Adams, P. D. et al. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr. D 58, 1948–1954 (2002)

    Article  Google Scholar 

  35. Jones, T. A., Zou, J. Y., Cowan, S. W. & Kjeldgaard, M. Improved methods for building protein models in eletron density maps and the location of errors in these models. Acta Crystallogr. A 47, 110–119 (1991)

    Article  Google Scholar 

  36. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004)

    Article  Google Scholar 

  37. Brunger, A. T. Version 1.2 of the Crystallography and NMR system. Nature Protocols 2, 2728–2733 (2007)

    Article  CAS  Google Scholar 

  38. 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 

Download references

Acknowledgements

We thank K. Sergiy for assistance with the in-house X-ray generator operation, and the staff at Advanced Photon Source beamlines 19ID and 23ID, Argonne National Laboratory, for help with data collection. We thank T. Townes and H. Wang for critical reading of the manuscript. This work was supported by a start-up fund (to J.-B.M.) and partly by a grant from the V Foundation for Cancer Research (to J.-B.M.) and a grant from the National Science Foundation (MCB-0718029 to X.C.). D.G.V. is supported by grants from National Institutes of Health (R01 GM074252 and R01 GM074840).

Author Contributions Y.H. expressed and purified proteins, grew crystals, solved structure and wrote the manuscript. J.-B.M. collected data, solved structure, performed crosslinking assays and wrote the manuscript. L.J. performed small RNA methyltransferase assays and wrote the manuscript. Q.H. and D.G.V. were involved in data processing and refinement. X.C. analysed data and wrote the manuscript. The overall project management and manuscript preparation were by Y.H., X.C. and J.-B.M.

Author information

Authors and Affiliations

  1. Department of Biochemistry and Molecular Genetics, Schools of Medicine and Dentistry, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA,

    Ying Huang, Qichen Huang, Dmitry G. Vassylyev & Jin-Biao Ma

  2. Department of Botany and Plant Sciences, Institute of Integrative Genome Biology, University of California, Riverside, California 92521, USA,

    Lijuan Ji & Xuemei Chen

  3. Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA ,

    Jin-Biao Ma

Authors
  1. Ying Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Lijuan Ji
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Qichen Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Dmitry G. Vassylyev
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Xuemei Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jin-Biao Ma
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jin-Biao Ma.

Additional information

Atomic coordinates and structural factors for the reported crystal structure have been deposited in the Protein Data Bank under access code 3HTX.

Supplementary information

Supplementary Information

This file contains Supplementary Notes, Supplementary References, Supplementary Tables 1-3 and Supplementary Figures S1-S10 with Legends. (PDF 7558 kb)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Cite this article

Huang, Y., Ji, L., Huang, Q. et al. Structural insights into mechanisms of the small RNA methyltransferase HEN1. Nature 461, 823–827 (2009). https://doi.org/10.1038/nature08433

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature08433

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