Article | Published:

Structures of human ADAR2 bound to dsRNA reveal base-flipping mechanism and basis for site selectivity

Nature Structural & Molecular Biology volume 23, pages 426433 (2016) | Download Citation


Adenosine deaminases acting on RNA (ADARs) are editing enzymes that convert adenosine to inosine in duplex RNA, a modification reaction with wide-ranging consequences in RNA function. Understanding of the ADAR reaction mechanism, the origin of editing-site selectivity, and the effect of mutations is limited by the lack of high-resolution structural data for complexes of ADARs bound to substrate RNAs. Here we describe four crystal structures of the human ADAR2 deaminase domain bound to RNA duplexes bearing a mimic of the deamination reaction intermediate. These structures, together with structure-guided mutagenesis and RNA-modification experiments, explain the basis of the ADAR deaminase domain's dsRNA specificity, its base-flipping mechanism, and its nearest-neighbor preferences. In addition, we identified an ADAR2-specific RNA-binding loop near the enzyme active site, thus rationalizing differences in selectivity observed between different ADARs. Finally, our results provide a structural framework for understanding the effects of ADAR mutations associated with human disease.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.


Referenced accessions

Protein Data Bank


  1. 1.

    Fine-Tuning of RNA Functions by Modification and Editing (Springer, 2005).

  2. 2.

    RNA editing by adenosine deaminases that act on RNA. Annu. Rev. Biochem. 71, 817–846 (2002).

  3. 3.

    Functions and regulation of RNA editing by ADAR deaminases. Annu. Rev. Biochem. 79, 321–349 (2010).

  4. 4.

    et al. ADAR1 regulates ARHGAP26 gene expression through RNA editing by disrupting miR-30b-3p and miR-573 binding. RNA 19, 1525–1536 (2013).

  5. 5.

    , & Regulation of alternative splicing by RNA editing. Nature 399, 75–80 (1999).

  6. 6.

    , , , & RNA editing changes the lesion specificity for the DNA repair enzyme NEIL1. Proc. Natl. Acad. Sci. USA 107, 20715–20719 (2010).

  7. 7.

    et al. A standardized nomenclature for adenosine deaminases that act on RNA. RNA 3, 947–949 (1997).

  8. 8.

    , , & A-to-I RNA editing and human disease. RNA Biol. 3, 1–9 (2006).

  9. 9.

    & Adenosine-to-inosine RNA editing and human disease. Genome Med. 5, 105 (2013).

  10. 10.

    et al. Mice with altered serotonin 2C receptor RNA editing display characteristics of Prader-Willi syndrome. Neurobiol. Dis. 39, 169–180 (2010).

  11. 11.

    et al. Mutations in ADAR1 cause Aicardi-Goutières syndrome associated with a type I interferon signature. Nat. Genet. 44, 1243–1248 (2012).

  12. 12.

    et al. Mutations of the RNA-specific adenosine deaminase gene (DSRAD) are involved in dyschromatosis symmetrica hereditaria. Am. J. Hum. Genet. 73, 693–699 (2003).

  13. 13.

    et al. Seven novel mutations of the ADAR gene in Chinese families and sporadic patients with dyschromatosis symmetrica hereditaria (DSH). Hum. Mutat. 23, 629–630 (2004).

  14. 14.

    et al. Recoding RNA editing of AZIN1 predisposes to hepatocellular carcinoma. Nat. Med. 19, 209–216 (2013).

  15. 15.

    RNA editing enters the limelight in cancer. Nat. Med. 19, 130–131 (2013).

  16. 16.

    et al. RNA editing of the GLI1 transcription factor modulates the output of Hedgehog signaling. RNA Biol. 10, 321–333 (2013).

  17. 17.

    , & ADAR proteins: structure and catalytic mechanism. Curr. Top. Microbiol. Immunol. 353, 1–33 (2012).

  18. 18.

    et al. Genome-wide identification of human RNA editing sites by parallel DNA capturing and sequencing. Science 324, 1210–1213 (2009).

  19. 19.

    et al. A transition state analogue for an RNA-editing reaction. J. Am. Chem. Soc. 126, 11213–11219 (2004).

  20. 20.

    et al. Recognition of duplex RNA by the deaminase domain of the RNA editing enzyme ADAR2. Nucleic Acids Res. 43, 1123–1132 (2015).

  21. 21.

    et al. Inositol hexakisphosphate is bound in the ADAR2 core and required for RNA editing. Science 309, 1534–1539 (2005).

  22. 22.

    & Mechanistic insights into editing-site specificity of ADARs. Proc. Natl. Acad. Sci. USA 109, E3295–E3304 (2012).

  23. 23.

    , & Substrate recognition by ADAR1 and ADAR2. RNA 7, 846–858 (2001).

  24. 24.

    , , & HhaI methyltransferase flips its target base out of the DNA helix. Cell 76, 357–369 (1994).

  25. 25.

    et al. HhaI DNA methyltransferase uses the protruding Gln237 for active flipping of its target cytosine. Structure 12, 1047–1055 (2004).

  26. 26.

    , & Cobalt hexammine induced tautomeric shift in Z-DNA: the structure of d(CGCGCA)*d(TGCGCG) in two crystal forms. Nucleic Acids Res. 32, 5945–5953 (2004).

  27. 27.

    et al. A nucleotide-flipping mechanism from the structure of human uracil-DNA glycosylase bound to DNA. Nature 384, 87–92 (1996).

  28. 28.

    , & Structural basis for recognition and repair of the endogenous mutagen 8-oxoguanine in DNA. Nature 403, 859–866 (2000).

  29. 29.

    , , , & Crystal structure of a human alkylbase-DNA repair enzyme complexed to DNA: mechanisms for nucleotide flipping and base excision. Cell 95, 249–258 (1998).

  30. 30.

    , , & Recent advances in the structural mechanisms of DNA glycosylases. Biochim. Biophys. Acta 1834, 247–271 (2013).

  31. 31.

    , & RNA-Seq analysis identifies a novel set of editing substrates for human ADAR2 present in Saccharomyces cerevisiae. Biochemistry 52, 7857–7869 (2013).

  32. 32.

    , & Predicting sites of ADAR editing in double-stranded RNA. Nat. Commun. 2, 319 (2011).

  33. 33.

    , & N2-Modified 2-aminopurine ribonucleosides as minor-groove-modulating adenosine replacements in duplex RNA. Org. Lett. 12, 1044–1047 (2010).

  34. 34.

    & Base flipping. Annu. Rev. Biochem. 67, 181–198 (1998).

  35. 35.

    & Cocrystal structure of a tRNA Psi55 pseudouridine synthase: nucleotide flipping by an RNA-modifying enzyme. Cell 107, 929–939 (2001).

  36. 36.

    et al. Base excision repair initiation revealed by crystal structures and binding kinetics of human uracil-DNA glycosylase with DNA. EMBO J. 17, 5214–5226 (1998).

  37. 37.

    , , & Crystal structures of restrictocin-inhibitor complexes with implications for RNA recognition and base flipping. Nat. Struct. Biol. 8, 968–973 (2001).

  38. 38.

    et al. Crystal structure of an initiation factor bound to the 30S ribosomal subunit. Science 291, 498–501 (2001).

  39. 39.

    et al. Structure, dynamics, and elasticity of free 16s rRNA helix 44 studied by molecular dynamics simulations. Biopolymers 82, 504–520 (2006).

  40. 40.

    , & Chemical trapping and crystal structure of a catalytic tRNA guanine transglycosylase covalent intermediate. Nat. Struct. Biol. 10, 781–788 (2003).

  41. 41.

    & Calculation of relative hydration free energy differences for heteroaromatic compounds: use in the design of adenosine deaminase and cytidine deaminase inhibitors. J. Am. Chem. Soc. 120, 3295–3304 (1998).

  42. 42.

    et al. The solution structure of the ADAR2 dsRBM-RNA complex reveals a sequence-specific readout of the minor groove. Cell 143, 225–237 (2010).

  43. 43.

    & Proteins binding to duplexed RNA: one motif, multiple functions. Trends Biochem. Sci. 25, 241–246 (2000).

  44. 44.

    et al. The RNA-editing enzyme ADAR1 controls innate immune responses to RNA. Cell Rep. 9, 1482–1494 (2014).

  45. 45.

    & Large-scale overexpression and purification of ADARs from Saccharomyces cerevisiae for biophysical and biochemical studies. Methods Enzymol. 424, 319–331 (2007).

  46. 46.

    et al. Matching active site and substrate structures for an RNA editing reaction. J. Am. Chem. Soc. 131, 11882–11891 (2009).

  47. 47.

    XDS. Acta Crystallogr. D Biol. Crystallogr. 66, 125–132 (2010).

  48. 48.

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

  49. 49.

    et al. Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr. D Biol. Crystallogr. 68, 352–367 (2012).

  50. 50.

    , , , & Click modification of RNA at adenosine: structure and reactivity of 7-ethynyl- and 7-triazolyl-8-aza-7-deazaadenosine in RNA. ACS Chem. Biol. 9, 1780–1787 (2014).

Download references


The authors acknowledge funding from the US National Institutes of Health (NIH) grant R01GM061115 (P.A.B.). A.I.S. was supported by NIH training grant T32 GM007377. C. Palumbo is acknowledged for technical assistance. Use of the Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, is supported by the US Department of Energy (DOE), Office of Science, Office of Basic Energy Sciences under contract no. DE-AC02-76SF00515. The SSRL Structural Molecular Biology Program is supported by the US Department of Energy, Office of Biological and Environmental Research, and by the NIH, US National Institute of General Medical Sciences (including P41GM103393). Part of this work is also based on research conducted at the Northeastern Collaborative Access Team beamlines, which are funded by the National Institute of General Medical Sciences from the NIH (P41 GM103403). The Pilatus 6M detector on the 24-ID-C beamline is funded by an NIH-ORIP HEI grant (S10 RR029205). This research used resources of the Advanced Photon Source, a DOE Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under contract no. DE-AC02-06CH11357. The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of NIGMS or NIH.

Author information

Author notes

    • Melissa M Matthews
    •  & Justin M Thomas

    These authors contributed equally to this work.


  1. Department of Chemistry, University of California, Davis, Davis, California, USA.

    • Melissa M Matthews
    • , Justin M Thomas
    • , Yuxuan Zheng
    • , Kiet Tran
    • , Kelly J Phelps
    • , Jocelyn Havel
    • , Andrew J Fisher
    •  & Peter A Beal
  2. Department of Molecular and Cellular Biology, University of California, Davis, Davis, California, USA.

    • Anna I Scott
    •  & Andrew J Fisher


  1. Search for Melissa M Matthews in:

  2. Search for Justin M Thomas in:

  3. Search for Yuxuan Zheng in:

  4. Search for Kiet Tran in:

  5. Search for Kelly J Phelps in:

  6. Search for Anna I Scott in:

  7. Search for Jocelyn Havel in:

  8. Search for Andrew J Fisher in:

  9. Search for Peter A Beal in:


J.M.T., M.M.M., A.I.S., and Y.Z. purified protein. K.J.P. and J.M.T. designed and purified RNA for crystallography and characterized protein-RNA binding. M.M.M. and A.I.S. conducted crystallization trials. M.M.M. and A.J.F. collected diffraction data and solved and refined the crystal structures. J.M.T., Y.Z., and J.H. measured enzyme reaction rates. K.T. synthesized 8-azanebularane phosphoramidite. J.M.T. and A.I.S. conducted mutagenesis. J.M.T., M.M.M., P.A.B. and A.J.F. analyzed the structures. P.A.B. wrote the initial manuscript draft. J.M.T., M.M.M., P.A.B., and A.J.F. edited the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Andrew J Fisher or Peter A Beal.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–8 and Supplementary Tables 1 and 2


  1. 1.

    RNA morph

    This file contains a morphing movie of the change in conformation of the RNA before and after hADAR2d binding (assuming an ideal A-form duplex RNA starting point).

  2. 2.

    hADAR2 morph

    This file contains a morphing movie of the change in hADAR2d conformation before and after RNA binding.

About this article

Publication history





Further reading