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Substrate-specific structural rearrangements of human Dicer

Nature Structural & Molecular Biology volume 20, pages 662–670 (2013)Cite this article

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Abstract

Dicer has a central role in RNA-interference pathways by cleaving double-stranded RNAs (dsRNAs) to produce small regulatory RNAs. Human Dicer can process long double-stranded and hairpin precursor RNAs to yield short interfering RNAs (siRNAs) and microRNAs (miRNAs), respectively. Previous studies have shown that pre-miRNAs are cleaved more rapidly than pre-siRNAs in vitro and are the predominant natural Dicer substrates. We have used EM and single-particle analysis of Dicer–RNA complexes to gain insight into the structural basis for human Dicer's substrate preference. Our studies show that Dicer traps pre-siRNAs in a nonproductive conformation, whereas interactions of Dicer with pre-miRNAs and dsRNA-binding proteins induce structural changes in the enzyme that enable productive substrate recognition in the central catalytic channel. These findings implicate RNA structure and cofactors in determining substrate recognition and processing efficiency by human Dicer.

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Figure 1: Domain architecture and cryo-EM structure of human Dicer.
Figure 2: A pre-siRNA spans human Dicer between the cap and branch while a pre-miRNA binds the platform of the enzyme.
Figure 3: RNA substrates induce structural rearrangements in human Dicer.
Figure 4: A domain reorganization of human Dicer in response to pre-miRNAs correlates with increased dicing efficiency.
Figure 5: dsRBPs promote conformational sampling of human Dicer for dsRNA substrate loading.
Figure 6: 3D reconstructions of major Dicer conformers show that branch position affects accessibility to the platform.
Figure 7: Model for small RNA processing by human Dicer.

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Acknowledgements

We thank W. Filipowicz (Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland) for antibodies to human Dicer; A. Giraldez (Department of Genetics, Yale University School of Medicine, New Haven, Connecticut, USA) for purified pre-miR-430; G. Lander, P. Grob and T. Houweling for expert EM and image-processing assistance; A. Brewster for help with creating the homology model of human Dicer; T. Albergo for help with particle picking; members of the Wang, Doudna, Nagayama and Nogales labs for helpful discussions; A. Giraldez, D. Cifuentes, A. Bazzini, S. Baserga, J. Steitz and members of the Giraldez and Baserga labs for discussion and expert technical assistance; H. Okawara and M. Ohara (Division of Nano-Structure Physiology, Okazaki Institute for Integrative Bioscience, Okazaki, Japan) for preparing the Zernike phase plates; and the Yale Center for Cellular and Molecular Imaging and Yale Center for High Performance Computation in Biology and Medicine. D.W.T. is supported as a US National Science Foundation (NSF) Graduate Research Fellow and as an NSF and Japan Society for the Promotion of Science East Asia and Pacific Summer Institute Fellow. This work was supported in part by US National Institutes of Health (NIH) molecular biophysics training grant 5 T32 GM008283 (D.W.T.), the Core Research for Evolutional Science and Technology of Japan Science and Technology Agency (K.N.), NIH 5R01GM073794 (J.A.D.), Human Frontiers in Science Program RPG0039/2008-C (E.N.), the Smith Family Awards Program for Excellence in Biomedical Research (H.-W.W.) and National Natural Science Foundation of China 31270765 (H.-W.W.). J.A.D. and E.N. are supported as Howard Hughes Medical Institute Investigators.

Author information

Author notes

  1. David W Taylor, Enbo Ma and Hideki Shigematsu: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, Connecticut, USA

    David W Taylor, Hideki Shigematsu & Hong-Wei Wang

  2. California Institute for Quantitative Biosciences, University of California, Berkeley, Berkeley, California, USA

    David W Taylor, Eva Nogales & Jennifer A Doudna

  3. Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California, USA

    Enbo Ma, Cameron L Noland, Eva Nogales & Jennifer A Doudna

  4. Biophysics Graduate Group, University of California, Berkeley, Berkeley, California, USA

    Michael A Cianfrocco

  5. Division of Nano-Structure Physiology, Okazaki Institute for Integrative Bioscience, Okazaki, Japan

    Kuniaki Nagayama

  6. National Institute for Physiological Sciences, National Institutes of Natural Sciences, Okazaki, Japan

    Kuniaki Nagayama

  7. Department of Physiological Sciences, Graduate University for Advanced Studies, Okazaki, Japan

    Kuniaki Nagayama

  8. Life Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA

    Eva Nogales

  9. Howard Hughes Medical Institute, University of California, Berkeley, Berkeley, California, USA

    Eva Nogales & Jennifer A Doudna

  10. Department of Chemistry, University of California, Berkeley, Berkeley, California, USA

    Jennifer A Doudna

  11. Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA

    Jennifer A Doudna

  12. Tsinghua-Peking Center for Life Sciences, Tsinghua University, Beijing, China

    Hong-Wei Wang

  13. Center for Structural Biology, School of Life Sciences, Tsinghua University, Beijing, China

    Hong-Wei Wang

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  1. David W Taylor
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Contributions

D.W.T. performed all E.M. and single-particle analysis. E.M. performed protein and RNA purification. H.S. performed ZPC-cryo-EM, supervised by K.N. M.A.C. and E.N. assisted with focused classification. C.L.N. purified Dicer–PACT complex. D.W.T., J.A.D. and H.-W.W. planned the experiments and wrote the manuscript. All authors analyzed and interpreted data and provided comments on the manuscript.

Corresponding authors

Correspondence to Jennifer A Doudna or Hong-Wei Wang.

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The authors declare no competing financial interests.

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Taylor, D., Ma, E., Shigematsu, H. et al. Substrate-specific structural rearrangements of human Dicer. Nat Struct Mol Biol 20, 662–670 (2013). https://doi.org/10.1038/nsmb.2564

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