Abstract
Targeted gene silencing by RNA interference (RNAi) requires loading of a short guide RNA (small interfering RNA (siRNA) or microRNA (miRNA)) onto an Argonaute protein to form the functional center of an RNA-induced silencing complex (RISC). In humans, Argonaute2 (AGO2) assembles with the guide RNA–generating enzyme Dicer and the RNA-binding protein TRBP to form a RISC-loading complex (RLC), which is necessary for efficient transfer of nascent siRNAs and miRNAs from Dicer to AGO2. Here, using single-particle EM analysis, we show that human Dicer has an L-shaped structure. The RLC Dicer's N-terminal DE×H/D domain, located in a short 'base branch', interacts with TRBP, whereas its C-terminal catalytic domains in the main body are proximal to AGO2. A model generated by docking the available atomic structures of Dicer and Argonaute homologs into the RLC reconstruction suggests a mechanism for siRNA transfer from Dicer to AGO2.
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References
Rana, T.M. Illuminating the silence: understanding the structure and function of small RNAs. Nat. Rev. Mol. Cell Biol. 8, 23–36 (2007).
Bernstein, E., Caudy, A.A., Hammond, S.M. & Hannon, G.J. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409, 363–366 (2001).
Chendrimada, T.P. et al. TRBP recruits the Dicer complex to Ago2 for microRNA processing and gene silencing. Nature 436, 740–744 (2005).
Rand, T.A., Petersen, S., Du, F.H. & Wang, X.D. Argonaute2 cleaves the anti-guide strand of siRNA during RISC activation. Cell 123, 621–629 (2005).
Matranga, C., Tomari, Y., Shin, C., Bartel, D.P. & Zamore, P.D. Passenger-strand cleavage facilitates assembly of siRNA into Ago2-containing RNAi enzyme complexes. Cell 123, 607–620 (2005).
Rivas, F.V. et al. Purified Argonaute2 and an siRNA form recombinant human RISC. Nat. Struct. Mol. Biol. 12, 340–349 (2005).
MacRae, I.J. et al. Structural basis for double-stranded RNA processing by dicer. Science 311, 195–198 (2006).
Song, J.J., Smith, S.K., Hannon, G.J. & Joshua-Tor, L. Crystal structure of Argonaute and its implications for RISC slicer activity. Science 305, 1434–1437 (2004).
Yuan, Y.R., Pei, Y., Chen, H.Y., Tuschl, T. & Patel, D.J. A potential protein-RNA recognition event along the RISC-loading pathway from the structure of A. aeolicus Argonaute with externally bound siRNA. Structure 14, 1557–1565 (2006.).
Yuan, Y.R. et al. Crystal structure of A. aeolicus argonaute, a site-specific DNA-guided endoribonuclease, provides insights into RISC-mediated mRNA cleavage. Mol. Cell 19, 405–419 (2005).
Wang, Y., Sheng, G., Juranek, S., Tuschl, T. & Patel, D.J. Structure of the guide-strand-containing argonaute silencing complex. Nature 456, 209–213 (2008).
Wang, Y. et al. Structure of an argonaute silencing complex with a seed-containing guide DNA and target RNA duplex. Nature 456, 921–926 (2008).
Radermacher, M., Wagenknecht, T., Verschoor, A. & Frank, J. Three-dimensional reconstruction from a single-exposure, random conical tilt series applied to the 50S ribosomal subunit of Escherichia coli. J. Microsc. 146, 113–136 (1987).
Penczek, P.A., Grassucci, R.A. & Frank, J. The ribosome at improved resolution: new techniques for merging and orientation refinement in 3D cryo-electron microscopy of biological particles. Ultramicroscopy 53, 251–270 (1994).
Ma, E., MacRae, I.J., Kirsch, J.F. & Doudna, J.A. Autoinhibition of human dicer by its internal helicase domain. J. Mol. Biol. 380, 237–243 (2008).
MacRae, I.J., Zhou, K. & Doudna, J.A. Structural determinants of RNA recognition and cleavage by Dicer. Nat. Struct. Mol. Biol. 14, 934–940 (2007).
Högbom, M. et al. Crystal structure of conserved domains 1 and 2 of the human DEAD-box helicase DDX3X in complex with the mononucleotide AMP. J. Mol. Biol. 372, 150–159 (2007).
MacRae, I.J., Ma, E., Zhou, M., Robinson, C.V. & Doudna, J.A. In vitro reconstitution of the human RISC-loading complex. Proc. Natl. Acad. Sci. USA 105, 512–517 (2008).
van Heel, M., Harauz, G., Orlova, E.V., Schmidt, R. & Schatz, M. A new generation of the IMAGIC image processing system. J. Struct. Biol. 116, 17–24 (1996).
Gao, H., Valle, M., Ehrenberg, M. & Frank, J. Dynamics of EF-G interaction with the ribosome explored by classification of a heterogeneous cryo-EM dataset. J. Struct. Biol. 147, 283–290 (2004).
Scheres, S.H. et al. Disentangling conformational states of macromolecules in 3D-EM through likelihood optimization. Nat. Methods 4, 27–29 (2007).
Kastner, B. et al. GraFix: sample preparation for single-particle electron cryomicroscopy. Nat. Methods 5, 53–55 (2008).
Herzog, F. et al. Structure of the anaphase-promoting complex/cyclosome interacting with a mitotic checkpoint complex. Science 323, 1477–1481 (2009).
Sasaki, T. & Shimizu, N. Evolutionary conservation of a unique amino acid sequence in human DICER protein essential for binding to Argonaute family proteins. Gene 396, 312–320 (2007).
Haase, A.D. et al. TRBP, a regulator of cellular PKR and HIV-1 virus expression, interacts with Dicer and functions in RNA silencing. EMBO Rep. 6, 961–967 (2005).
Kok, K.H., Ng, M.H.J., Ching, Y.P. & Jin, D.Y. Human TRBP and PACT directly interact with each other and associate with dicer to facilitate the production of small interfering RNA. J. Biol. Chem. 282, 17649–17657 (2007).
Schwarz, D.S. et al. Asymmetry in the assembly of the RNAi enzyme complex. Cell 115, 199–208 (2003).
Tomari, Y., Matranga, C., Haley, B., Martinez, N. & Zamore, P.D. A protein sensor for siRNA asymmetry. Science 306, 1377–1380 (2004).
Preall, J.B., He, Z., Gorra, J.M. & Sontheimer, E.J. Short interfering RNA strand selection is independent of dsRNA processing polarity during RNAi in Drosophila. Curr. Biol. 16, 530–535 (2006).
Tomari, Y., Du, T. & Zamore, P.D. Sorting of Drosophila small silencing RNAs. Cell 130, 299–308 (2007).
Lee, Y. et al. The role of PACT in the RNA silencing pathway. EMBO J. 25, 522–532 (2006).
Ludtke, S.J., Baldwin, P.R. & Chiu, W. EMAN: semiautomated software for high-resolution single-particle reconstructions. J. Struct. Biol. 128, 82–97 (1999).
Wriggers, W. & Birmanns, S. Using situs for flexible and rigid-body fitting of multiresolution single-molecule data. J. Struct. Biol. 133, 193–202 (2001).
Pettersen, E.F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).
Acknowledgements
We thank I. MacRae at the Scripps Institute for the purified Dicer and un–cross-linked RLC samples used in the initial stages of this work, P. Gabriel for help with particle picking, M. Jinek and S. Chakravarthy for help with sample preparation, members of the Nogales and Doudna laboratories for valuable insights and technical support, A. Fischer for tissue culture assistance and the Keck MacroLab and the Unger and Baserga laboratories at Yale University for the use of their resources. This work was supported in part by grants from the US National Institutes of Health (J.A.D.) and the Human Frontier Science Program (E.N.). J.A.D. and E.N. are Howard Hughes Medical Institute investigators.
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H.-W.W. planned the experiments, performed EM, analyzed and interpreted the data and wrote the paper; C.N. purified the proteins, prepared the cross-linked RLC specimen, performed EM and analyzed the data; B.S. prepared the specimen of un–cross-linked RLC, performed EM and analyzed the data; D.W.T. prepared the specimen of GraFix-prepared RLC, performed EM and analyzed the data; E.M. purified the proteins and reconstituted the RLC; K.F. prepared the specimen of human Dicer, performed EM and analyzed the data; J.A.D. planned the experiments, interpreted the data and wrote the paper; E.N. planned the experiments, analyzed and interpreted the data and wrote the paper.
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Supplementary Figures 1–9 and Supplementary Methods (PDF 1113 kb)
Supplementary Video 1
RLC reconstruction and Argonaute docking: reconstruction of the GraFix prepared RLC (solid gray isosurface that converts to a transparent gray isosurface). The Dicer reconstruction (gray wire map) is substracted to generate a difference map (transparent yellow isosurface). The atomic model of Argonaute (ribbon, color coding as in Figure 3b) is docked in the largest part of the difference map. (MOV 7958 kb)
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Wang, HW., Noland, C., Siridechadilok, B. et al. Structural insights into RNA processing by the human RISC-loading complex. Nat Struct Mol Biol 16, 1148–1153 (2009). https://doi.org/10.1038/nsmb.1673
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DOI: https://doi.org/10.1038/nsmb.1673
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