Abstract
MicroRNAs (miRNAs) are short non-coding RNAs that inhibit the expression of target genes by directly binding to their mRNAs. In animals, pri-miRNAs are cleaved by Drosha to generate pre-miRNAs, which are subsequently cleaved by Dicer to generate mature miRNAs. Instead of being cleaved by two different enzymes, both cleavages in plants are performed by Dicer-like 1 (DCL1). With a similar domain architecture as human Dicer, it is mysterious how DCL1 recognizes pri-miRNAs and performs two cleavages sequentially. Here, we report the single-particle cryo-electron microscopy structures of Arabidopsis DCL1 complexed with a pri-miRNA and a pre-miRNA, respectively, in cleavage-competent states. These structures uncover the plasticity of the PAZ domain, which is critical for the recognition of both pri-miRNA and pre-miRNA. These structures suggest that the helicase module serves as an engine that transfers the substrate between two sequential cleavage events. This study lays a foundation for dissecting the regulation mechanism of miRNA biogenesis in plants and provides insights into the dicing state of human Dicer.
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Data availability
The accession codes for the cryo-EM density maps reported in this paper are Electron Microscopy Data Bank: EMD-31181 (https://www.emdataresource.org/EMD-31181) (DCL1–pri-miRNA complex) and EMD-31182 (https://www.emdataresource.org/EMD-31182) (DCL1–pre-miRNA complex). The accession codes for the atomic coordinates reported in this paper are Protein Data Bank: 7ELD (https://doi.org/10.2210/pdb7ELD/pdb) (DCL1–pri-miRNA complex) and 7ELE (https://doi.org/10.2210/pdb7ELE/pdb) (DCL1–pre-miRNA complex). The accession codes for the atomic coordinates used in this study are PDB 4NHA (https://doi.org/10.2210/pdb4NHA/pdb), PDB 5ZAK (https://doi.org/10.2210/pdb5ZAK/pdb), PDB 5ZAL (https://doi.org/10.2210/pdb5ZAL/pdb), PDB 6BU9 (https://doi.org/10.2210/pdb6BU9/pdb) and PDB 6LXD (https://doi.org/10.2210/pdb6LXD/pdb). Source data are provided with this paper. Other data are available from the corresponding author on reasonable request..
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Acknowledgements
We thank S. Chang at the Center of Cryo Electron Microscopy in Zhejiang University School of Medicine for help with cryo-EM data collection. We are thankful for technical support by the Core Facilities, Zhejiang University School of Medicine. This work was funded by National Natural Science Foundation of China (grant nos. 31970040 to Y.F. and 32000025 to J.S.) and Natural Science Foundation of Zhejiang Province (grant no. LR21C010002 to Y.F.).
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X.W., H.K., A.W., B.G. and J.S. performed the experiments. Y.F. supervised the experiments. All authors contributed to the analysis of the data and the interpretation of the results. Y.F. wrote the manuscript with contributions from the other authors.
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Peer review information Nature Plants thanks Seong Wook Yang, Peng Zhang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Extended data
Extended Data Fig. 1 Purification and characterization of Arabidopsis DCL1.
a, SDS–PAGE of full-length (FL) and nuclear localization signal truncated (ΔNLS) DCL1. Experiments were repeated independently three times with similar results. b, Electrophoretic mobility shift assay shows that Arabidopsis DCL1 binds to pri-miRNA 166f. The assay is performed in 20 μL reaction mixtures containing 1 μM DCL1, 1 μM pri-miRNA, 20 mM Tris-HCl, pH 7.0, 50 mM NaCl and 2 mM DTT. Experiments were repeated independently three times with similar results. c, Arabidopsis DCL1 cuts pri-miRNA 166f efficiently. The positions of the substrate and cleavage products are indicated. Bottom, schematic illustration of the final cleavage products of pri-miRNA 166f. Cleavage sites are indicated by black triangles. Experiments were repeated independently three times with similar results.
Extended Data Fig. 2
Data processing pipeline for the dataset of DCL1–pri-miRNA complex.
Extended Data Fig. 3 Data validation for DCL1–pri-miRNA complex.
a, A representative micrograph of DCL1–pri-miRNA complex. 4,759 micrographs were collected with similar results. b, Typical 2D class averages of DCL1–pri-miRNA complex. 100 classes were obtained with similar results. c, The gold-standard FSC of DCL1–pri-miRNA complex. The gold-standard FSC is calculated by comparing the two independently determined half-maps from RELION. The dashed line represents the 0.143 FSC cutoff. d, Cryo-EM density map coloured by local resolution. View orientations as in Fig. 1c. e, Angular distribution of particle projections. View orientations as in Fig. 1c.
Extended Data Fig. 4
Data processing pipeline for the dataset of DCL1–pre-miRNA complex.
Extended Data Fig. 5 Data validation for DCL1–pre-miRNA complex.
a, A representative micrograph of DCL1–pre-miRNA complex. 2,145 micrographs were collected with similar results. b, Typical 2D class averages of DCL1–pre-miRNA complex. 100 classes were obtained with similar results. c, The gold-standard FSC of DCL1–pre-miRNA complex. The gold-standard FSC is calculated by comparing the two independently determined half-maps from RELION. The dashed line represents the 0.143 FSC cutoff. d, Cryo-EM density map coloured by local resolution. View orientations as in Fig. 1d. e, Angular distribution of particle projections. View orientations as in Fig. 1d.
Extended Data Fig. 6 Sequence alignment of the RNase III domains.
At, Arabidopsis thaliana; Hs, Homo sapiens; Dm, Drosophila melanogaster; Gi, Giardia intestinalis; Aa, Aquifex aeolicus. The sequences were aligned using Clustal Omega and the figure was prepared using ESPript 3.0. Black triangles indicate the conserved catalytic residues.
Extended Data Fig. 7 Representative gel images of in vitro cleavage assay and electrophoretic mobility shift assay.
a, Representative gel images of in vitro cleavage assay with pri-miRNA. Experiments were repeated independently three times with similar results. b, Representative gel images of in vitro cleavage assay with pre-miRNA. Experiments were repeated independently three times with similar results. c, Representative gel images of electrophoretic mobility shift assay with pri-miRNA. Experiments were repeated independently three times with similar results. d, Representative gel images of electrophoretic mobility shift assay with pre-miRNA. Experiments were repeated independently three times with similar results.
Extended Data Fig. 8 Sequence alignment of DCL1 PAZ domains.
At, Arabidopsis thaliana; Os, Oryza sativa; Tu, Triticum urartu; Zm, Zea mays; Mp, Mucuna pruriens; Na, Nicotiana attenuate; Sl, Solanum lycopersicum; Ht, Helianthus tuberosus; Sm, Salvia miltiorrhiza; Dc, Dendrobium catenatum; Pt, Pinus tabuliformis. The sequences were aligned using Clustal Omega and the figure was prepared using ESPript 3.0. Black triangles indicate the conserved residues of the internal loop binding groove.
Extended Data Fig. 9 Sequence alignment of the RIG-I helicase module.
At, Arabidopsis thaliana; Hs, Homo sapiens; Dm, Drosophila melanogaster; Ap, Anas platyrhynchos. The sequences were aligned using Clustal Omega and the figure was prepared using ESPript 3.0. Black triangles indicate the conserved Walker A and Walker B residues.
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Supplementary Tables 1 and 2.
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Statistical source data.
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Statistical source data.
Source Data Extended Data Fig. 1
Unprocessed gels.
Source Data Extended Data Fig. 7
Unprocessed gels.
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Wei, X., Ke, H., Wen, A. et al. Structural basis of microRNA processing by Dicer-like 1. Nat. Plants 7, 1389–1396 (2021). https://doi.org/10.1038/s41477-021-01000-1
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DOI: https://doi.org/10.1038/s41477-021-01000-1
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