Abstract
Small RNAs, including microRNA, small interfering RNA (siRNA) and PIWI-interacting RNA, are regulatory RNAs that play critical roles in gene regulation, development, viral defence and environmental response1. The biogenesis of microRNA and siRNA relies on the Dicer family ribonucleases to capture, measure and cleave their double-stranded RNA substrates2,3. In Arabidopsis, DICER-LIKE 4 (DCL4) produces 21-nucleotide siRNA in association with Double-Stranded RNA-Binding Protein 4 (DRB4) for post-transcriptional gene silencing4,5,6,7,8,9,10,11. Here we determined the structures of the DCL4–RNA complex in a dicing-competent conformation and the DCL4–DRB4–RNA complex in a pre-dicing conformation. DCL4 measures 21 nucleotides along RNA between its PAZ and RNase III domains to determine the product siRNA length. A DCL4-specific loop locates the second double-stranded RNA binding domain of DCL4 and DRB4 to a distal position of the substrate RNA, yielding a preference for long RNA substrates. Our studies demonstrate the molecular basis of substrate recognition, length measurement and long RNA preference by the DCL4–DRB4 complex for 21-nucleotide siRNA biogenesis in plants.
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Data availability
The structures have been deposited in the PDB with the accession codes 9KBY and 9KBZ. The cryo-EM maps have been deposited in the Electron Microscopy Data Bank with accession codes EMD-62234 and EMD-62235. Source data are provided with this paper.
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Acknowledgements
We thank the staff members at SUSTech Cryo-EM Center and PKU-IAAS Cryo-EM Center for cryo-EM data collection. This work was supported by the National Natural Science Foundation of China (grant nos 92581101 to J.D., 32325008 to J.D., 32261160572 to H.G. and 32301080 to Q.W.), the Guangdong Basic and Applied Basic Research Foundation (grant no. 2023B1515120048 to J.D.), the Shenzhen Science and Technology Program (grant nos RCJC20221008092720004 to J.D., 20231120201445001 to J.D. and ZDSYS20230626091659010 to H.G. and J.D.) and the New Cornerstone Science Foundation (grant no. NCI202235 to H.G.). J.D. is an investigator at the SUSTech Institute for Biological Electron Microscopy.
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C.W., C.C., J. Zhao and Q.W. performed the structural studies and biochemical assays. N.W., Z.Z., K.J., Y. Li and Y.X. contributed to data analysis and discussion. Y. Liu, P.W., J. Zhai and H.G. provided functional insights. J.D. supervised the project and wrote the manuscript.
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Extended data
Extended Data Fig. 1 Protein purification and in vitro activity assay.
a, The purification of Arabidopsis DCL4 (left) and DCL4-DRB4 complex (right). b, In vitro dicing assay of DCL4 and DCL4-DRB4 complex showing that DCL4-DRB4 has a stronger activity than DCL4 alone. The in vitro assay was repeated at least three times independently with similar results.
Extended Data Fig. 2 Cryo-EM image processing workflow of DCL4-hairpin RNA complex.
a, A representative cryo-EM image of DCL4-hairpin RNA complex. b, Flowchart of cryo-EM data processing. c, Typical 2D class averages of DCL4-dsRNA complex. d, Gold-standard Fourier shell correlation (GSFSC) of the final map of DCL4-hairpin RNA complex in dicing state. e, Angular distribution heat map calculated in cryoSPARC for particle projections contributing to the local resolution of DCL4-hairpin RNA complex.
Extended Data Fig. 3 The comparison of DCL4 and DCL3 reveals potential molecular mechanisms for measuring RNA length by Dicers.
a, Superimpositions of DCL4 (colored) and DCL3 (PDB code: 7VG2, in silver) based on the positions of their RIIIa/b domains. The directions of the conformational changes of platform-PAZ-connector cassette around the RNA duplex from DCL4 to DCL3 are shown by arrows. The red and black boxes denote the guide strand 5′-end and complementary strand 3′-end recognition regions, which are detailed in panel b-c and d-e, respectively. b-c, Recognition of the guide strand 5′-end first nucleotides by DCL4 (b) and DCL3 (c). Interacting residues and hydrogen bonds are highlighted in sticks and dashed lines, respectively. d-e, Recognition of 3′-end of the complementary strand by DCL4 (d) and DCL3 (e). f, The dsRBD1 domain uses a positively charged surface to bind to the RNA backbone. g, Structural comparison of DCL4-DRB4-RNA complex (colored) and DCL3-RNA complex (PDB code: 7VG2, in sliver) shows the different orientations of their second dsRBDs.
Extended Data Fig. 4 Multiple sequence alignment of the Platform-PAZ domains of DCLs.
Structure-based sequence alignment of the Platform-PAZ domains of DCL4 from multiple species, along with DCL1, DCL2, and DCL3. The key residues involved in the recognition of the 5′-end of the guide strand RNA, including the charged interactions with the phosphate group and the stacking interaction with the first base, are all conserved in DCL4 and are highlighted by red triangles. The secondary structure diagram of DCL4 is shown on top.
Extended Data Fig. 5 Multiple sequence alignment of the RNase IIIa, and RNase IIIb of DCLs.
Structure-based sequence alignment of DCL4 from multiple species, along with DCL1, DCL2, and DCL3, reveals that the R1135, Y1339, and S1343 of Arabidopsis DCL4, which are important residues of the RIII domains involved in hydrogen bonding interaction with the LinkerRBDs, are conserved in the DCL4 proteins. The secondary structure diagram of DCL4 is shown on top.
Extended Data Fig. 6 Multiple sequence alignment of the dsRBDs and LinkerRBDs of DCLs.
Structure-based sequence alignment of DCL4 from multiple species, along with DCL1, DCL2, and DCL3, reveals that only the DCL4 clade DCL proteins have the LinkerRBDs long enough to extend dsRBD2 to a distal position on RNA. The secondary structure diagram for DCL4 is shown on top.
Extended Data Fig. 7 Cryo-EM image processing workflow of DCL4-DRB4CTD-dsRNA complex.
a, A representative cryo-EM image of DCL4-DRB4CTD-dsRNA complex. b, Flowchart of cryo-EM data processing. c, 2D class averages. d, Gold-standard Fourier shell correlation (GSFSC) of the final map. e, Angular distribution heat map calculated in cryoSPARC for particle projections contributing to the local resolution. f, Cryo-EM maps showing the fitting of the representative protein and RNA regions.
Extended Data Fig. 8 A working model for the long RNA processing and 21-nt siRNA biogenesis by the DCL4-DRB4 complex.
a, A proposed model illustrating DRB4’s role in assisting DCL4 with long dsRNA processing. The DRB4CTD-DCL4dsRBD2 interaction may position the DRB4dsRBD1/2 (modeled from PDB: 2N3F) to a distal position of the dsRNA, achieving the long RNA substrate preference of the DCL4-DRB4 complex. b, A working model of the long RNA processing by the DCL4-DRB4 complex to produce phasiRNA by continuously dicing. The red arrow and stars indicate the direction of RNA translocation and the cleavage site, respectively. The DRB4dsRBD1/2 may help DCL4 search and capture long RNA substrates. Once the RNA is bound by the DCL4-DRB4 complex, DCL4 helicase domain could pump the RNA towards the platform-PAZ domain cassette, resulting in RNA bending. Upon the platform-PAZ domain cassette capturing the RNA termini, the pumping may push the RNA to further bend to approach the catalytic center of DCL4 RIIIs for cleavage. After cleavage, the 21-nt siRNA duplex is released, and the remaining RNA fragment, which is held by the helicase domain, could further be pumped by the helicase to start the next dicing cycle.
Extended Data Fig. 9 In vitro dicing assay of DCL4 and DCL4-DRB4 complex towards RNA substrates with different lengths.
a, The sequences of three RNA duplexes used in this assay. b, In vitro dicing assay of DCL4 and DCL4-DRB4 complex to process RNAs with different lengths (30/32-nt, 55/57-nt, and 107/109-nt). The schematic view of the RNA length is shown on the right. The cutting sites on RNA are denoted by triangles. The in vitro assay was repeated at least three times independently with similar results.
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Supplementary Table 1.
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Unmodified gels for the three repeats of Fig. 1h.
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Unmodified gels for Extended Data Fig. 1a and four repeats of Extended Data Fig. 1b.
Source Data Extended Data Fig. 9b (download PDF )
Unmodified gels for the three repeats of Extended Data Fig. 9b.
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Wang, C., Chi, C., Liu, Y. et al. Molecular basis of DRB4-assisted long RNA processing and 21-nucleotide siRNA biogenesis by DCL4 in plants. Nat. Plants 12, 512–519 (2026). https://doi.org/10.1038/s41477-026-02236-5
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DOI: https://doi.org/10.1038/s41477-026-02236-5
