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Structure of the human DICER–pre-miRNA complex in a dicing state

Nature volume 615, pages 331–338 (2023)Cite this article

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Abstract

Dicer has a key role in small RNA biogenesis, processing double-stranded RNAs (dsRNAs)1,2. Human DICER (hDICER, also known as DICER1) is specialized for cleaving small hairpin structures such as precursor microRNAs (pre-miRNAs) and has limited activity towards long dsRNAs—unlike its homologues in lower eukaryotes and plants, which cleave long dsRNAs. Although the mechanism by which long dsRNAs are cleaved has been well documented, our understanding of pre-miRNA processing is incomplete because structures of hDICER in a catalytic state are lacking. Here we report the cryo-electron microscopy structure of hDICER bound to pre-miRNA in a dicing state and uncover the structural basis of pre-miRNA processing. hDICER undergoes large conformational changes to attain the active state. The helicase domain becomes flexible, which allows the binding of pre-miRNA to the catalytic valley. The double-stranded RNA-binding domain relocates and anchors pre-miRNA in a specific position through both sequence-independent and sequence-specific recognition of the newly identified ‘GYM motif’3. The DICER-specific PAZ helix is also reoriented to accommodate the RNA. Furthermore, our structure identifies a configuration of the 5′ end of pre-miRNA inserted into a basic pocket. In this pocket, a group of arginine residues recognize the 5′ terminal base (disfavouring guanine) and terminal monophosphate; this explains the specificity of hDICER and how it determines the cleavage site. We identify cancer-associated mutations in the 5′ pocket residues that impair miRNA biogenesis. Our study reveals how hDICER recognizes pre-miRNAs with stringent specificity and enables a mechanistic understanding of hDICER-related diseases.

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Fig. 1: Cryo-EM structure of hDICER in complex with a pre-miRNA in a dicing state.
Fig. 2: Sequence-specific and non-specific binding to RNA by the dsRBD and RIIID domains.
Fig. 3: The PAZ helix reorganizes to accommodate pre-miRNA in a dicing state.
Fig. 4: Cancer-associated mutations of the 5′ pocket affect miRNA biogenesis.
Fig. 5: Model of the structural transition and substrate recognition of hDICER during the pre-miRNA processing cycle.

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Data availability

The structural models and density maps have been deposited in the PDB under the accession codes 7XW3 (apo-hDICER) and 7XW2 (hDICER–pre-let-7a-1GYM), as listed in Extended Data Table 1. The raw images have been deposited in the Electron Microscopy Data Bank (EMDB) under the accession codes EMD-33490 (apo-hDICER) and EMD-33489 (hDICER–pre-let-7a-1GYM), as listed in Extended Data Table 1. Other structural models cited in this study for analysis (5ZAL, 5ZAK, 2EZ6, 7VG2, 7VG3, 4NGD, 4NHA and 4NH6) are also accessible through the PDB. The rescue data were deposited to the Gene Expression Omnibus (GSE215867).

Code availability

Custom analysis codes are available at https://github.com/haedongkim615/dicer_dicing_state_structure.

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Acknowledgements

We thank J.-S. Woo for the mammalian cell transfection protocol and Y.-G. Choi, S.-M. Ji, J. Yang, D.-E. Choi, S. Bang and E. Kim for technical assistance. This research was supported by the Institute for Basic Science from the Ministry of Science and ICT of Korea (IBS-R008-D1 to Y.-Y.L., H.K. and V.N.K.); BK21 research fellowships from the Ministry of Education of Korea (to Y.-Y.L. and H.K.); and the National Research Foundation of Korea (NRF-2018-Global Ph.D. Fellowship Program to Y.-Y.L. and NRF-2015-Global Ph.D. Fellowship Program to H.K). S.-H.R. acknowledges financial support from the Creative-Pioneering Researchers Program through Seoul National University, NRF grants (2019M3E5D6063871, 2019R1C1C1004598, 2020R1A5A1018081 and 2021M3A9I4021220) and the SUHF Foundation. Computing resources were used in the CMCI at SNU and the Global Science Experimental Data Hub Center (GSDC) at Korea Institute of Science and Technology Information (KISTI).

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  1. These authors contributed equally: Young-Yoon Lee, Hansol Lee, Haedong Kim

Authors and Affiliations

  1. Center for RNA Research, Institute for Basic Science (IBS), Seoul, Republic of Korea

    Young-Yoon Lee, Haedong Kim & V. Narry Kim

  2. School of Biological Sciences, Seoul National University, Seoul, Republic of Korea

    Young-Yoon Lee, Hansol Lee, Haedong Kim, V. Narry Kim & Soung-Hun Roh

  3. Institute of Molecular Biology and Genetics, Seoul National University, Seoul, Republic of Korea

    Hansol Lee & Soung-Hun Roh

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  1. Young-Yoon Lee
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Contributions

All of the authors conceived the project. V.N.K. and S.-H.R. collected financial support. Y.-Y.L. performed protein purification and cryo-EM sample preparation. Y.-Y.L. and H.K. performed biochemical and cellular experiments. H.L. and S.-H.R. performed structural studies. H.K. performed bioinformatic analyses.

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Correspondence to V. Narry Kim or Soung-Hun Roh.

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Extended data figures and tables

Extended Data Fig. 1 Purification of hDICER and the hDICER–RNA complex.

a, The sequence of pre-let-7a-1GYM used for structural determination. b, SDS–PAGE of wild-type and mutant hDICER proteins. c, Size-exclusion chromatography of purified proteins. d, In vitro processing of pre-let-7a-1 by purified hDICER. e, Size-exclusion chromatography of the hDICER–pre-let-7a-1GYM complex. f, SDS–PAGE of the hDICER–pre-let-7a-1GYM complex visualized by Coomassie blue staining. Protein concentration for each fraction was estimated by Bradford protein assay, and the same amount of protein was loaded for each fraction. g, Urea-PAGE of the hDICER–pre-let-7a-1GYM complex visualized by SYBR gold staining. RNA concentration for each fraction was estimated by absorbance at 260 nm, and the same amount of RNA was loaded for each fraction. For gel source data, see Supplementary Fig. 1.

Extended Data Fig. 2 Cryo-EM image processing procedure for apo-hDICER.

a, Overview of the image processing procedure (see Methods). b, Representative micrograph and 2D class averages of the apo-hDICER (scale bar, 50 nm). c, Gold-standard FSC at 0.143 of the apo-hDICER. d, Angular particle distribution heat map. e, Consensus map of apo-hDICER. Each domain is indicated in a different colour. f, Local-resolution analysis shown in rainbow scale. g, Domain organization of hDICER with colour code for each domain. Schematics for the apo state shows amino acid residues included (solid lines) or not included (dashed lines) in the model. h, Atomic model fitting to the map of apo-hDICER.

Extended Data Fig. 3 Cryo-EM image processing procedure for hDICER–pre-let-7a-1GYM.

a, Overview of the image processing procedure (see Methods). b, Representative micrograph and 2D class averages of hDICER–pre-let-7a-1GYM (scale bar, 50 nm). c, Gold-standard FSC at 0.143 of the hDICER–pre-let-7a-1GYM. d, Angular particle distribution heat map. e, Consensus map of hDICER–pre-let-7a-1GYM. Each domain is indicated in a different colour. f, Local-resolution analysis shown in rainbow scale. g, Domain organization of hDICER with colour code for each domain. Schematics for the dicing state shows amino acid residues included (solid lines) or not included (dashed lines) in the model. h, Sequence of pre-let-7a-1GYM in the model. i, Atomic model fitting to the map of hDICER–pre-let-7a-1GYM.

Extended Data Fig. 4 Overall structure of hDICER in a dicing state.

a, Cryo-EM map of the catalytic site created by RIIIDa. b, Cryo-EM map of the catalytic site created by RIIIDb. c, B-factor and Q-score plots for active site residues in the hDICER–let-7a-1GYM complex structure. Q-scores for each residue were derived from MapQ of Segger tool plugged in Chimera v.1.15. B-factor values were derived from real space refinement in Phenix ISOLDE v.1.1.0. d, Superposition of RIIID domains of hDICER (this study) and Aa RNase III (PDB: 2EZ6, grey)30. e, Active sites of hDICER and Aa RNase III (PDB:2EZ6, grey)30. f, Buried surface area of hDICER in a pre-dicing state (PDB: 5ZAL)24 and a dicing state. g, RMSD of hDICER–pre-let-7a-1GYM (this study) compared to hDICER–TRBP-pre-let-7a-1mutant (PDB: 5ZAL)24. Residues not resolved in the dicing state are coloured in grey.

Extended Data Fig. 5 The structure of the helicase domain.

a, Interdomain interactions in apo-hDICER. b, Steric clash between pre-let-7a-1GYM and apo-hDICER. c, Cryo-EM map of the hDICER–pre-miR-3121GYM complex in a dicing state. d, Selected 2D class averages and 3D maps showing heterogeneity in the helicase domain. White arrowhead indicates the location of the helicase domain in 2D averages. Bound RNA density is indicated in orange. e, Urea-PAGE of hDICER–pre-let-7a-1GYM complex incubated with or without MgCl2 for 10 min at room temperature, visualized by SYBR gold staining. For gel source data, see Supplementary Fig. 1. f, Selected 2D class averages and 3D maps showing heterogeneity of the helicase domain of hDICER–pre-let-7a-1GYM complex.

Extended Data Fig. 6 The structure of dsRBD in different states.

a, Conformational changes of the dsRBD in the apo (this study), dicing (this study) and pre-dicing states (PDB: 5ZAL)24. b, Superposition of the dsRBDs of hDICER and AtDCL3 (PDB: 7VG2)9. c, Surface charge of the dsRBD, with the dsRNA–dsRBD interface in dicing and pre-dicing states (PDB: 5ZAL)24. d, Cryo-EM map and model of the hDICER dsRBD with dsRNA.

Extended Data Fig. 7 The PAZ helix rearranges to interact with pre-miRNA in a cleavage-competent position.

a, Superposition of hDICER PAZ–platform domain in the cryo-EM structure (this study) and in the crystal structure (PDB: 4NHA, grey)20. b, Changes in the position of the pre-miRNA in a dicing state (this study) and a pre-dicing state (PDB: 5ZAL)24. c, In vitro processing of pre-let-7a-1 with a 2-nt 3′ overhang. *, radiolabelled 5′ phosphate. d, In vitro processing of pre-let-7a-1 with a 1-nt 3′ overhang (lanes 1–5) or a 3-nt 3′ overhang (lanes 6–10). Relative cleavage was calculated by quantifying the band intensity (1 − uncleaved/input). Squares indicate mean (n = 2, independent experiments). *, radiolabelled 5′ phosphate. For gel source data, see Supplementary Fig. 1.

Extended Data Fig. 8 End recognition mechanism of hDICER.

a, Cryo-EM map of the 5′ pocket. b, Cryo-EM map of the 3′ pocket. c, Superposition of hDICER–pre-let-7a-1GYM and Platform–PAZ–Connector Helix (PDB: 4NHA, 4NGD and 4NH6)20. d, Superposition of dsRNAs complexed with hDICER and AtDCL3 (PDB: 7VG2)9. e, Close-up view of the ends of the dsRNAs complexed with hDICER and AtDCL3 (PDB: 7VG2, grey)9. f, Superposition of hDICER in a dicing state and AtDCL3-pre-siRNA complex (PDB: 7VG3, magenta)9. g, 5′ terminal base recognition by AtDCL3 via PAZ region corresponding to the PAZ helix of hDICER. h, In vitro DICER processing of pre-miRNA-like duplex with a 2-nt or 3-nt 3′ overhang. The base opposite to the varying sequence is A on the 3p strand. For gel source data, see Supplementary Fig. 1. *, radiolabelled 5′ phosphate. i, Schematic outline of the rescue experiment (n = 2, biological replicates). j, Changes in cleavage accuracy, estimated with the fold change of the proportion of the major 5′-isomiR. For a given miRNA, the most abundant 5′-isomiR was identified in the wild-type sample. Grey, unannotated strand. k, DROSHA/DICER cleavage sites dictated by 5′ ends of mature miRNAs.

Extended Data Fig. 9 Rescue experiments with DICER 5′ pocket mutants.

a, Predicted structural effect of the 5′ end base substitutions on the interaction with the 5′ pocket. b, Examples of altered processing sites observed in the rescue experiments. Note that the DICER cleavage sites can be inferred from the 5′ end of 3p miRNAs. miRNA isoforms beginning at the indicated position are plotted with circles, with the size of the circle reflecting the proportion of the cleavage-site usage at the given position.

Extended Data Fig. 10 Distinct functions and evolution of Dicer proteins in two small RNA pathways.

a, Comparison of the substrate RNA movement during DICER processing between two small RNA pathways. In the miRNA pathway, a hairpin-shaped small RNA (pre-miRNA) is bound to DICER by the helicase and PAZ domains. For cleavage, the helicase domain becomes flexible to accommodate the pre-miRNA into the catalytic centre. By contrast, in the siRNA pathway, a long dsRNA comes into DICER by passing through the helicase domain. The ATP-dependent translocation by the helicase domain leads to processive cleavage of long dsRNAs. b, A phylogenetic tree of Dicer homologues. The scale bar indicates the length for the indicated frequency of amino acid variation.

Extended Data Table 1 Cryo-EM data collection, refinement and validation statistics
Full size table

Supplementary information

Supplementary Figure 1

Uncropped gels.

Reporting Summary

Supplementary Table 1

DICER rescue experiment in HCT116 cells. Read counts, spike-in-normalized abundances and the proportions of the main 5′-isomiR identified in the WT samples of rescued HCT116 cells.

Peer Review File

Supplementary Video 1

Stem recognition by dsRBD and RIIID. The C-terminal dsRBD of DICER shows a large conformational change to accommodate dsRNA in the catalytic valley. Near the cleavage sites, this major groove of the RNA helix is expanded and sandwiched between dsRBD and RIIIDa. The mismatch of the GYM motif is recognized by R1855 of dsRBD.

Supplementary Video 2

PAZ helix reorients to accommodate RNA in a dicing state. In a closed conformation, the PAZ helix sterically prevents the substrate from accessing the RNase III domains. The PAZ helix reorients to allow a simultaneous recognition of the 5′ and 3′ termini. Positively charged residues in the PAZ helix interact with the negatively charged dsRNA backbone.

Supplementary Video 3

Model of pre-miRNA processing cycle by hDICER. Pre-miRNA binds to DICER, initially in a closed conformation where the helicase domain, the dsRBD and the PAZ helix sterically prevent the substrate from accessing the RNase III domains. From this closed form, DICER undergoes a large conformational change in the C-terminal dsRBD, the PAZ helix, and the helicase domain to allow the substrate to bind to the RNase III domains for cleavage.

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Lee, YY., Lee, H., Kim, H. et al. Structure of the human DICER–pre-miRNA complex in a dicing state. Nature 615, 331–338 (2023). https://doi.org/10.1038/s41586-023-05723-3

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