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Structural insights into RNA cleavage by PIWI Argonaute

Nature volume 639, pages 250–259 (2025)Cite this article

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Abstract

Argonaute proteins are categorized into AGO and PIWI clades. Across most animal species, AGO-clade proteins are widely expressed in various cell types, and regulate normal gene expression1. By contrast, PIWI-clade proteins predominantly function during gametogenesis to suppress transposons and ensure fertility1,2. Both clades use nucleic acid guides for target recognition by means of base pairing, crucial for initiating target silencing, often through direct cleavage. AGO-clade proteins use a narrow channel to secure a tight guide–target interaction3. By contrast, PIWI proteins feature a wider channel that potentially allows mismatches during pairing, broadening target silencing capability4,5. However, the mechanism of PIWI-mediated target cleavage remains unclear. Here we demonstrate that after target binding, PIWI proteins undergo a conformational change from an ‘open’ state to a ‘locked’ state, facilitating base pairing and enhancing target cleavage efficiency. This transition involves narrowing of the binding channel and repositioning of the PIWI-interacting RNA–target duplex towards the MID-PIWI lobe, establishing extensive contacts for duplex stabilization. During this transition, we also identify an intermediate ‘comma-shaped’ conformation, which might recruit GTSF1, a known auxiliary protein that enhances PIWI cleavage activity6. GTSF1 facilitates the transition to the locked state by linking the PIWI domain to the RNA duplex, thereby expediting the conformational change critical for efficient target cleavage. These findings explain the molecular mechanisms underlying PIWI–PIWI-interacting RNA complex function in target RNA cleavage, providing insights into how dynamic conformational changes from PIWI proteins coordinate cofactors to safeguard gametogenesis.

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Fig. 1: Conformational dynamics of MILI ternary complexes during the transition to the locked state.
Fig. 2: Recognition of piRNA–target duplex clamped in the MILI ternary complex.
Fig. 3: Docking of target segment t9–t13 into the nucleic acid-binding channel.
Fig. 4: Cryo-EM structures identified an intermediate state during the open-to-locked conformational transition.
Fig. 5: Effect of the EmGTSF1–EfPiwi interface on target cleavage.

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Data availability

The cryo-EM density maps for MILI–piRNA–target (8 nt), MILI–piRNA–target (26 nt), MILIK852A–piRNA–target (26 nt), MILIK853A–piRNA–target (26 nt), EfPiwi–piRNA–target (25 nt, intermediate), MILI–piRNA–target (22 nt, intermediate), MILI–piRNA–target (22 nt, locked), EfPiwi–piRNA–target (25 nt, locked) have been deposited in the Electron Microscopy Data Bank (EMDB) under accession codes EMD-60611, EMD-60614, EMD-60615, EMD-60616, EMD-60610, EMD-60613, EMD-60612 and EMD-60609, respectively. Corresponding atomic models have been deposited in the Protein Data Bank (PDB) under accession IDs 9ij0, 9ij3, 9ij4, 9ij5, 9iiz, 9ij2, 9ij1 and 9iiy, respectively. The EfPiwi–piRNA–targets (25 nt) in the presence of EmGTSF1Q22A/R25A/K32A/K35A and EmGTSF1W100A/W109A have been deposited in the EMDB under accession numbers EMD-61765 and EMD-61764, respectively. Source data are provided with this paper.

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Acknowledgements

We thank C. C. Mello, P. D. Zamore, D. Li, H. Gao and C.-Qing Song for insightful suggestions; members of Shen and Wu laboratories for discussions; F. Xu at the Westlake University High-Throughput Core Facility for assistance in data collection; and the Cryo-EM Facility of Westlake University for providing support for cryo-EM data collection. This work was supported by Westlake Center for Genome Editing, programme no. 21200000A992210/004, Westlake Education Foundation, Zhejiang Provincial Foundation of China (grant no. 2021R01013), National Natural Science Foundation of China (grant no. NSFC32070628), Westlake Education Foundation (grant no. 041010140118), Zhejiang Provincial Key Laboratory Construction Project and the Westlake Laboratory of Life Sciences to E.-Z.S., the National Natural Science Foundation of China (grant no. 32271261), Zhejiang Provincial Natural Science Foundation of China (grant no. LR22C050003), Westlake University (grant no. 1011103860222B1) and an Institutional Startup grant from the Westlake Education Foundation (grant no. 101486021901) to J.W.

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Author notes

  1. These authors contributed equally: Zhiqing Li, Qikui Xu, Jing Zhong, Yan Zhang, Tianxiang Zhang, Xiaoze Ying

Authors and Affiliations

  1. Key Laboratory of Growth Regulation and Translational Research of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou, China

    Zhiqing Li, Jing Zhong, Yan Zhang, Tianxiang Zhang, Xiaoze Ying, Junchao Xue & En-Zhi Shen

  2. Westlake Laboratory of Life Sciences and Biomedicine, Hangzhou, China

    Zhiqing Li, Qikui Xu, Jing Zhong, Yan Zhang, Tianxiang Zhang, Xiaoze Ying, Xiaoyi Li, Li Wan, Junchao Xue, Jing Huang, Ying Zhen, Jianping Wu & En-Zhi Shen

  3. Institute of Biology, Westlake Institute for Advanced Study, Hangzhou, China

    Zhiqing Li, Qikui Xu, Jing Zhong, Yan Zhang, Tianxiang Zhang, Xiaoze Ying, Xiaoli Lu, Xiaoyi Li, Li Wan, Junchao Xue, Jing Huang, Ying Zhen, Jianping Wu & En-Zhi Shen

  4. Key Laboratory of Structural Biology of Zhejiang Province, School of Life Sciences, Westlake University, Hangzhou, China

    Qikui Xu, Xiaoli Lu, Xiaoyi Li, Li Wan, Jing Huang, Ying Zhen & Jianping Wu

  5. Duke University School of Medicine, Department of Pharmacology and Cancer Biology, Durham, NC, USA

    Zhao Zhang

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Contributions

E.-Z.S. and Z.L. conceived and designed the study. Z.L., J.Z., T.Z. and X.Y. performed protein expression and purification, mutagenesis studies, cleavage assays and target binding assays. Z.L., T.Z. and L.W. prepared grids and collected the cryo-EM data. Q.X. and J.W. calculated the cryo-EM maps and built atomic models. Y. Zhang and Z.L. refined the atomic models and analysed the cryo-EM data. Y. Zhang, X. Lu, J.H. and Z.L. predicted structures of protein complexes. X. Li and Y. Zhen contributed to protein sequence analysis. E.-Z.S. wrote the manuscript with contributions from all coauthors. Z.Z., J.W. Z.L. and J.X. reviewed and edited the manuscript. E.-Z.S. and J.W. supervised the project.

Corresponding authors

Correspondence to Jianping Wu or En-Zhi Shen.

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Extended data figures and tables

Extended Data Fig. 1 Conformational transition differences in MILI ternary complexes compared to hAGO2 and TtAGO ternary complexes.

a,b, Stereo views of ternary complexes of mouse PIWI Argonaute MILI and 26-nt guide RNA with target RNAs of 15 nt (PDB: 7yfy) (a, left) and 26 nt (full complementarity) (b, left), human AGO2 (hAGO2) and 21-nt guide RNA with target RNAs of 16 nt (PDB: 6n4o) (a, middle) and 21 nt (mismatches at position 10-11) (PDB: 6mdz) (b, middle), T. thermophilus AGO (TtAGO) and 21-nt guide DNA with target RNAs of 15 nt (PDB: 4n41) (a, right) and 19 nt (PDB: 4ncb) (b, right). c, Superposition of MILI ternary complexes with 15-nt-target and 26-nt-target conformations (c, left), hAGO2 ternary complexes with 16-nt-target and 21-nt-target conformations (c, middle), TtAGO ternary complexes with 15-nt-target and 19-nt-target conformations (c, right). d,e, Cross-sectional views of the MILI ternary complexes with 15-nt-target (d, left) and 26-nt-target conformations (e, left), hAGO2 ternary complexes with 16-nt-target (d, middle) and 21-nt-target conformations (e, middle), TtAGO ternary complexes with 15-nt-target (d, right) and 19-nt-target conformations (e, right). f, Superposition of cross-sectional views showing the rotation of PAZ-domain in MILI ternary complexes with 15-nt-target and 26-nt-target conformations (f, left), hAGO2 ternary complexes with 16-nt-target and 21-nt-target conformations (f, middle), TtAGO ternary complexes with 15-nt-target and 19-nt-target conformations (f, right). Arrows indicate major shifts of PAZ and N domain in two different conformations.

Extended Data Fig. 2 Structural comparison between MILI-guide-target complexes and hAGO2/TtAGO-guide-target complexes.

a, d, g, Conformational superposition of MILI-guide RNA complex binding target RNAs with complementarity to nucleotides g2-g15 and g2-g26 (a), hAGO2-guide RNA complex binding target RNAs with complementarity to nucleotides g2-g16 and g2-g21 (d), TtAGO-guide DNA complex binding target RNAs with complementarity to nucleotides g2-g15 and g2-g19 (g). b, e, h, Schematic of major contacts between the guide (red) and target (blue) and MILI in the g2-g15 base-paired conformation (b), and hAGO2 in the g2-g16 base-paired conformation (e), and TtAGO in the g2-g15 base-paired conformation (h). c, f, i, Schematic of major contacts between the guide (red) and target (blue) and MILI in the g2-g26 base-paired conformation (c), and hAGO2 in the g2-g21 base-paired (g10-g11 mispairing, colored by light blue) conformation (f), and TtAGO in the g2-g19 base-paired conformation (i). Residues colored by domain, as in Fig. 2. Disordered nucleotides labelled by dashed circle.

Extended Data Fig. 3 Biochemical determination of target cleavage by MILI mutants.

a-f, Fraction of 2-26 target RNA (10 nM) cleaved by wild-type (100 nM) or MILI mutants versus time. L1-domain (S379A, H380A, K381A) mutant (a), PAZ-domain (T424A, T499A) mutant (b), L1- and PAZ-domain (S379A, H380A, K381A,T424A, T499A) mutant (c), L1-hairpin (R339G, N340G) mutant (d), PIWI-cS7 (S816A, D817A, Q819A) mutant (e), both N-domain (K252A, D273A, S275A (termed as N1)) and (Q240A, H242A, T307A, K308A (termed as N2)) mutants (f). g, t10-t11 docking in the active site of MILIWT-piRNA-target (26 nt) complex (left) and MILIR853A-piRNA-target (26 nt) complex (right). Plotted data are represented as mean ± s.d. from independent triplicates.

Source Data

Extended Data Fig. 4 Cryo-EM Structural comparison of EfPiwi-piRNA-target (25 nt) and MILI-piRNA-target (26 nt) complexes in the “comma-shaped” and “locked” conformations.

a,b, Cryo-EM structures of EfPiwi-piRNA bound to target RNA (25 nt) (a) and MILI-piRNA complex bound to target RNA (26 nt) (b). c,d, Superposition of guide-target RNA duplex and adjacent PIWI-MID domain in the EfPiwi ternary complex with 25 nt (purple) in the “comma-shaped” conformation and in the MILI ternary complex with 26-nt target RNA (blue) in the “locking” conformation (c). Insert shows the conformational difference within catalytic pocket (d). Black arrow indicates the shift of the RNA duplex and the scissile phosphate from the “comma-shaped” to the “locked” ternary complexes, respectively.

Extended Data Fig. 5 The EfPiwi-piRNA-target RNA (25 nt) ternary complex undergoes the conformational transition from the “comma-shaped” to “locked” state after adding EmGTSF1WT.

a-f, Reconstructions of the EfPiwi-piRNA complex (a), EfPiwi-piRNA-target (16 nt) complex (b), EfPiwi-piRNA-target (25 nt) complex in the “comma-shaped” state (c) and in the “locked” state after the EmGTSF1WT addition (d), and still in the “comma-shaped” state after the EmGTSF1Q22A/R25A/K32A/K35A (e) or EmGTSF1W100A/W109A (f) addition.

Extended Data Fig. 6 AlphaFold structure prediction of the quaternary complex consisting of PIWI, piRNA, Target RNA, GTSF1 shows convergence of models.

a, AlphaFold 3 predicts a quaternary complex consisting of PIWI, piRNA-Target RNA duplex and GTSF1. AlphaFold EfPiwi complex: EfPiwi residues 220–987, piRNA-target RNA duplex 25 base-pairings and EmGTSF1 residues 1-129 (a, left); AlphaFold MILI complex: MILI residues 209-971, piRNA-target RNA duplex 26 base-pairings and MmGTSF1 residues 1-129 (a, right). EfPiwi in light brown and MILI in yellow, piRNA in red, target in blue and EmGTSF1 in green and MmGTSF1 in cyan. b,c, The best of the five predicted models is colored per chain (b) or per pLDDT score (c), which reports on the model confidence. Blue, yellow and orange indicate high, middle and low model confidence, respectively. d,e, Sequence alignment of GTSF1 protein from various species (d) and their corresponding PIWI proteins (e), colored boxes showing that the residues involved in GTSF1-PIWI interactions are conserved across species. Species abbreviations as below, Hs: Homo sapiens; Pt: Pan troglodytes; Mm: Mus musculus; Xt: Xenopus tropicalis; Dr: Danio rerio; Ef: Ephydatia fluviatilis; Dm: Drosophila melanogaster; Siwi is from silkworm.

Extended Data Fig. 7 Effect of the MmGTSF1-MILI interface on target cleavage.

a-c, AlphaFold3-predicted structure of a quaternary MILI-piRNA-target (26 nt)-GTSF1 complex. The best of five predicted models is shown as a cartoon in two different orientations (a, b) and surface representation (c). d-f, Residues involved in the interaction between MmGTSF1 and MILI-piRNA-target (26 nt) ternary complex are magnified and shown as a stick representation in (d), (e) and (f). g,i,k Target cleavage by MILI at the presence of wild-type and mutant MmGTSF1. These mutated positions are predicted to interact with MILI. Data are representative of three independent experiments. h, Target cleavage by the wild-type and mutant MILI in the presence of wild-type MmGTSF1. Data are representative of three independent experiments. j, Target cleavage by MILI in the presence of wild-type and two MmGTSF1 mutants (MmGTSF1E2A/D3A/T4A, MmGTSF1D43A/V44A/N46A), which are not predicted to interact with MILI. Data are representative of three independent experiments. l,m, Fraction of representative cleaved target by MILI in the absence and presence of wild-type MmGTSF1, where targets used for cleavage are complementary to nucleotides g2-g15, g2-g16, g2-g17, g2-g18, g2-g19, g2-g20, g2-g21, g2-g22, g2-g23, g2-g24, g2-g25 (l). Gel is representative of n = 3 independent experiments. Quantification of the in vitro cleavage assays (m). Data are represented as mean ± s.d. from independent triplicates.

Source Data

Extended Data Fig. 8 Target cleavage by EfPiwi at the presence of wild-type EmGTSF1 and mutants.

a,b, Fraction of representative cleaved target by EfPiwi in the presence of RNA binding mutant EmGTSF1Q22A/R25A/K32A/K35A (a), EmGTSF1V2A/E3A/S4A and EmGTSF1S39A/S40A/G41A/I42A (b), which are not predicted to interact with EfPiwi. Gel is representative of n = 3 independent experiments.

Extended Data Fig. 9 Comparison of overall structures.

a, Superposition of PIWI-clade Argonautes’ structures (HILI, PDB: 7yfx; MILI, PDB: 7ygn; EfPiwi, PDB: 7kx7; Siwi, PDB: 5guh; DmPiwi, PDB: 6kr6). b, Superposition of AGO-clade Argonautes’ structures (hAgo1, PDB: 4kxt; hAgo2, PDB: 4w5n; hAgo3, PDB: 5vm9; hAgo4, PDB: 6oon). c, Superposition of MILI (blue) and hAgo2 (green). d, Superposition of MILI (blue) and TtAgo (light brown) (PDB: 3dlh). Structural differences in their N and PAZ domains are indicated by red arrows.

Extended Data Table 1 Statistics for data collection and structural refinement
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Supplementary information

Supplementary Information

This file contains Supplementary Figs. 1–9, Table 1 and legends for the videos.

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Supplementary Video 1

Transition of protein components from MILI–piRNA–target (15 nt) (grey) to MILI–piRNA–target (26 nt) (blue) structures.

Supplementary Video 2

Transitions between Extended Data Fig. 1a and b.

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Li, Z., Xu, Q., Zhong, J. et al. Structural insights into RNA cleavage by PIWI Argonaute. Nature 639, 250–259 (2025). https://doi.org/10.1038/s41586-024-08438-1

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