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Mechanistic insights into RNA cleavage by human Argonaute2–siRNA complex

Cell Research volume 35, pages 453–464 (2025)Cite this article

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Abstract

In animals, AGO-clade Argonaute proteins utilize small interfering RNAs (siRNAs) as guides to recognize target with complete complementarity, resulting in target RNA cleavage that is a critical step for target silencing. These proteins feature a constricted nucleic acid-binding channel that limits base pairing between the guide and target beyond the seed region. How the AGO–siRNA complexes overcome this structural limitation and achieve efficient target cleavage remains unclear. We performed cryo-electron microscopy of human AGO–siRNA complexes bound to target RNAs of increasing lengths to examine the conformational changes associated with target recognition and cleavage. Initially, conformational transition propagates from the opening of the PAZ domain and extends through a repositioning of the PIWI–L1–N domain toward the binding channel, facilitating the capture of siRNA–target duplex. Subsequent extension of base pairing drives the downward movement of the PIWI–L1–N domain to enable catalytic activation. Finally, further base pairing toward the 3′ end of siRNA destabilizes the PAZ–N domain, resulting in a “uni-lobed” architecture, which might facilitate the multi-turnover action of the AGO–siRNA enzyme complex. In contrast to PIWI-clade Argonautes, the “uni-lobed” structure of the AGO complex makes multiple contacts with the target in the central region of the siRNA–target duplex, positioning it within the catalytic site. Our findings shed light on the stepwise mechanisms by which the AGO–siRNA complex executes target RNA cleavage and offer insights into the distinct operational modalities of AGO and PIWI proteins in achieving such cleavage.

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Fig. 1: Conformational dynamics of hAGO2 ternary complexes as complementarity extended towards the siRNA 3ʹ end.
Fig. 2: Structure of hAGO2–siRNA complex with complementary target of 12-nt.
Fig. 3: Structure of hAGO2–siRNA complex with complementary target of 14-nt.
Fig. 4: PAZ–N loop interaction is required to stimulate efficient slicing by extensive base pairing.
Fig. 5: Catalytic conformation of hAGO2–siRNA complex.
Fig. 6: Contacts in the central region are required to activate target cleavage.
Fig. 7: Structural comparison between MILI–piRNA–target (22-nt), EfPiwi–piRNA–target (25-nt) and hAGO2–siRNA–target (21-nt).

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Acknowledgements

We thank Craig C. Mello, Phillip D. Zamore, Dangsheng Li for insightful suggestions; members of Shen’s lab for discussions; Cryo-EM Facility of Westlake University for providing support on cryo-EM data collection. This work was supported by Westlake Education Foundation, Zhejiang Provincial Foundation of China (2021R01013), the National Natural Science Foundation of China (32070628), Westlake Education Foundation (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 (32271261), Zhejiang Provincial Natural Science Foundation of China (LR22C050003), Westlake University (1011103860222B1), and Institutional Startup Grant from the Westlake Education Foundation (101486021901) to J.W.

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  1. These authors contributed equally: Zhenzhen Li, Qikui Xu, Yan Zhang.

Authors and Affiliations

  1. Fudan University, Shanghai, China

    Zhenzhen Li & Qikui Xu

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

    Zhenzhen Li, Yan Zhang, Jing Zhong, Tianxiang Zhang, Junchao Xue, Shuxian Liu & En-Zhi Shen

  3. Westlake Laboratory of Life Sciences and Biomedicine, Hangzhou, Zhejiang, China

    Zhenzhen Li, Qikui Xu, Yan Zhang, Jing Zhong, Tianxiang Zhang, Junchao Xue, Shuxian Liu, Haishan Gao, Jianping Wu & En-Zhi Shen

  4. Institute of Biology, Westlake Institute for Advanced Study, Hangzhou, Zhejiang, China

    Zhenzhen Li, Qikui Xu, Yan Zhang, Jing Zhong, Tianxiang Zhang, Junchao Xue, Shuxian Liu, Haishan Gao, Jianping Wu & En-Zhi Shen

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

    Qikui Xu & Jianping Wu

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

    Z. Z. Zhao Zhang

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Contributions

E.Z.S. conceived and designed the study. Z.L., Y.Z., Q.X., J.Z., T.Z., J.X. and S.L. performed experiments and analyzed the data. E.Z.S. wrote the manuscript with help from all authors; H.G., Z.Z., J.W. and E.Z.S. reviewed and edited the manuscript. E.Z.S. and J.W. supervised the project.

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Correspondence to Jianping Wu or En-Zhi Shen.

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41422_2025_1114_MOESM14_ESM.mp4

Supplementary Video S1. Overview of cryo-EM density and the structural model of hAGO2D669A-siRNA-target (12-nt). The map and model are colored as in Fig. 1.

41422_2025_1114_MOESM15_ESM.mp4

Supplementary Video S2. Overview of cryo-EM density and the structural model of hAGO2D669A-siRNA-target (14-nt, sesqui-lobed).

41422_2025_1114_MOESM16_ESM.mp4

Supplementary Video S3. Overview of cryo-EM density and the structural model of hAGO2D669A-siRNA-target (14-nt, uni-lobed).

Supplementary Video S4. Overview of cryo-EM density and the structural model of hAGO2D669A-siRNA-target (19-nt).

Supplementary Video S5. Overview of cryo-EM density and the structural model of hAGO2D669A-siRNA-target (21-nt).

41422_2025_1114_MOESM19_ESM.mp4

Supplementary Video S6. Conformational dynamics of hAGO2 ternary complexes as complementarity extended towards the siRNA 3ʹ end.

41422_2025_1114_MOESM20_ESM.mp4

Supplementary Video S7. Conformational dynamics of MILI ternary complexes as complementarity extended towards the piRNA 3ʹ end.

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Li, Z., Xu, Q., Zhang, Y. et al. Mechanistic insights into RNA cleavage by human Argonaute2–siRNA complex. Cell Res 35, 453–464 (2025). https://doi.org/10.1038/s41422-025-01114-7

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