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Structural basis for RNA slicing by a plant Argonaute

Nature Structural & Molecular Biology volume 30, pages 778–784 (2023)Cite this article

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Abstract

Argonaute (AGO) proteins use small RNAs to recognize transcripts targeted for silencing in plants and animals. Many AGOs cleave target RNAs using an endoribonuclease activity termed ‘slicing’. Slicing by DNA-guided prokaryotic AGOs has been studied in detail, but structural insights into RNA-guided slicing by eukaryotic AGOs are lacking. Here we present cryogenic electron microscopy structures of the Arabidopsis thaliana Argonaute10 (AtAgo10)–guide RNA complex with and without a target RNA representing a slicing substrate. The AtAgo10–guide–target complex adopts slicing-competent and slicing-incompetent conformations that are unlike known prokaryotic AGO structures. AtAgo10 slicing activity is licensed by docking target (t) nucleotides t9–t13 into a surface channel containing the AGO endoribonuclease active site. A β-hairpin in the L1 domain secures the t9–t13 segment and coordinates t9–t13 docking with extended guide–target pairing. Results show that prokaryotic and eukaryotic AGOs use distinct mechanisms for achieving target slicing and provide insights into small interfering RNA potency.

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Fig. 1: Structure of the AtAgo10–guide RNA complex without target RNA.
Fig. 2: AtAgo10–guide–target complex captured in two functional states.
Fig. 3: Catalytic-competent conformation of the AtAgo10–guide–target complex.
Fig. 4: Structural basis for RNA slicing.

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Data availability

Maps for the AtAgo10–guide, AtAgo10–guide–target (central-duplex) and AtAgo10–guide–target (bent-duplex) complexes were deposited in the EMDB under accession IDs EMD-25446, EMD-25472 and EMD-25482, respectively. Corresponding atomic models were deposited in the PDB under accession IDs PDB-7SVA, PDB-7SWF and PDB-7SWQ.

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Acknowledgements

We are grateful to W. He and Y. Hou for plasmids containing Arabidopsis thaliana AGO cDNAs and to MacRae lab members, G. Riddihough and J. Williamson, for advice. The research was funded by NIH grants R35GM127090 (I.J.M.), R01GM092740 (T.O.) and S10OD021634.

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Authors and Affiliations

  1. Department of Integrative Structural and Computational Biology, The Scripps Research Institute, La Jolla, CA, USA

    Yao Xiao, Shintaro Maeda, Takanori Otomo & Ian J. MacRae

  2. San Diego Biomedical Research Institute, San Diego, CA, USA

    Takanori Otomo

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  1. Yao Xiao
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Contributions

Y.X. prepared AtAgo10 samples, performed biochemical experiments, produced high-resolution reconstructions, built AtAgo10 models, analyzed data and co-wrote the manuscript. S.M. prepared cryo-EM grids and collected cryo-EM data. T.O. advised cryo-EM data collection and production of high-resolution reconstructions and provided mechanistic insights. I.J.M. provided structural and mechanistic insights, analyzed data and co-wrote the manuscript.

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Correspondence to Ian J. MacRae.

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Nature Structural & Molecular Biology thanks Ailong Ke and Dinshaw Patel for their contribution to the peer review of this work. Sara Osman was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Imaging and processing of the AtAgo10-guide RNA complex.

Representative cryo-EM micrograph (one of 2,656 micrographs in total) and cryo-EM data processing workflow. Particles isolated from micrographs were sorted by reference-free 2D classification. Only particles containing high-resolution features for the intact complex were selected for downstream processing. 3D classification was used to further remove low-resolution or damaged particles, and the remaining particles were refined to obtain a 3.26 Å resolution reconstruction.

Extended Data Fig. 2 Reconstruction of the AtAgo10-guide complex.

a. The final 3D map for the AtAgo10-guide RNA complex colored by local resolution values, where the majority of the map was resolved between 3.3 Å and 3.7 Å, and the map covering the more mobile PAZ and N domains has lower resolution. b. Angular distribution plot showing the Euler angle distribution of the AtAgo10-guide particles in the final reconstruction. The position of each cylinder corresponds to the 3D angular assignments and their height and color (blue to red) correspond to the number of particles in that angular orientation. c. Directional Fourier Shell Correlation (FSC) plot representing 3D resolution anisotropy in the reconstructed map, with the red line showing the global FSC, green dashed lines correspond to ±1 standard deviation from mean of directional resolutions, and the blue histograms correspond to percentage of directional resolution over the 3D FSC. d. EM density (mesh) quality of the guide RNA (sticks). e. Individual domains of AtAgo10 fit into the EM density, EM density shown in mesh; molecular models (colored as in Fig. 1) shown in cartoon representation with side chains shown as sticks.

Extended Data Fig. 3 Details of AtAgo10 structural analysis.

a. Alignment of ß-finger sequences from clade I AGOs from diverse plants, algae, and animals. The insertion can be found in clade I AGOs in all plant phyla but is not apparent in animals or algae, indicating β-finger arose around the emergence of land plants and has been broadly maintained in the clade I AGOs over the last ~500 million years. b. All-against-all correspondence analysis45 of known eukaryotic AGO and PIWI guide-bound structures (target-bound structures not included). c. Cα alignment of AtAgo10 with known human AGO (PDB: 4KRF, 4KXT, 4OLA, 4W5N, 5JS1, 5VM9, 6OON), PIWI (PDB: 5GUH, 6KR6, 7KX7), and yeast AGO (PDB: 4F1N) guide-bound structures.

Extended Data Fig. 4 Imaging and processing of the AtAgo10-guide-target RNA complex.

Representative cryo-EM micrograph (one out of 2,049 micrographs collected in total) in upper left, proceeded by cryo-EM data processing workflow. Particles isolated from micrographs were sorted by reference-free 2D classification. Only particles containing high-resolution features for the intact complex were selected for downstream processing. 3D classification was used to further remove low-resolution or damaged particles, and isolate classes with distinct RNA conformations. 3D classification and particles in the two major classes were further refined to obtain two 3.79 Å resolution reconstructions. Conformation-1 is the ‘central-duplex’, catalytic conformation. Conformation-2 corresponds to the ‘bent-duplex’, inactive family of conformations.

Extended Data Fig. 5 Reconstruction of the AtAgo10-guide-target bent-duplex conformation.

a. Final 3D map for Conformation-2 (see Extended Data Fig. 4) of the AtAgo10-guide-target RNA complex colored by local resolution values. b. Angular distribution plot showing the Euler angle distribution of the AtAgo10-guide-target particles in the final reconstruction. The position of each cylinder corresponds to the 3D angular assignments and their height and color (blue to red) corresponds to the number of particles in that angular orientation. c. Directional Fourier Shell Correlation (FSC) plot representing 3D resolution anisotropy in the reconstructed map, with the red line showing the global FSC, green dashed lines correspond to ±1 standard deviation from mean of directional resolutions, and the blue histograms correspond to percentage of directional resolution over the 3D FSC. d. Guide-target duplex (shown as sticks) and individual AtAgo10 domains (shown in cartoon representation with side chains shown as sticks) fit into the EM density (shown in mesh). e. The bent-duplex conformation of AtAgo10 strongly resembles a previous bent duplex structure of HsAgo2 (PDB 6MDZ), with an RMSD of 1.5 Å for 634 equivalent Cα atoms.

Extended Data Fig. 6 Reconstruction of the AtAgo10-guide-target central-duplex conformation.

a. Final 3D map for Conformation-1 (see Extended Data Fig. 4) of the AtAgo10-guide-target RNA complex colored by local resolution values. b. Angular distribution plot showing the Euler angle distribution of the AtAgo10-guide-target particles in the final reconstruction. The position of each cylinder corresponds to the 3D angular assignments and their height and color (blue to red) corresponds to the number of particles in that angular orientation. c. Directional Fourier Shell Correlation (FSC) plot representing 3D resolution anisotropy in the reconstructed map, with the red line showing the global FSC, green dashed lines correspond to ±1 standard deviation from mean of directional resolutions, and the blue histograms correspond to percentage of directional resolution over the 3D FSC. d. Guide-target duplex (shown as sticks) and individual AtAgo10 domains (shown in cartoon representation with side chains shown as sticks) fit into the EM density (shown in mesh). e. Close-up view of density surrounding the scissile phosphate (yellow).

Extended Data Fig. 7 Bent-duplex conformation of AtAgo10 cannot bind a central guide-target duplex with pairing beyond t13.

a. Surface representation of the AtAgo10 central-duplex structure with the guide-target RNA duplex shown as sticks. Inset shows no steric clashes between AtAgo10 and the end of the RNA duplex. b. Surface representation of AtAgo10 in the bent-duplex conformation with a modeled continuous guide-target RNA duplex (taken from the central-duplex structure). Inset shows steric clashes (white dashed circles) between t14/t16 of the modeled RNA and the L1 hairpin/N domain of AtAgo10.

Extended Data Fig. 8 Comparison of catalytic and non-catalytic AtAgo10-guide-target structures.

a. Superposition of catalytic (central duplex) and non-catalytic (bent duplex) conformations. Arrow indicates the magnitude of movement traversed by the guide RNA 3’ end between conformations. b. Schematic of major contacts between AtAgo10 and the guide (red) and target (blue) RNAs in the non-catalytic (bent duplex) conformation. c. Schematic of major contacts between AtAgo10 and the RNAs in the catalytic (central duplex) conformation. Residues colored by domain, as in Fig. 1.

Extended Data Fig. 9 L1-hairpin-mut AtAgo10 is impaired in target-slicing but not target-binding.

a. Blots from an RNA filter-binding experiment using L1-hairpin-mut AtAgo10 (10 nM) and target RNAs (2 nM) used in this study (shown in Fig. 4b). Complexes were formed with 32P-labeled target RNAs under conditions used for slicing experiments (except Mg2+ was omitted to prevent target cleavage) and passed through a nitrocellulose membrane (to capture protein-RNA complexes) followed by a nylon membrane (to capture RNAs not retained on the nitrocellulose). b. Quantitation of data in panel a. Plotted data are the values from n = 1 experiments. c. Fraction of 2–21 target RNA (2 nM) cleaved by wild-type (10 nM) or L1-hairpin-mut AtAgo10 (10 nM) versus time. Data points are the mean values of n = 3 independent experiments. Error bars indicate SEM.

Extended Data Fig. 10 Comparison of TtAgo and AtAgo10 PIWI domain conformational changes.

a. Superimposition of TtAgo PIWI domains from guide-only (grey, PDB: 3DLH) and slicing competent (purple, PDB: 4NCB) conformations. Top panel: an overview of the TtAgo PIWI domain with active site residues shown as sticks and labeled. Bottom panel: Detailed views showing the formation of the catalytic conformation involves three rearranged loops and flipping+translation of a ß-strand connected to loop-1 in the TtAgo PIWI domain. Arrows indicate major shifts from the guide-only to the slicing-competent conformation. b. Superimposition of the AtAgo10 PIWI domains from guide-only (grey) and slicing competent (central-duplex) conformations (green) shows relatively few changes and no ß-strand repositioning.

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Xiao, Y., Maeda, S., Otomo, T. et al. Structural basis for RNA slicing by a plant Argonaute. Nat Struct Mol Biol 30, 778–784 (2023). https://doi.org/10.1038/s41594-023-00989-7

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