PIWI proteins use PIWI-interacting RNAs (piRNAs) to identify and silence transposable elements and thereby maintain genome integrity between metazoan generations1. The targeting of transposable elements by PIWI has been compared to mRNA target recognition by Argonaute proteins2,3, which use microRNA (miRNA) guides, but the extent to which piRNAs resemble miRNAs is not known. Here we present cryo-electron microscopy structures of a PIWI–piRNA complex from the sponge Ephydatia fluviatilis with and without target RNAs, and a biochemical analysis of target recognition. Mirroring Argonaute, PIWI identifies targets using the piRNA seed region. However, PIWI creates a much weaker seed so that stable target association requires further piRNA–target pairing, making piRNAs less promiscuous than miRNAs. Beyond the seed, the structure of PIWI facilitates piRNA–target pairing in a manner that is tolerant of mismatches, leading to long-lived PIWI–piRNA–target interactions that may accumulate on transposable-element transcripts. PIWI ensures targeting fidelity by physically blocking the propagation of piRNA–target interactions in the absence of faithful seed pairing, and by requiring an extended piRNA–target duplex to reach an endonucleolytically active conformation. PIWI proteins thereby minimize off-targeting cellular mRNAs while defending against evolving genomic threats.
This is a preview of subscription content, access via your institution
Open Access articles citing this article.
Programmable RNA targeting by bacterial Argonaute nucleases with unconventional guide binding and cleavage specificity
Nature Communications Open Access 08 August 2022
Nature Open Access 30 June 2022
BMC Genomics Open Access 08 June 2022
Subscribe to Nature+
Get immediate online access to the entire Nature family of 50+ journals
Subscribe to Journal
Get full journal access for 1 year
only $3.90 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Get time limited or full article access on ReadCube.
All prices are NET prices.
Maps for the EfPiwi–piRNA and EfPiwi–piRNA-target complexes have been deposited in the EMDB under accession numbers EMD-23061 and EMD-23063, respectively. Corresponding atomic models have been deposited in the PDB under accession numbers 7KX7 and 7KX9. The EfPiwi(MID/Piwi)–piRNA–long-target complex map has been deposited in the EMDB under accession number EMD-23062. Source data are provided with this paper.
Ozata, D. M., Gainetdinov, I., Zoch, A., O'Carroll, D. & Zamore, P. D. PIWI-interacting RNAs: small RNAs with big functions. Nat. Rev. Genet. 20, 89–108 (2019).
Shen, E. Z. et al. Identification of piRNA binding sites reveals the Argonaute regulatory landscape of the C. elegans germline. Cell 172, 937–951.e18 (2018).
Gou, L. T. et al. Pachytene piRNAs instruct massive mRNA elimination during late spermiogenesis. Cell Res. 24, 680–700 (2014).
Grimson, A. et al. Early origins and evolution of microRNAs and Piwi-interacting RNAs in animals. Nature 455, 1193–1197 (2008).
Lewis, B. P., Shih, I. H., Jones-Rhoades, M. W., Bartel, D. P. & Burge, C. B. Prediction of mammalian microRNA targets. Cell 115, 787–798 (2003).
Wee, L. M., Flores-Jasso, C. F., Salomon, W. E. & Zamore, P. D. Argonaute divides its RNA guide into domains with distinct functions and RNA-binding properties. Cell 151, 1055–1067 (2012).
Salomon, W. E., Jolly, S. M., Moore, M. J., Zamore, P. D. & Serebrov, V. Single-molecule imaging reveals that Argonaute reshapes the binding properties of its nucleic acid guides. Cell 162, 84–95 (2015).
Chandradoss, S. D., Schirle, N. T., Szczepaniak, M., MacRae, I. J. & Joo, C. A dynamic search process underlies microRNA targeting. Cell 162, 96–107 (2015).
Parker, J. S., Parizotto, E. A., Wang, M., Roe, S. M. & Barford, D. Enhancement of the seed-target recognition step in RNA silencing by a PIWI/MID domain protein. Mol. cell 33, 204–214 (2009).
Schirle, N. T. & MacRae, I. J. The crystal structure of human Argonaute2. Science 336, 1037–1040 (2012).
Schirle, N. T., Sheu-Gruttadauria, J. & MacRae, I. J. Structural basis for microRNA targeting. Science 346, 608–613 (2014).
Sheu-Gruttadauria, J., Xiao, Y., Gebert, L. F. & MacRae, I. J. Beyond the seed: structural basis for supplementary microRNA targeting by human Argonaute2. EMBO J. 38, e101153 (2019).
Friedman, R. C., Farh, K. K., Burge, C. B. & Bartel, D. P. Most mammalian mRNAs are conserved targets of microRNAs. Genome Res. 19, 92–105 (2009).
Zhang, D. et al. The piRNA targeting rules and the resistance to piRNA silencing in endogenous genes. Science 359, 587–592 (2018).
Goh, W. S. et al. piRNA-directed cleavage of meiotic transcripts regulates spermatogenesis. Genes Dev. 29, 1032–1044 (2015).
Halbach, R. et al. A satellite repeat-derived piRNA controls embryonic development of Aedes. Nature 580, 274–277 (2020).
Zhang, P. et al. MIWI and piRNA-mediated cleavage of messenger RNAs in mouse testes. Cell Res. 25, 193–207 (2015).
Nozawa, M. et al. Evolutionary transitions of microRNA–target pairs. Genome Biol. Evol. 8, 1621–1633 (2016).
Brennecke, J. et al. Discrete small RNA-generating loci as master regulators of transposon activity in Drosophila. Cell 128, 1089–1103 (2007).
Wang, J. et al. piRBase: a comprehensive database of piRNA sequences. Nucleic Acids Res. 47, D175–D180 (2019).
Matsumoto, N. et al. Crystal structure of silkworm PIWI-clade Argonaute Siwi bound to piRNA. Cell 167, 484–497.e9 (2016).
Yamaguchi, S. et al. Crystal structure of Drosophila Piwi. Nat. Commun. 11, 858 (2020).
Funayama, N., Nakatsukasa, M., Mohri, K., Masuda, Y. & Agata, K. Piwi expression in archeocytes and choanocytes in demosponges: insights into the stem cell system in demosponges. Evol. Dev. 12, 275–287 (2010).
Alie, A. et al. The ancestral gene repertoire of animal stem cells. Proc. Natl Acad. Sci. USA 112, E7093–E7100 (2015).
Wynant, N., Santos, D. & Vanden Broeck, J. The evolution of animal Argonautes: evidence for the absence of antiviral AGO Argonautes in vertebrates. Sci. Rep. 7, 9230 (2017).
Reuter, M. et al. Miwi catalysis is required for piRNA amplification-independent LINE1 transposon silencing. Nature 480, 264–267 (2011).
Wu, P. H. et al. The evolutionarily conserved piRNA-producing locus pi6 is required for male mouse fertility. Nat. Genet. 52, 728–739 (2020).
Arif, A. et al. The tiny, conserved zinc-finger protein GTSF1 helps PIWI proteins achieve their full catalytic potential. Preprint at https://doi.org/10.1101/2021.05.04.442675 (2021).
Herzog, V. A. et al. Thiol-linked alkylation of RNA to assess expression dynamics. Nat. Methods 14, 1198–1204 (2017).
Sienski, G., Donertas, D. & Brennecke, J. Transcriptional silencing of transposons by Piwi and Maelstrom and its impact on chromatin state and gene expression. Cell 151, 964–980 (2012).
Le Thomas, A. et al. Piwi induces piRNA-guided transcriptional silencing and establishment of a repressive chromatin state. Genes Dev. 27, 390–399 (2013).
Aravin, A. A. et al. A piRNA pathway primed by individual transposons is linked to de novo DNA methylation in mice. Mol. Cell 31, 785–799 (2008).
Kuramochi-Miyagawa, S. et al. DNA methylation of retrotransposon genes is regulated by Piwi family members MILI and MIWI2 in murine fetal testes. Genes Dev. 22, 908–917 (2008).
Nott, T. J. et al. Phase transition of a disordered nuage protein generates environmentally responsive membraneless organelles. Mol. Cell 57, 936–947 (2015).
Sheu-Gruttadauria, J. & MacRae, I. J. Phase transitions in the assembly and function of human miRISC. Cell 173, 946–957.e16 (2018).
Flores-Jasso, C. F., Salomon, W. E. & Zamore, P. D. Rapid and specific purification of Argonaute–small RNA complexes from crude cell lysates. RNA 19, 271–279 (2013).
Suloway, C. et al. Automated molecular microscopy: the new Leginon system. J. Struct. Biol. 151, 41–60 (2005).
Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).
Lander, G. C. et al. Appion: an integrated, database-driven pipeline to facilitate EM image processing. J. Struct. Biol. 166, 95–102 (2009).
Kimanius, D., Forsberg, B. O., Scheres, S. H. & Lindahl, E. Accelerated cryo-EM structure determination with parallelisation using GPUs in RELION-2. eLife 5, e18722 (2016).
Rohou, A. & Grigorieff, N. CTFFIND4: Fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216–221 (2015).
Goddard, T. D., Huang, C. C. & Ferrin, T. E. Visualizing density maps with UCSF Chimera. J. Struct. Biol. 157, 281–287 (2007).
Pettersen, E. F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).
Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296 (2017).
Tan, Y. Z. et al. Addressing preferred specimen orientation in single-particle cryo-EM through tilting. Nat. Methods 14, 793–796 (2017).
Waterhouse, A. et al. SWISS-MODEL: homology modelling of protein structures and complexes. Nucleic Acids Res. 46, W296–W303 (2018).
Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010).
Liebschner, D. et al. Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix. Acta Crystallogr. D 75, 861–877 (2019).
Williams, C. J. et al. MolProbity: More and better reference data for improved all-atom structure validation. Protein Sci. 27, 293–315 (2018).
Goddard, T. D. et al. UCSF ChimeraX: Meeting modern challenges in visualization and analysis. Protein Sci. 27, 14–25 (2018).
Buchan, D. W. A. & Jones, D. T. The PSIPRED protein analysis workbench: 20 years on. Nucleic Acids Res. 47, W402–W407 (2019).
Jones, D. T. Protein secondary structure prediction based on position-specific scoring matrices. J. Mol. Biol. 292, 195–202 (1999).
Nakanishi, K. et al. Eukaryote-specific insertion elements control human ARGONAUTE slicer activity. Cell Rep. 3, 1893–1900 (2013).
Park, M. S. et al. Human Argonaute3 has slicer activity. Nucleic Acids Res. 45, 11867–11877 (2017).
Park, M. S. et al. Multidomain convergence of Argonaute during RISC assembly correlates with the formation of internal water clusters. Mol. Cell 75, 725–740.e6 (2019).
Martin, M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet.journal 17, 10–12 (2011).
We thank N. Funayama for the E. fluviatilis piwi-a cDNA clone, Y. Tomari for the Siwi cDNA clone, and I. H. Segel for advice about measuring binding reactions with very slow off-rates. The research of G.C.L. is supported by NIH grant R21AG067594 and an Amgen Young Investigator Award. The research of I.J.M. is supported by NIH grant R35GM127090.
The authors declare no competing interests.
Peer review information Nature thanks Hong-Wei Wang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
a, Coomassie-stained SDS PAGE of piRNA-loaded PIWI proteins captured using an immobilized complementary oligonucleotide. Input shows partially purified protein samples that were incubated with capture resin. Unbound shows protein that did not bind the resin. Captured shows protein retained on the resin after washing (eluted by boiling in SDS). After elution shows protein retained on the resin after incubation with the competitor oligonucleotide (eluted by boiling in SDS). b, Capture-purified PIWI proteins before and after anion exchange purification. Input fraction shows samples after elution by competitor oligonucleotide in capture-purification step. Purified indicates the final purification products. Note: Δseed-gate Siwi was captured at such low levels that it was unclear whether any active Siwi was obtained until observing the sample’s ability to specifically bind 32P-labeled target RNAs. c, Phylogenetic tree of PIWI proteins shows EfPiwi belongs to the ancient Drosophila Ago3-like branch.
Extended Data Fig. 2 Imaging and processing of the EfPiwi-piRNA complex (and EfPiwi-piRNA-long target complex).
a, Representative cryo-EM micrograph (1,765 micrographs collected in total). Input sample contained EfPiwi-piRNA and a long target RNA (complementary to piRNA nucleotides g2–g25). b, Cryo-EM data processing workflow. The data set contained two populations of well resolved particles, one for the binary EfPiwi-piRNA complex and another for the ternary EfPiwi-piRNA-long target complex. 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 3.8 Å reconstruction for the EfPiwi-guide complex, and 8.6 Å for the ternary EfPiwi-piRNA-long target complex. c, The final 3D map for the EfPiwi-piRNA complex coloured by local resolution values, where the majority of the map was resolved between 3.5 Å and 4 Å with the flexible PAZ and N domains having lower resolution. d, Angular distribution plot showing the Euler angle distribution of the EfPiwi-piRNA particles in the final reconstruction. The position of each cylinder corresponds to the 3D angular assignments and their height and colour (blue to red) corresponds to the number of particles in that angular orientation. e, 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. f, EM density quality of EfPiwi-piRNA complex. Individual domains of EfPiwi fit into the EM density, EM density shown in mesh; molecular models (coloured as in Fig. 2) shown in cartoon representation with side chains shown as sticks; piRNA shown in stick representation.
a, Surface of hAGO2 (left) and EfPiwi (right), highlighting g5-g6 nucleotide-binding loops. Superimposing g5-g6 nucleotides (red sticks) from hAGO2 onto EfPiwi results in steric clashes. b, g5-g6 loop in AGO structures (left) is kinked, enabling pre-organization of seed 3' end. Equivalent loop in Piwi structures (right) cannot kink due to bulky residues (labeled positions 1 and 2), conserved in Piwi family. c, Close up superposition of central-gate and seed-gate structures in AGO and PIWI proteins, respectively. d, Superposition of seed-gate regions from all known Piwi (left) and AGO (right) structures, with secondary structure schematics shown above. e, Secondary structure predictions indicate the α6 extension is a defining feature of the Piwi family. Predictions were by PSIPRED 4.0. f, L1-L2 interface near seed-gate in EfPiwi. Hydrophobic residues buried at the L1-L2 interface are shown. g, Sequence alignment shows L1-L2 interface residues in EfPiwi are broadly conserved in Piwis (green) and distinct from the equivalent residues in AGOs (blue).
a, Schematic of pairing between guide RNAs and seed-matched target RNAs used in main text Fig. 2b, c. b, Schematic of pairing between 22 nt guide RNA and target RNAs spanning the seed and central regions. c. Release of 32P-labeled target RNAs from EfPiwi-22 nt guide in the presence of excess unlabeled target RNA over time. d, Release rates of target RNAs from hAGO2-22 nt guide (data from Fig. 2d, left) and EfPiwi-22nt guide (c). Results show hAGO2 and EfPiwi create distinct binding properties for the same guide RNA. All plotted data are the mean values of triplicate measurements. Error bars indicate SD. e, Ribbon representation of hAGO2, EfPiwi, and an overlay illustrating relative positions of the central-gate and seed-gate. In c, d, n = 3 independent experiments, data are mean ± s.d.
a, Representative cryo-EM micrograph of EfPiwi-piRNA-target complex (1,881 micrographs collected in total). b, Workflow for processing EfPiwi-piRNA-target complex dataset. 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 3.5 Å map. c, The EfPiwi-piRNA-target complex map coloured by local resolution. d, Euler angle distribution plot for the EfPiwi-piRNA-target complex particles. e, Directional Fourier Shell Correlation (FSC) plot representing 3D resolution anisotropy in the reconstructed map. Red line shows global FSC; green dashed lines ±1 standard deviation from mean of directional resolutions; blue histograms indicate percentage of directional resolution over the 3D FSC. f, EM density quality of EfPiwi-piRNA-target complex. Individual domains of EfPiwi and RNAs fit into the EM density; EM density shown in mesh; protein models shown in cartoon representation (coloured as in Fig. 1) with side chains shown as sticks; RNAs shown in stick representation.
a, Raw data for kon values shown in Fig. 3f. Plots of target RNAs with mismatches (sequences shown in Extended Data Fig. 6c) binding to EfPiwi-piRNA complexes over time. Protein concentrations used in each experiment are indicated at top of each graph. 95% confidence limits of observed association rates (kobs) and kon values indicated. WT EfPiwi (black), ∆seed-gate EfPiwi (red). b, Raw data for koff values shown in Extended Data Fig. 7e. Plots of target RNAs with mismatches (mm) dissociating from EfPiwi-piRNA complexes over time. All data were fit to a plateau value of 0.15. 95% confidence limits of kon values indicated. All data points were measured three times. Error bars indicate SEM. Center line indicates best fit to data. Surrounding lines indicate 95% confidence limits. In all panels, n = 3 independent experiments, data are mean ± s.d.
a, Guide-target pairing schematic for select mismatched targets binding Siwi-piRNA complexes. Mismatches coloured gold. b, Association rates of target RNAs (shown in a) with wild-type Siwi (left) and ∆seed-gate Siwi. Indicated p-values from two-sided t-test are 6.11 x 10−5 and 0.205 for wild-type and ∆seed-gate Siwi, respectively. c, Guide-target pairing schematic for mismatched targets used in main text Fig. 3f, and panels d and e here. Mismatches coloured gold. d, Dissociation rates of 32P-labeled target RNAs with three consecutive mismatches from wild-type EfPiwi. Most mismatched segments had moderate (~10-fold) effects on koff, except 14–16 mismatches, which increased koff ~70 fold. e, Dissociation constants (KD) calculated from kon and koff values for target RNAs binding wild-type EfPiwi-guide complex. f, Surface representation of the modeled piRNA-target duplex. piRNA nucleotides numbered at the Watson-Crick face. Non-hydrogen RNA atoms positioned ≤ 4 Å from an EfPiwi atom coloured purple. In b, d, and e, n = 3 independent experiments, data are mean ± s.d.
a, Denaturing gels showing cleavage of g2-g21 matched 32P-labeled target RNA by EfPiwi, hAGO2 or Siwi in the presence of various divalent cations (2 mM each). Schematic of piRNA-target pairing shown (top). Gels are representative results for n = 3 independent experiments for EfPiwi and hAGO2, and n = 2 independent experiments for Siwi. b, Time course showing cleavage of g2-g21 paired 32P-labeled target RNA by EfPiwi in the presence of Mg2+, Mn2+, or both at approximate physiological divalent cation concentrations. c, Cleavage of g2-g21 matched 32P-labeled target RNA by EfPiwi at various temperatures shows activity over the full physiological range (17–30 °C). Gel is representative of n = 3 independent experiments. d, Quantification of results (and replicates) in c. e, Cleavage of target RNAs with varying degrees of 3' complementarity by EfPiwi at 30 °C. In b, d and e, n = 3 independent experiments, data are mean ± s.d.
a, Guide-target pairing schematic for targets with varying degrees of complementarity to piRNA 3' end used in Fig. 4a and b, e. b, Quantification target RNAs (1 nM) cleaved after treating with excess (100 nM) EfPiwi loaded with a 22 or 25 nt guide or hAGO2 loaded with a 22 nt guide for 1 h. n = 3 independent experiments, data are mean ± s.d. c, Guide-target pairing schematic for targets with 3 nt mismatched regions. d, Quantification mismatched target RNAs (1 nM) cleaved after treating with excess (100 nM) EfPiwi loaded with a 22 or 25 nt guide or hAGO2 loaded with a 22 nt guide for 1 h. n = 3 independent experiments, data are mean ± s.d. e, Cleavage of targets with varying degrees of complementarity to piRNA 3' end (shown panel a) by Siwi (2 mM MnCl2, 37 °C). Gel is representative of n = 3 independent experiments, with data plotted as mean ± s.d. shown below. f, From Fig. 4c: sequences of the 26 target RNAs, with 0–8 mismatches opposite piRNA nucleotides g11–g18, that were most readily cleaved by EfPiwi (listed in order of cleavage product abundance). Mismatched nucleotides coloured yellow. Greyed out sequences indicate constant regions shared by all target RNAs. Triangle indicates cleavage site.
Unprocessed and uncropped gel images. a, Unprocessed gel scans corresponding to gel in Fig. 4a and Extended Data Fig. 9b. Corresponding panels or data indicated above gels. Dotted lines indicate where the gel was cropped. b, Unprocessed gel scans corresponding to gels in Extended Data Fig. 8a. Corresponding panels indicated above gels. Dotted lines indicate where the gel was cropped. c, Unprocessed gel scan corresponding to data in Extended Data Fig. 8b. Dotted lines indicate separation between samples. d, Unprocessed gel scan corresponding to gel in Extended Data Fig. 8c and data in Extended Data Fig. 8d. Dotted lines indicate where the gel was cropped. e, Unprocessed gel scan corresponding to data in Extended Data Fig. 8e. Dotted line indicates separation between samples. f, Unprocessed gel scan corresponding to gel and data in Extended Data Fig. 9e. Dotted lines indicate where the gel was cropped. g, Unprocessed gel scan corresponding to data in Extended Data Fig. 9d. Dotted line indicates separation between samples.
A list of oligonucleotides used in this study.
About this article
Cite this article
Anzelon, T.A., Chowdhury, S., Hughes, S.M. et al. Structural basis for piRNA targeting. Nature 597, 285–289 (2021). https://doi.org/10.1038/s41586-021-03856-x
BMC Genomics (2022)
Nature Reviews Molecular Cell Biology (2022)
Programmable RNA targeting by bacterial Argonaute nucleases with unconventional guide binding and cleavage specificity
Nature Communications (2022)