Crystal structure of Drosophila Piwi

Abstract

PIWI-clade Argonaute proteins associate with PIWI-interacting RNAs (piRNAs), and silence transposons in animal gonads. Here, we report the crystal structure of the Drosophila PIWI-clade Argonaute Piwi in complex with endogenous piRNAs, at 2.9 Å resolution. A structural comparison of Piwi with other Argonautes highlights the PIWI-specific structural features, such as the overall domain arrangement and metal-dependent piRNA recognition. Our structural and biochemical data reveal that, unlike other Argonautes including silkworm Siwi, Piwi has a non-canonical DVDK tetrad and lacks the RNA-guided RNA cleaving slicer activity. Furthermore, we find that the Piwi mutant with the canonical DEDH catalytic tetrad exhibits the slicer activity and readily dissociates from less complementary RNA targets after the slicer-mediated cleavage, suggesting that the slicer activity could compromise the Piwi-mediated co-transcriptional silencing. We thus propose that Piwi lost the slicer activity during evolution to serve as an RNA-guided RNA-binding platform, thereby ensuring faithful co-transcriptional silencing of transposons.

Fig. 1. Overall structure.

a Domain organization of Piwi. NLS, nuclear localization signal; ID, intrinsically disordered region. b Crystal structure of the Piwi–piRNA complex.

Fig. 2. Structural comparison.

a, b Crystal structures of Siwi (PDB: 5GUH) a and hAgo2 (PDB: 4W5N) b. c, d Superimposition of Piwi on Siwi (blue) c and hAgo2 (red) d, based on their PIWI domains. Note that the Piwi PAZ domain is not well resolved in the density map, suggesting its flexibility. e–g N domain interfaces of Piwi e, Siwi f, and hAgo2 g.

Fig. 3. Surface conservation.

The residues conserved among Piwi, Aub, and Ago3 are colored yellow on the molecular surface of Piwi.

Fig. 4. Recognition of the piRNA 5′ end.

a–c Guide RNA recognitions by Piwi a, Siwi (PDB: 5GUH) b, and hAgo2 (PDB: 4W5O) c (stereo view). The zinc and magnesium ions are shown as magenta spheres in a and b, respectively. Hydrogen bonds and electrostatic interactions are indicated by cyan dashed lines.

Fig. 5. Recognition of the piRNA 3′ end.

a, b ITC experiments for the binding of the SUMO-tagged Piwi PAZ domain a or the SUMO protein b to the 8-mer RNA containing the 2′-O-methyl group at its 3′ end.

Fig. 6. Catalytic tetrad.

a, b DEDH tetrads of hAgo2 (PDB: 4W5N) a and Siwi (PDB: 5GUH) b. c Slicer activities of Siwi. The immunopurified FLAG-tagged wild-type Siwi and mutants were incubated with the internally ³²P-labeled substrate RNA (piRNA-4 target), and the reaction products were analyzed by denaturing urea-PAGE. d DVDK tetrad of Piwi. e Sequences of the target RNA (flam target) and the piRNAs (flam-piRNA-1 and flam-piRNA-2). f Slicer activities of Piwi. The immunopurified FLAG-tagged wild-type Piwi or slicer-Piwi was incubated with the internally ³²P-labeled substrate RNA (flam target), and the reaction products were analyzed by denaturing urea-PAGE. HSEH, the slicer-Piwi K617H/A625S/V653E/K818H mutant. Source data are provided as a Source Data file.

Fig. 7. Transposon silencing.

a Piwi-mediated silencing of the mdg1 transposon. FLAG-tagged wild-type Piwi or slicer-Piwi was expressed in endogenous Piwi-depleted OSCs, and the expression levels of the mdg1 transposon were examined by quantitative RT-PCR (n = 3; error bars indicate SEM). Empty, empty vector control; Slicer, the slicer-Piwi K617H/A625S/V653E/K818H mutant. b Binding of Piwi to Arx. FLAG-tagged wild-type Piwi or slicer-Piwi was expressed in endogenous Piwi-depleted OSCs, and the proteins were then immunoprecipitated using anti-FLAG beads. The cell lysates and immunoprecipitates were analyzed by western blotting, using the indicated antibodies. H3 was used as a loading control. c Requirement of Arx for Piwi-mediated silencing. FLAG-tagged wild-type Piwi or slicer-Piwi was expressed in endogenous Piwi/Arx-depleted OSCs, and the expression levels of the mdg1 transposon were examined by quantitative RT-PCR (n = 3; error bars indicate SEM). d Binding of slicer-Piwi to the cleavage products. FLAG-tagged wild-type Piwi or slicer-Piwi was immunopurified from OSCs, and the proteins were then incubated with the internally ³²P-labeled substrate RNA (flam target). The supernatant and beads fractions were analyzed by denaturing urea-PAGE. Signal intensities of the fragments are shown on the right. e Binding of Piwi to partially complementary targets. The immunopurified FLAG-tagged wild-type Piwi and slicer-Piwi were incubated with the 5′ ³²P-labeled substrate RNA, and the supernatant and beads fractions were then analyzed by denaturing urea-PAGE. Sequences of the target RNAs (WT, mt1, and mt2) and the piRNA (mdg1-piRNA) are shown below. Source data are provided as a Source Data file.