Crystal structure of Drosophila Piwi

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.

Piwi protein, identified in 1997 as a critical gene for germline stem cell division, binds piRNA to form piRNA-induced silencing complexes that silence transposons and maintain genome integrity. PIWI and AGO are two clades of Argonaute proteins. The structures of AGO proteins have been well studied in the last 15 years. However, the structural information of Piwi protein is very limited due to difficulties in sample preparation. To date, only one Piwi protein Siwi structure has been reported.
Yamaguchi et al. solved the crystal structure of Drosophila Piwi protein in complex with endogenous piRNAs. This is the second structure of the Piwi protein. Although the overall structure of Piwi is similar to Siwi protein, the structure of the Piwi-piRNA complex reveals some PIWI specific structural features including the overall domain arrangement and metal-dependent piRNA binding. Authors find that the Piwi protein has a non-canonical DVDK tetrad and lacks the slicer activity, but the DEDH mutant has the slicer activity. The authors purpose that Piwi protein lack of slicer activity severs as a piRNA-guided RNA binding platform, ensuring faithful co-transcriptional silencing of transposons. Collectively, this study provides significant insights into the molecular mechanism of the Piwi-mediated transposon silencing.
Piwi proteins and prokaryotic Ago proteins share several structural features. 1. The binding of the 5'-phosphate of piRNA and siRNA are both metal-dependent. 2. The Glu finger from both the Piwi and prokaryotic Ago proteins are located away from the other three catalytic residues. Authors compared the structures of Piwi, Siwi, and hAgo2, but comparisons between Piwi and pfAgo and TtAgo are limited. Additional comparisons between the Piwi and pf/TtAgo are suggested.  We thank the reviewer for appreciating the importance of the Drosophila Piwi structure. We have addressed the points raised by the reviewer as follows. We appreciate the helpful comments. According to the reviewer's comment, we examined whether the isolated PAZ domain of Piwi binds an 8-mer RNA containing a 2′-O-methyl group at its 3′ end, using isothermal titration calorimetry (ITC). We found that the SUMO-tagged PAZ domain binds the ssRNA with a K d of 4.0 µM (new Fig. 5), indicating that the PAZ domain of Piwi recognizes the piRNA 3′ end, as in the other Argonaute proteins. Consistent with this, the residues that recognize the piRNA 3′ end in the other PIWIs are highly conserved in Piwi (new Fig. S6).
We have added the ITC data and the sequence alignment in the revised manuscript.
During the revision, we reported that the Piwi PAZ mutation (Y327A/Y328A) reduces the amounts of mature piRNAs in OSCs, highlighting the importance of the PAZ-mediated piRNA recognition for the piRNA maturation (Yamashiro et al., EMBO Rep, 2019). Intriguingly, the length distribution of the Piwi-bound piRNAs (~23-30 nt with the peak of 26 nt) (Saito et al., Nature, 2009) is wider than that of the Siwi-bound piRNAs (~27-29 nt with the peak of 28 nt) (Nishida et al., Cell Rep, 2015) (Fig. L1). These observations suggest that the flexibility of the Piwi PAZ domain contributes to the accommodation of piRNAs with a wider range of lengths. We have added these statements with the references in the revised manuscript.

Phenotype of 'slicer-Piwi'
The authors need to comment on their earlier findings (2006 Genes Dev)  We appreciate the important suggestion. In this study, we purified FLAG-tagged Piwi from OSCs using anti-FLAG antibody beads, and measured its cleavage activity toward the ssRNA substrate. We found that Piwi does not cleave the ssRNA substrate under our experimental conditions. Importantly, we also found, in contrast to wild-type Piwi, the slicer-Piwi mutant cleaves the ssRNA substrate with efficiency comparable to that of Siwi. Based on these results, we concluded that Piwi is not a slicer. In our previous study, we purified GST-tagged Piwi from E. coli using Glutathione Sepharose resin, and used an excess amount (~1 μg) of the partially purified Piwi for in vitro cleavage experiments (Saito et al. Genes Dev, 2006). Thus, it is likely that we detected the negligible, if any, slicer activity by Piwi in the previous study. We added these statements in the revised manuscript.
The crystal structure together with biochemical analysis presented here proves that nicely. The fact that the authors are able to generate 'slicer-Piwi' through the mutation of key residues demonstrates that the inactivity of Piwi indeed is based on the altered catalytic tetrad and its environment. This is an interesting experiment. In their rescue assay in OSC cells, the authors demonstrate that 'slicer-Piwi' does complement a Piwi knockdown and that 'slicer-Piwi' is able to initiate co-transcriptional transposon silencing (this is shown only indirectly as silencing is still Gtsf1 dependent). Based on in vitro experiments using target RNA that has impaired complementarity, the authors state that "slicing activity could compromise the . To strengthen their case, the authors should consider analyzing the effect of 'slicer-Piwi' in a fly model, rather than relying on the fairly rough rescue assay in cell culture. Only a fly rescue experiment will allow a strong statement about whether or not slicing does affect Piwi function.
Thank you for the critical comment. We agree with the reviewer that an analysis of slicer-Piwi in a fly model will be required to fully clarify the biological significance of the slicer activity.
Nonetheless, as the editor suggested, an analysis in a fly model seems beyond the scope of this work, and we hope that future work using a fly model will clarify the effects of the slicer activity on the Piwi-mediated co-transcriptional silencing. We appreciate the positive comments by the reviewer. Thank you for the comment. We have corrected it.

Figures 2C, 2D and S4 should be optimized to improve their clarity.
According to the reviewer's comment, we have modified Figs. 2C, 2D, and S4 to improve their clarity.