Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan.
Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo, 113-0032, Japan.
Nat Commun. 2020 Feb 12;11(1):858. doi: 10.1038/s41467-020-14687-1.
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 结构域 Argonaute 蛋白与 PIWI 相互作用 RNA（piRNA）结合，并在动物生殖腺中沉默转座子。在这里，我们报告了果蝇 PIWI 结构域 Argonaute Piwi 与内源性 piRNA 复合物的晶体结构，分辨率为 2.9 Å。Piwi 与其他 Argonautes 的结构比较突出了 PIWI 特异性的结构特征，例如整体结构域排列和金属依赖性 piRNA 识别。我们的结构和生化数据表明，与其他 Argonautes（包括家蚕 Siwi）不同，Piwi 具有非典型的 DVDK 四联体，并且缺乏 RNA 引导的 RNA 切割切割酶活性。此外，我们发现具有典型 DEDH 催化四联体的 Piwi 突变体表现出切割酶活性，并且在切割酶介导的切割后容易从互补性较低的 RNA 靶标上解离，这表明切割酶活性可能会损害 Piwi 介导的共转录沉默。因此，我们提出 Piwi 在进化过程中失去了切割酶活性，作为 RNA 引导的 RNA 结合平台，从而确保转座子的忠实共转录沉默。
