Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo 113-0032, Japan.
Department of Molecular Biology, Keio University School of Medicine, Tokyo 162-8582, Japan.
Nature. 2018 Mar 8;555(7695):260-264. doi: 10.1038/nature25788. Epub 2018 Feb 28.
PIWI-interacting RNAs (piRNAs) are small regulatory RNAs that bind to PIWI proteins to control transposons and maintain genome integrity in animal germ lines. piRNA 3' end formation in the silkworm Bombyx mori has been shown to be mediated by the 3'-to-5' exonuclease Trimmer (Trim; known as PNLDC1 in mammals), and piRNA intermediates are bound with PIWI anchored onto mitochondrial Tudor domain protein Papi. However, it remains unclear whether the Zucchini (Zuc) endonuclease and Nibbler (Nbr) 3'-to-5' exonuclease, both of which have pivotal roles in piRNA biogenesis in Drosophila, are required for piRNA processing in other species. Here we show that the loss of Zuc in Bombyx had no effect on the levels of Trim and Nbr, but resulted in the aberrant accumulation of piRNA intermediates within the Papi complex, and that these were processed to form mature piRNAs by recombinant Zuc. Papi exerted its RNA-binding activity only when bound with PIWI and phosphorylated, suggesting that complex assembly involves a hierarchical process. Both the 5' and 3' ends of piRNA intermediates within the Papi complex showed hallmarks of PIWI 'slicer' activity, yet no phasing pattern was observed in mature piRNAs. The loss of Zuc did not affect the 5'- and 3'-end formation of the intermediates, strongly supporting the idea that the 5' end of Bombyx piRNA is formed by PIWI slicer activity, but independently of Zuc, whereas the 3' end is formed by the Zuc endonuclease. The Bombyx piRNA biogenesis machinery is simpler than that of Drosophila, because Bombyx has no transcriptional silencing machinery that relies on phased piRNAs.
PIWI 相互作用 RNA（piRNAs）是与 PIWI 蛋白结合以控制转座子并维持动物生殖系基因组完整性的小调控 RNA。已经表明，在桑蚕 Bombyx mori 中，piRNA 的 3' 端形成是由 3'-5' 外切酶 Trimmer（在哺乳动物中称为 PNLDC1）介导的，并且 piRNA 中间产物与锚定在线粒体 Tudor 结构域蛋白 Papi 上的 PIWI 结合。然而，尚不清楚在果蝇中对 piRNA 生物发生具有关键作用的 Zucchini（Zuc）内切核酸酶和 Nibbler（Nbr）3'-5' 外切酶是否需要在其他物种中进行 piRNA 加工。在这里，我们表明，Bombyx 中 Zuc 的缺失对 Trim 和 Nbr 的水平没有影响，但导致 Papi 复合物内 piRNA 中间产物的异常积累，并且这些中间产物被重组 Zuc 加工形成成熟的 piRNA。只有当与 PIWI 结合并磷酸化时，Papi 才发挥其 RNA 结合活性，这表明复合物组装涉及一个层次过程。Papi 复合物内 piRNA 中间产物的 5' 和 3' 端均显示出 PIWI“slicer”活性的特征，但在成熟的 piRNA 中未观察到相位模式。Zuc 的缺失不影响中间产物的 5' 和 3' 端形成，这强烈支持 Bombyx piRNA 的 5' 端是由 PIWI slicer 活性形成的，但独立于 Zuc，而 3' 端是由 Zuc 内切核酸酶形成的观点。Bombyx 的 piRNA 生物发生机制比果蝇的更简单，因为 Bombyx 没有依赖于相化 piRNA 的转录沉默机制。
