Hierarchical roles of mitochondrial Papi and Zucchini in Bombyx germline piRNA biogenesis
Hierarchical roles of mitochondrial Papi and Zucchini in Bombyx germline piRNA biogenesis
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DOI：
10.1038/nature25788
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发表时间：
2018-03-08
期刊：
影响因子：
64.8
通讯作者：
Siomi, Mikiko C.
中科院分区：
文献类型：
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作者：
Nishida, Kazumichi M.;Sakakibara, Kazuhiro;Siomi, Mikiko C.
PIWI相互作用RNA（piRNA）是与PIWI蛋白结合以控制转座子并维持动物生殖系中基因组完整性的小调控RNA 1 -3。家蚕中的皮尔纳3'末端形成已被证明是由3'至5'核酸外切酶Trimmer（Trim;在哺乳动物中称为PNLDC 1）4介导的，并且皮尔纳中间体与锚定在线粒体Tudor结构域蛋白Papi上的PIWI结合（5）。然而，尚不清楚在果蝇中的皮尔纳生物发生中均具有关键作用的小西葫芦（Zucchini，Zuc）内切核酸酶和Nibbler（Nbr）3 '至5'外切核酸酶（6-8）是否是其他物种中的皮尔纳加工所需的。在这里，我们表明，Zuc在Patix中的丢失对Trim和Nbr的水平没有影响，但导致Papi复合物内皮尔纳中间体的异常积累，并且这些中间体被重组Zuc加工形成成熟的piRNA。Papi只有在与PIWI结合并磷酸化时才发挥其RNA结合活性，这表明复杂的组装涉及一个分层过程。Papi复合物内的皮尔纳中间体的5'和3'末端均显示PIWI“切片器”活性的标志，但在成熟piRNA中未观察到定相模式。Zuc的缺失不影响中间体的5 '和3'末端的形成，这强烈支持了以下观点，即pMx皮尔纳的5'末端是由PIWI切割酶活性形成的，但独立于Zuc，而3'末端是由Zuc内切核酸酶形成的。Apex皮尔纳的生物发生机制比果蝇简单，因为Apex没有依赖于阶段性piRNA的转录沉默机制。
PIWI-interacting RNAs (piRNAs) are small regulatory RNAs that bind to PIWI proteins to control transposons and maintain genome integrity in animal germ lines1-3. 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) 4, and piRNA intermediates are bound with PIWI anchored onto mitochondrial Tudor domain protein Papi(5). 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(6-8), 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'-and3'-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.