Recruitment of Armitage and Yb to a transcript triggers its phased processing into primary piRNAs in <i>Drosophila</i> ovaries

Small RNAs called PIWI -interacting RNAs (piRNAs) are essential for transposon control and fertility in animals. Primary processing is the small RNA biogenesis pathway that uses long single-stranded RNA precursors to generate millions of individual piRNAs, but the molecular mechanisms that identify a transcript as a precursor are poorly understood. Here we demonstrate that artificial tethering of the piRNA biogenesis factor, Armi, to a transcript is sufficient to direct it into primary processing in Drosophila ovaries and in an ovarian cell culture model. In the fly ovarian somatic follicle cells, the transcript becomes cleaved in a stepwise manner, with a 5′→3′ directionality, liberating U1-containing ~24 nt piRNAs that are loaded into Piwi. Although uridines are preferred for generation of piRNA 5′ ends, processing takes place even in their absence, albeit at a lower efficiency. We show that recombinant Armi has 5′→3′ helicase activity, and mutations that abolish this activity also reduce piRNA processing in vivo. Another somatic piRNA pathway factor Yb, an interactor of Armi, is also able to trigger piRNA biogenesis when tethered to a transcript. Tethering-mediated primary piRNA biogenesis is also functional in the fly ovarian germline and loads all the three PIWI proteins present in this environment. Our study finds a broad correlation between piRNA processing and localization of the tethered factors to the cytoplasmic perinuclear ribonucleoprotein granules called germline nuage or somatic Yb bodies. We conclude that transcripts bound by Armi and Yb are identified as piRNA precursors, resulting in localization to cytoplasmic processing granules and their subsequent engagement by the resident piRNA biogenesis machinery.被称为PIWI互作RNA（PIWI-interacting RNAs）的小型RNA对于动物体内的转座子调控与生殖力维持至关重要。初级加工通路是一类以长单链RNA前体为底物，生成数百万条独立piRNAs的小型RNA生物合成途径，但目前人们对识别转录本为piRNA前体的分子机制尚不清楚。本研究证实，将piRNA生物合成因子Armi人工锚定至某一转录本，即可使其在果蝇（Drosophila）卵巢及卵巢细胞培养模型中进入初级加工途径。在果蝇卵巢体细胞滤泡细胞中，该转录本会以逐步方式被切割，且切割方向为5′→3′，最终释放出含U的约24 nt piRNAs，这些piRNAs会被装载进入Piwi蛋白。尽管piRNA 5′端的生成偏好使用尿嘧啶核苷酸，但即便缺乏尿嘧啶核苷酸，加工过程仍可发生，只是效率较低。我们的实验表明，重组Armi蛋白具有5′→3′解旋酶活性，而丧失该活性的突变体在体内会减弱piRNA的加工过程。另一类体细胞piRNA通路因子Yb作为Armi的互作蛋白，当被锚定至转录本时，同样能够触发piRNA的生物合成。锚定介导的初级piRNA生物合成在果蝇卵巢生殖系中同样具有功能，且可装载该环境中存在的全部三种PIWI蛋白。本研究发现，piRNA加工过程与锚定因子的定位存在广泛关联：锚定因子会被转运至细胞质核周核糖核蛋白颗粒——这类颗粒在生殖系中被称为nuage颗粒，在体细胞中则被称为Yb小体。综上，被Armi与Yb结合的转录本会被识别为piRNA前体，进而被转运至细胞质加工颗粒中，并由其中固有的piRNA生物合成复合物完成后续加工。