Aub, Vasa and Armi concentration in phase separated nuage is dispensable for piRNA biogenesis and transposon silencing

The piRNA biogenesis machinery localizes to phase-separated nuage granules, but nuage function is not well understood. We therefore assayed nuage composition, piRNA expression, and transposon silencing in Drosophila mutants that disrupt piRNA precursor production and nuclear export, ping-pong amplification, and phased piRNA biogenesis. These mutations destabilize the genome and activate Chk2 signaling, and chk2/mnk double mutants were therefore analyzed in parallel. Aub and Vasa are required for ping-pong amplification, and Armi promotes phased piRNA processing. We show that Chk2 activation releases Aub and Vasa from nuage and that piRNA precursors are required for nuage localization of the ping-pong and phased biogenesis machinery. However, this analysis also indicates that Vasa, Aub, and Armi concentration in nuage is dispensable for piRNA production and transposon silencing, indicating that dispersed cytoplasmic proteins can drive these processes. We speculate that nuage sequesters silencing effectors, which are released by Chk2 in response to transposon mobilization. Overall design: We evaluated long RNA transcript expression and small RNA expression patterns in various piRNA pathway mutants and corresponding mnk (Chk2) double mutants.

Piwi互作RNA（piRNA）的生成系统定位于相分离的nuage颗粒，但目前对于nuage的功能尚不清楚。为此，我们针对破坏piRNA前体生成与核输出、乒乓扩增以及阶段性piRNA生成的果蝇突变体，检测了nuage的组成、piRNA的表达模式以及转座子沉默情况。这类突变会导致基因组不稳定并激活Chk2（Checkpoint激酶2）信号通路，因此我们同时对chk2/mnk双突变体开展了分析。Aub（Aubergine）与Vasa（Vasa蛋白）是乒乓扩增所必需的，而Armi（Armitage）则促进阶段性piRNA的加工过程。我们的研究表明，Chk2的激活会将Aub与Vasa从nuage中释放出来，且piRNA前体是乒乓扩增与阶段性生成系统定位于nuage所必需的。不过，本分析同时也显示，nuage中Vasa、Aub与Armi的浓度对于piRNA生成与转座子沉默并非必需，这说明分散在细胞质中的蛋白质即可驱动这些过程。我们推测，nuage会隔离沉默效应因子，而当转座子发生转座时，Chk2会释放这些因子。实验设计概述：我们对多种piRNA通路突变体及其对应的mnk（Chk2）双突变体的长链RNA转录本表达与小RNA表达模式进行了评估。