A transcriptome-wide RNAi screen in the Drosophila ovary reveals factors of the germline piRNA pathway [RNA-Seq]. Drosophila melanogaster

The Drosophila piRNA pathway provides an RNA-based immune system that defends the germline genome against selfish genetic elements. Two inter-related branches of the piRNA system exist: somatic cells that support oogenesis only employ Piwi, whereas germ cells utilize a more elaborated pathway centered on the three gonad-specific Argonaute proteins Piwi, Aubergine, and Argonaute3. While several key factors of each branch have been identified, our current knowledge is insufficient to explain the complex workings of the piRNA machinery. Here, we report a reverse genetic screen spanning the ovarian transcriptome in an attempt to uncover the full repertoire of genes required for piRNA-mediated transposon silencing in the female germline. Our screen reveals new key factors of piRNA-mediated transposon silencing, including the novel piRNA biogenesis factors, CG2183 (GASZ) and Deadlock. Last, our data uncovers a previously unanticipated set of factors preferentially required for repression of different transposons types. Overall design: Examination of total RNA levels from nos-GAL4 or tj-GAL4 driven UAS-dsRNA knockdowns of control genes and piRNA pathway components in ovaries of Drosophila melanogaster by deep sequencing (using Illumina HiSeq2000).

果蝇piRNA通路（piRNA pathway）是一类基于RNA的免疫系统，可保护生殖系基因组免受自私遗传元件的侵袭。该piRNA系统存在两个相互关联的分支：支持卵子发生的体细胞仅表达Piwi蛋白（Piwi），而生殖细胞则采用以三种性腺特异性Argonaute蛋白（Argonaute）——Piwi、Aubergine与Argonaute3——为核心的更为复杂的通路。尽管目前已鉴定出两个分支中的多个关键因子，但我们当前的认知仍不足以阐明piRNA复合物的复杂运作机制。
本研究针对果蝇卵巢转录组开展反向遗传筛选，以期全面鉴定雌性生殖系中piRNA介导的转座子沉默所需的全套基因。本次筛选鉴定出多个piRNA介导转座子沉默的新型关键因子，包括新发现的piRNA生物发生因子CG2183（GASZ）与Deadlock。此外，本研究数据还揭示了一组此前未被预见的因子，它们可优先参与不同类型转座子的沉默抑制。
实验设计概述：通过深度测序（采用Illumina HiSeq2000平台），检测黑腹果蝇卵巢中由nos-GAL4或tj-GAL4驱动的UAS-dsRNA介导的对照基因与piRNA通路组分敲低样本的总RNA水平。