Multiple roles for Piwi in silencing Drosophila transposons
Multiple roles for Piwi in silencing Drosophila transposons
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DOI：
10.1101/gad.209767.112
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发表时间：
2013-02-15
影响因子：
10.5
通讯作者：
Hannon, Gregory J.
中科院分区：
文献类型：
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作者：
Rozhkov, Nikolay V.;Hammell, Molly;Hannon, Gregory J.
果蝇卵巢转座子的沉默依赖于三种Piwi家族蛋白——Piwi、Aubergine （Aub）和ago3——与它们的小RNA向导Piwi相互作用RNA （piRNAs）协同作用。Aub和Ago3存在于生殖细胞细胞质中，它们在乒乓循环中消耗转座子mrna。在生殖和体细胞卵泡细胞中，核Piwi蛋白是转座子沉默所必需的，但Piwi作用的确切机制在很大程度上仍不清楚。我们通过结合细胞类型特异性敲低与稳态转座子mRNA水平、新生RNA合成、染色质状态和小RNA丰度的测量来研究Piwi的作用。在体细胞中，Piwi缺失导致对新生转录物和转座子mrna的协同作用，表明Piwi通过转录基因沉默（transcriptional gene silencing， TGS）起作用。在生殖细胞中，Piwi缺失对稳态RNA水平表现出不成比例的影响，表明它也对转录后基因沉默（PTGS）产生影响。Piwi敲低影响生殖细胞可能与Aub和Ago3结合的pirna水平，这可能解释了其转录后的影响。总的来说，我们的研究结果表明Piwi在piRNA通路中发挥多种作用，部分通过影响局部染色质状态和转录来强制转座子抑制，但也参与生殖细胞piRNA的生物发生。
Silencing of transposons in the Drosophila ovary relies on three Piwi family proteins-Piwi, Aubergine (Aub), and Ago3-acting in concert with their small RNA guides, the Piwi-interacting RNAs (piRNAs). Aub and Ago3 are found in the germ cell cytoplasm, where they function in the ping-pong cycle to consume transposon mRNAs. The nuclear Piwi protein is required for transposon silencing in both germ and somatic follicle cells, yet the precise mechanisms by which Piwi acts remain largely unclear. We investigated the role of Piwi by combining cell type-specific knockdowns with measurements of steady-state transposon mRNA levels, nascent RNA synthesis, chromatin state, and small RNA abundance. In somatic cells, Piwi loss led to concerted effects on nascent transcripts and transposon mRNAs, indicating that Piwi acts through transcriptional gene silencing (TGS). In germ cells, Piwi loss showed disproportionate impacts on steady-state RNA levels, indicating that it also exerts an effect on post-transcriptional gene silencing (PTGS). Piwi knockdown affected levels of germ cell piRNAs presumably bound to Aub and Ago3, perhaps explaining its post-transcriptional impacts. Overall, our results indicate that Piwi plays multiple roles in the piRNA pathway, in part enforcing transposon repression through effects on local chromatin states and transcription but also participating in germ cell piRNA biogenesis.