Regulation of piRNA pathway by the Nuclear Pore Complex

项目总结/摘要 转座因子（transposable elements，TE），也被称为跳跃基因，是移动的DNA元件，从 基因组中的一个基因组位置到另一个。TEs的活性可以引起基因组的重大变化 结构和必须限制，以防止发育缺陷，衰老，神经退行性疾病， 癌TE移动性在种系发育期间被沉默，以防止基因组变化被传递 传给后代在种系中，piRNA途径使转座子沉默。piRNA的主要功能 该途径由PIWI进化枝蛋白介导，其在果蝇中由Piwi、Aubergine（Aub）和 Argonaute 3（Ago3）蛋白。这些蛋白质结合piRNA（长度约26个核苷酸）并通过以下方式靶向TE： 序列特异性互补性。所有三种蛋白质在沉默TE中具有非冗余功能。te 种系中的沉默被划分。Piwi是核的，是转录沉默所必需的。 TEs。Aub和Ago3是细胞质的，并且是TE的转录后沉默所需要的。大多数 piRNA由Aub和Ago3产生，涉及发生在nuage中的乒乓扩增， 核周颗粒在生殖系细胞中，Aub和Ago3参与乒乓循环以产生piRNA， 这些piRNA被加载到Piwi上;然而，无论是机制还是参与偶联的蛋白质， 利用piRNA加载到Piwi上的piRNA生物发生是已知的。Aub和Ago3需要相互作用以获得piRNA 生物起源;然而，Aub和Ago3相互作用的机制尚不清楚。无缝网络需要 存在于核孔复合物（NPC）和核之间，以确保Piwi-piRNA复合物 组装在nuage易位到细胞核和沉默转座子;然而，该机制， NPC可能调节Piwi核功能的机制尚不清楚。我们的初步数据首次显示， Nup358是NPC细胞质丝的关键组分，与Piwi相互作用，并且是a）Piwi's 进入细胞核，B）TE沉默，c）将piRNA加载到Piwi上，d）piRNA生物发生，和e）Aub-Ago 3 互动这些数据表明，Nup358是piRNA途径中的关键参与者，并且通过表征Nup358如何参与piRNA途径， Nup358调节piRNA通路，我们将揭示NPC如何促进piRNA的重要见解 生物发生、TE沉默和基因组稳定性。这一建议的科学前提是， 证据表明Nup358是piRNA途径中的关键参与者，但是Nup358实现piRNA途径的机制是不确定的。 同样难以捉摸。根据初步数据，我们假设Nup358将Piwi招募到核 膜，并将piRNA生物合成与piRNA加载到Piwi上相结合。为了验证这个假设，我们将 生物化学定义Piwi-Nup358相互作用（Aim I）并阐明Nup358偶联的机制 将piRNA加载到Piwi上的piRNA生物发生（Aim II）。

Project Summary/Abstract Transposable elements (TEs), also known as jumping genes are mobile DNA elements that move from one genomic location to the other in the genome. The activity of TEs can cause major changes in genome structure and must be restricted to prevent developmental defects, aging, neurodegenerative diseases and cancer. TE mobility is silenced during germline development to prevent genome changes from being passed on to the progeny. In the germline, piRNA pathway silences transposons. The primary function of the piRNA pathway is mediated by PIWI clade proteins which in Drosophila are represented by Piwi, Aubergine (Aub) and Argonaute 3 (Ago3) proteins. These proteins bind piRNAs (~26 nucleotides in length) and target TEs through sequence-specific complementarity. All three proteins have non-redundant function in silencing TE. TE silencing in the germline is compartmentalized. Piwi is nuclear and is needed for transcriptional silencing of TEs. Aub and Ago3 are cytoplasmic and are needed for post-transcriptional silencing of TEs. Majority of piRNAs are made by Aub and Ago3 involved ping-pong amplification that occurs in the nuage, a RNA-rich perinuclear granule. In the germline cells, Aub and Ago3 participate in ping-pong cycle to produce piRNAs and these piRNAs are loaded onto Piwi; however, neither the mechanism nor the proteins involved in coupling piRNA biogenesis with piRNA loading onto Piwi are known. Aub and Ago3 need to interact for piRNA biogenesis; however the mechanism by which Aub and Ago3 interact is not known. A seamless network needs to exist between the nuclear pore complex (NPC) and the nuage to ensure that Piwi-piRNA complexes assembled in the nuage translocate into the nucleus and silence transposons; however the mechanism by which the NPC might regulate Piwi nuclear function is elusive. Our preliminary data shows for the first time that Nup358, a key component of cytoplasmic filaments of the NPC, interacts with Piwi and is required for a) Piwi's entry into the nucleus, b) TE silencing, c) loading of piRNAs onto Piwi, d) piRNA biogenesis, and e) Aub-Ago3 interaction. These data suggest that Nup358 is a key player in piRNA pathway and by characterizing how Nup358 regulates piRNA pathway, we will reveal significant insights into how the NPC promotes piRNA biogenesis, TE silencing and genome stability. The scientific premise of this proposal is that there is sufficient evidence that Nup358 is a key player in piRNA pathway, but the mechanism by which Nup358 achieves the same is elusive. Based on the preliminary data, we hypothesize that Nup358 recruits Piwi to the nuclear membrane and couples piRNA biogenesis with piRNA loading onto Piwi. To test this hypothesis, we will biochemically define Piwi-Nup358 interaction (Aim I) and unravel the mechanism by which Nup358 couples piRNA biogenesis with piRNA loading onto Piwi (Aim II).