Regulation of piRNA pathway by the Nuclear Pore Complex

项目摘要/摘要 转座元件(TES)，也称为跳跃基因，是一种可移动的DNA元件，从 基因组中一个基因组位置与另一个基因组位置的对应关系。TES的活性可以引起基因组的重大变化 结构，必须加以限制，以防止发育缺陷、衰老、神经退行性疾病和 癌症。在生殖系发育期间，TE的流动性被沉默，以防止基因组变化被传递 传到后代身上。在生殖系中，piRNA途径沉默转座子。PiRNA的主要功能 途径是由PIWI分支蛋白介导的，在果蝇中PIWI分支蛋白由Piwi，Aubergine(Aub)和 ArgAerte 3(Ago3)蛋白。这些蛋白质结合piRNAs(长度约26个核苷酸)，并通过 序列特异性互补性。这三种蛋白质都具有沉默TE的非冗余功能。TE 生殖系中的沉默是被分割的。Piwi是有核的，是转录沉默所必需的 特斯。AUB和Ago3是细胞质，是转录后沉默TES所必需的。大多数人 PiRNAs是由aub和ago3参与的乒乓球扩增所产生的，这种乒乓球扩增发生在nuage中，一种富含rna的物质。 核周颗粒。在生殖系细胞中，Aub和Ago3参与乒乓循环，产生piRNAs和 这些piRNAs被加载到piwi上；然而，无论是机制还是参与偶联的蛋白质 将piRNA装载到piwi上的piRNA生物发生是已知的。AuB和Ago3需要与piRNA相互作用 生物发生；然而，Aub和Ago3相互作用的机制尚不清楚。无缝网络需求 存在于核孔复合体(NPC)和核之间，以确保Piwi-piRNA复合体 在核中组装的转座子移位到核中并沉默转座子；然而，通过 全国人大可能会对Piwi核功能进行哪些监管是难以捉摸的。我们的初步数据首次显示， Nup358是鼻咽癌细胞质细丝的关键成分，与Piwi相互作用，是Piwi‘s所必需的 进入细胞核，b)TE沉默，c)将piRNA装载到Piwi上，d)piRNA生物发生，以及e)Aub-Ago3 互动。这些数据表明，Nup358是piRNA途径中的关键参与者，并通过表征 Nup358调节piRNA途径，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。这一提议的科学前提是，有足够的 有证据表明Nup358是piRNA途径中的关键角色，但Nup358实现 同样的情况也难以捉摸。根据初步数据，我们假设Nup358招募Piwi到核 膜，并将piRNA生物生成与piwi上的piRNA负载结合起来。为了检验这一假设，我们将 生物化学定义Piwi-Nup358相互作用(Aim I)并解开Nup358偶联的机制 将piRNA负载到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).