Regulation of piRNA pathway by the Nuclear Pore Complex

核孔复合物对 piRNA 途径的调节

基本信息

批准号：
10404495
负责人：
VAMSI GANGARAJU
金额：
$ 31.4万
依托单位：
MEDICAL UNIVERSITY OF SOUTH CAROLINA
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-08-01 至 2024-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10404495
关键词：
Address Aging Applications Grants Aubergine Binding Proteins Biochemical Biogenesis Cell Nucleus Cells Complex Couples Coupling Cytoplasmic Filaments Cytoplasmic Granules DNA Transposable Elements Data Defect Development Drosophila genus Elements Ensure Gene Silencing Genetic Genome Genome Stability Genomics Jumping Genes Length Location Maintenance Malignant Neoplasms Mass Spectrum Analysis Mediating Modeling Molecular Mutate Neurodegenerative Disorders Nuclear Nuclear Envelope Nuclear Pore Nuclear Pore Complex Nucleotides Pathway interactions Pore Proteins Proteins Publishing RNA RNA Binding RNA Interference Regulation Reproductive Health Role Sterility Structure Testing Time Transcriptional Silencer Elements Transgenic Organisms base design fly genome integrity in vivo insight knock-down mutant novel piRNA prevent recruit transposon/insertion element

项目摘要

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).

项目概要/摘要转座元件 (TE)，也称为跳跃基因，是从基因组中一个基因组位置与另一个基因组位置的关系。 TE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。在种系发育过程中，TE 的流动性被抑制，以防止基因组变化被传递到后代。在种系中，piRNA 途径使转座子沉默。 piRNA的主要功能该途径由 PIWI 分支蛋白介导，在果蝇中以 Piwi、茄子 (Aub) 和 Argonaute 3 (Ago3) 蛋白。这些蛋白质结合 piRNA（长度约 26 个核苷酸）并通过序列特异性互补性。所有三种蛋白均具有非冗余的 TE 沉默功能。 TE 种系中的沉默是区室化的。 Piwi 是核蛋白，是转录沉默所必需的 TE。 Aub 和 Ago3 位于细胞质中，是 TE 转录后沉默所必需的。大多数 piRNA 由 Aub 和 Ago3 产生，涉及在 nuage（富含 RNA 的细胞）中发生的乒乓扩增核周颗粒。在生殖细胞中，Aub 和 Ago3 参与乒乓循环以产生 piRNA 和这些 piRNA 被加载到 Piwi 上；然而，无论是机制还是参与偶联的蛋白质都没有将piRNA加载到Piwi上的piRNA生物发生是已知的。 Aub 和 Ago3 需要相互作用才能生成 piRNA 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物 (NPC) 和 nuage 之间，以确保 Piwi-piRNA 复合物在 nuage 中组装易位到细胞核并沉默转座子；然而该机制通过 NPC 可能会调节 Piwi 核功能，目前尚不清楚。我们的初步数据首次表明 Nup358 是 NPC 细胞质丝的关键成分，与 Piwi 相互作用，并且是 a) Piwi 所必需的进入细胞核，b) TE 沉默，c) 将 piRNA 加载到 Piwi 上，d) piRNA 生物发生，以及 e) Aub-Ago3 相互作用。这些数据表明 Nup358 是 piRNA 通路中的关键参与者，并通过表征如何 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。这个提议的科学前提是有足够的有证据表明 Nup358 是 piRNA 途径中的关键参与者，但 Nup358 实现这一作用的机制同样是难以捉摸的。根据初步数据，我们假设 Nup358 将 Piwi 招募到核中膜并将 piRNA 生物发生与加载到 Piwi 上的 piRNA 偶联。为了检验这个假设，我们将从生物化学角度定义 Piwi-Nup358 相互作用（目标 I）并揭示 Nup358 偶联机制 piRNA 生物合成，piRNA 加载到 Piwi 上（目标 II）。