Regulation of piRNA pathway by the Nuclear Pore Complex

核孔复合物对 piRNA 途径的调节

• 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
• 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

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).

项目摘要/摘要 转座元素（TES），也称为跳跃基因是移动DNA元素，从 一个基因组中的一个基因组位置。 TE的活性会导致基因组的重大变化 结构，必须限制以防止发育缺陷，衰老，神经退行性疾病和 癌症。在种系开发过程中，迁移率是沉默的，以防止基因组变化通过 到后代。在种系中，piRNA途径静音转座子。 PIRNA的主要功能 途径是由Piwi进化枝蛋白介导的，果蝇中由Piwi，Aubergine（Aub）和 Argonaute 3（AGO3）蛋白质。这些蛋白质结合PIRNA（长度约为26个核苷酸），并通过 序列特定的互补性。这三种蛋白质在沉默的TE中具有非冗余功能。 TE 种系中的沉默被分隔了。 Piwi是核的，是需要转录沉默的 TES。 AUB和AGO3是细胞质的，是TES转录后沉默所必需的。多数 PIRNA由AUB制造，Ago3涉及在富含RNA的NUAGE中发生的乒乓放大 核周颗粒。在种系细胞中，AUB和AGO3参与PING-PONG循环以产生PIRNA和 这些PIRNA被加载到Piwi上；但是，偶联的机制和蛋白质均不 已知的PIRNA生物发生在PIWI上载有PIRNA。 AUB和AGO3需要与Pirna相互作用 生物发生；但是，尚不清楚AUB和AGO3相互作用的机制。无缝网络需求 存在于核孔复合物（NPC）和分布之间，以确保Piwi-Pirna复合物 组装在细胞核和沉默的转座子中；但是机制是 NPC可能会调节Piwi核功能是难以捉摸的。我们的初步数据首次显示 NUP358是NPC的细胞质细丝的关键组成部分，与PIWI相互作用，是PIWI所必需的 进入细胞核，b）沉默，c）将PIRNA载入PIWI，d）PIRNA生物发生，E）aub-ago3 相互作用。这些数据表明NUP358是PIRNA途径的关键参与者，并通过表征如何 NUP358调节了Pirna途径，我们将揭示有关NPC如何促进PIRNA的重要见解 生物发生，沉默和基因组稳定性。该提议的科学前提是有足够的 NUP358是Pirna Pathway的关键参与者的证据，但是NUP358实现的机制 同样难以捉摸。根据初步数据，我们假设NUP358将PIWI招募到核 膜和伴侣Pirna生物发生，将piRNA载入PIWI。为了检验这一假设，我们将 生化定义Piwi-Nup358相互作用（AIM I）并解开NUP358夫妇的机制 piRNA生物发生，piRNA载入PIWI（AIM II）。