Mitotic roles of the Nuclear Transport Machinery

核运输机械的有丝分裂作用

• 批准号: 10267570
• 负责人: MARY C. DASSO
• 金额: $ 208.59万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10267570
• 关键词: Acute; Amyotrophic Lateral Sclerosis; Aneuploidy; Auxins; Binding; Biological; CRISPR/Cas technology; Cell Line; Cell Nucleus; Cell Survival; Cell physiology; Cells; Chromatin; Chromosome Segregation; Chromosomes; Complex; Cytoplasm; Cytoplasmic Filaments; Data; Defect; Dependence; Development; Developmental Process; Drosophila genus; Enzymes; Filament; GTPase-Activating Proteins; Gene Expression; Gene Expression Profile; Genes; Genetic Transcription; Genome; Goals; Guanine Nucleotide Exchange Factors; Guanosine Triphosphate; Guanosine Triphosphate Phosphohydrolases; Health; Hematologic Neoplasms; Hour; Housekeeping; Human; Individual; Interphase; Kidney Diseases; Kinetochores; MXD1 gene; Maintenance; Mental Retardation; Messenger RNA; Methods; Mitosis; Mitotic; Mitotic Chromosome; Mitotic spindle; Modeling; Modification; Molecular; Mutation; Neurodegenerative Disorders; Nuclear; Nuclear Envelope; Nuclear Export; Nuclear Import; Nuclear Pore; Nuclear Pore Complex; Nuclear Pore Complex Proteins; Organism; Pathology; Pathway interactions; Phenotype; Physiological; Play; Process; Proteins; RNA; RNA Interference; Regulation; Role; Side; Solid Neoplasm; Spontaneous abortion; Steroid-resistant idiopathic nephrotic syndrome; Structure; System; Time; Tissues; Work; base; cell growth; chromatin remodeling; chromosome missegregation; experimental study; human disease; human tissue; in vivo; mRNA Export; member; mutant; novel; nuclear pore complex protein 96; nuclear pore complex protein p107; nucleocytoplasmic transport; receptor; recruit; response; scaffold; tissue/cell culture; trafficking; transcriptome sequencing; transcriptomics

Exchange of molecules between the cytoplasm and the nucleus occurs through conduits called nuclear pore complexes (NPCs), which consist of roughly 30 distinct proteins (nucleoporins), forming a central channel with filaments extending into the nucleus and cytoplasm. Beyond macromolecular trafficking, nucleoporins participate in the control of gene expression via interactions with the genome, as well as in chromatin maintenance and mitotic progression. Their roles in these diverse processes offer a rich variety of possible mechanisms for biological regulation and coordination amongst cellular functions. Recent findings have documented many developmental stage- or tissue-specific phenotypes that result from nucleoporin perturbation, consistent with complex roles that extend beyond simple housekeeping functions. Moreover, human diseases in which nucleoporin function is compromised show remarkably tissue-specific phenotypes, as in neurodegenerative diseases like amyotrophic lateral sclerosis (ALS) or in renal diseases like steroid-resistant nephrotic syndromes (SRNS). However, understanding the roles of individual nucleoporins in vertebrate cells is limited because their manipulation by standard methods (e.g., RNAi) has been problematic due to their abundance and their multiple essential roles for cell viability: vertebrate nucleoporin depletion can cause highly pleiotropic phenotypes, many of which may be secondary consequences of extended incubations with sub-physiological nucleoporin levels. To circumvent this problem, we are systematically targeting nucleoporin genes using CRISPR/Cas9 gene editing to create cell lines wherein endogenous nucleoporins have Auxin Inducible Degron (AID) tags, allowing their degradation in a rapid and regulated manner. We are using this approach to analyze the function of individual nucleoporins in a variety of contexts. A major goal of this work is to decipher the specific mechanisms and cellular processes that underlie nucleoporin-based developmental phenotypes and tissue-specific pathologies. We are currently focused on three domains of the NPC. First, NUP153, TPR, and NUP50 localize to nucleoplasmic filaments, and they are collectively called the basket nucleoporins. The nucleoplasmic filaments have been proposed to serve as a platform for RNA modification and export, as well as for chromatin remodeling. AID-tagged basket nucleoporins localize correctly, are functional within NPCs and are rapidly degraded upon Auxin addition (<2 hours). To assess the role of each nucleoporin, we followed cell growth in the absence and presence of Auxin, as well as nuclear trafficking and the immediate response in gene expression profile (RNA-sequencing). Moreover, we assessed the interdependence of the basket components, and associated with the basket proteins (SENP1, SENP2, MAD1) on each other, on the stability of the assembled nuclear pore, and ability to reform the nuclear pore post mitosis. Our data show that individual basket nucleoporins play distinct roles in nuclear function and gene expression, and that this system provides us the capacity to dissect these roles at a molecular level. Acute depletion of TPR in particular caused rapid and pronounced changes in transcriptomic profiles. These changes were dissimilar to shifts observed after loss of NUP153 or NUP50, but closely related to changes caused by depletion of mRNA export receptor NXF1 or the GANP subunit of the TRanscription-EXport-2 (TREX-2) mRNA export complex. Moreover, TPR depletion disrupts association of TREX-2 subunits (GANP, PCID2, ENY2) to NPCs and results in abnormal RNA transcription and export. Our findings demonstrate a unique and pivotal role of TPR in gene expression through TREX-2- and/or NXF1-dependent mRNA turnover. Second, the central domain of NPCs consists of three co-axial rings that each display a lattice-like arrangement, and that are called the cytoplasmic ring, inner ring, and nucleoplasmic ring, respectively. The Nup107-160 complex contains nine core nucleoporins (Nup37, Nup85, Seh1, Sec13, Nup96, Nup107, Nup133 and Nup160), with a tenth subunit called ELYS required for chromatin recruitment. The Nup107-160 complex forms the scaffold underlying the cytoplasmic and nuclear rings. The Nup107-160 complex also associates with kinetochores in metazoan mitosis, where it plays a transport-independent role in spindle assembly and chromosome segregation. Earlier efforts at in vivo analysis of individual vertebrate Nup107-160 complex members during interphase and mitosis have been problematic because their abundance and stability makes them difficult to deplete by RNAi: The extended time required for depletion causes progressive defects in both interphase and mitotic functions that can produce adverse secondary consequences. Moreover, the levels of non-targeted subunits decrease during extended RNAi depletion, possibly suggesting that they become unstable when the larger complex is absent. AID-tagged Nup107-160 complex nucleoporins assemble into functional NPCs, and they are degraded rapidly (<4 hours) after auxin addition, with minimal impact on the stability of other Nup107-160 complex members. We have assessed the roles of Nup107-160 complex subunits in nuclear trafficking through comparison of nuclear import and export in the absence and presence of auxin. We are now examining how individual complex members contribute to the structural stability of NPCs, and the inter-dependence between subunits for Nup107-160 complex persistence at existing NPCs, as well as for spindle function and post-mitotic NPC assembly. Third, we are investigating nucleoporins associated with the cytoplasmic filaments (CFs), which include RanBP2 (also known as Nup358). The Ran GTP/RanGDP cellular gradient is critical for nuclear-cytoplasmic transport, nuclear envelope (NE) assembly and mitotic chromosome segregation. This gradient is established by the activities of asymmetrically localized Rans GTP exchange factor, which is chromatin-bound, and cytosolic localization of Rans GTPase activating protein, RanGAP. Mammalian RanBP2 binds the SUMO1-modified form of the RanGAP (RanGAP1-SUMO1), and the SUMO conjugating enzyme Ubc9 in a stable complex (RRSU complex). During mitosis, the RRSU complex associates to mitotic kinetochores in a Crm1- and Ran-dependent manner, and this recruitment is important for the formation of spindle-kinetochore attachments. While RanGAP is tethered to the cytoplasmic side of NE in multicellular organism, the functional consequences of its localization remain unknown. To investigate this issue, we used human tissue culture cells and Drosophila. Disruption of RanGAP1 NE localization surprisingly had neither an obvious impact on tissue culture cell viability nor did it cause defects in nucleocytoplasmic transport of a model substrate. We then focused on Drosophila and identified a region within the nucleoporin dmRanBP2 that is required for direct, SUMO-independent tethering of dmRanGAP to the NPC. We have analyzed the developmental phenotype of mutants in which this interaction is disrupted. Collectively, our results indicate that while the localization of dmRanGAP to the NE is widely conserved in multicellular organisms, the targeting mechanisms are not. Further, we find a requirement for this localization to be critical during tissue developmental processes. These experiments collectively indicate that we are now able to assess the function of individual nucleoporins in vital cellular processes during both interphase and mitosis, and to dissect these processes at a molecular level. This offers an excellent opportunity to assess novel mechanisms of cellular function and how they result in the diverse developmental phenotypes associated with mutations in nucleoporin genes.

细胞质和细胞核之间的分子交换通过称为核孔复合体 (NPC) 的管道进行，该管道由大约 30 种不同的蛋白质（核孔蛋白）组成，形成一个中央通道，其中的细丝延伸到细胞核和细胞质中。除了大分子运输之外，核孔蛋白还通过与基因组的相互作用参与基因表达的控制，以及染色质的维持和有丝分裂的进展。它们在这些不同过程中的作用为细胞功能之间的生物调节和协调提供了丰富多样的可能机制。最近的研究结果记录了许多由核孔蛋白扰动引起的发育阶段或组织特异性表型，这与超出简单内务功能的复杂作用一致。此外，核孔蛋白功能受损的人类疾病表现出明显的组织特异性表型，如肌萎缩侧索硬化症（ALS）等神经退行性疾病或类固醇抵抗性肾病综合征（SRNS）等肾脏疾病。然而，了解单个核孔蛋白在脊椎动物细胞中的作用是有限的，因为通过标准方法（例如RNAi）对其进行操作一直存在问题，因为它们的丰度及其对细胞活力的多种重要作用：脊椎动物核孔蛋白的消耗可导致高度多效性表型，其中许多可能是亚生理核孔蛋白水平延长孵育的继发后果。为了解决这个问题，我们使用 CRISPR/Cas9 基因编辑系统地靶向核孔蛋白基因，以创建细胞系，其中内源核孔蛋白具有生长素诱导降解子 (AID) 标签，从而使其能够以快速且受调控的方式降解。我们正在使用这种方法来分析各个核孔蛋白在各种情况下的功能。这项工作的一个主要目标是破译基于核孔蛋白的发育表型和组织特异性病理的具体机制和细胞过程。我们目前主要关注 NPC 的三个领域。 首先，NUP153、TPR和NUP50定位于核质丝，它们统称为篮核孔蛋白。核质丝被提议作为 RNA 修饰和输出以及染色质重塑的平台。 AID 标记的篮子核孔蛋白正确定位，在 NPC 内发挥作用，并在添加生长素后迅速降解（<2 小时）。为了评估每种核孔蛋白的作用，我们跟踪了生长素不存在和存在下的细胞生长，以及核运输和基因表达谱的即时反应（RNA 测序）。此外，我们评估了篮子成分的相互依赖性，以及篮子蛋白（SENP1、SENP2、MAD1）之间的相互依赖性、组装核孔的稳定性以及有丝分裂后重组核孔的能力。我们的数据表明，单个篮子核孔蛋白在核功能和基因表达中发挥着不同的作用，并且该系统使我们能够在分子水平上剖析这些作用。 TPR 的急性耗竭尤其会导致转录组谱发生快速而明显的变化。这些变化与 NUP153 或 NUP50 丢失后观察到的变化不同，但与 mRNA 输出受体 NXF1 或 TRanscription-EXport-2 (TREX-2) mRNA 输出复合物的 GANP 亚基耗尽引起的变化密切相关。此外，TPR 缺失会破坏 TREX-2 亚基（GANP、PCID2、ENY2）与 NPC 的关联，导致 RNA 转录和输出异常。我们的研究结果表明，TPR 通过 TREX-2 和/或 NXF1 依赖性 mRNA 更新在基因表达中发挥独特且关键的作用。 其次，NPC 的中心域由三个同轴环组成，每个环均呈格子状排列，分别称为细胞质环、内环和核质环。 Nup107-160 复合体包含九个核心核孔蛋白（Nup37、Nup85、Seh1、Sec13、Nup96、Nup107、Nup133 和 Nup160），其中第十个亚基称为 ELYS，是染色质招募所需的。 Nup107-160 复合物形成细胞质环和核环下方的支架。 Nup107-160 复合体还与后生动物有丝分裂中的动粒相关，在纺锤体组装和染色体分离中发挥独立于运输的作用。早期对个体脊椎动物 Nup107-160 复合体成员在间期和有丝分裂期间的体内分析一直存在问题，因为它们的丰度和稳定性使它们难以被 RNAi 耗尽：耗尽所需的时间延长会导致间期和有丝分裂功能的渐进性缺陷，从而产生不利的继发后果。此外，非靶向亚基的水平在长期 RNAi 消耗期间下降，这可能表明当较大的复合物不存在时它们会变得不稳定。 AID 标记的 Nup107-160 复合体核孔蛋白组装成功能性 NPC，并且在添加生长素后迅速降解（<4 小时），对其他 Nup107-160 复合体成员的稳定性影响极小。我们通过比较不存在和存在生长素的情况下的核进出口，评估了 Nup107-160 复合亚基在核贩运中的作用。我们现在正在研究单个复合体成员如何促进 NPC 的结构稳定性，以及现有 NPC 中 Nup107-160 复合体持久性的亚基之间的相互依赖性，以及纺锤体功能和有丝分裂后 NPC 组装的相互依赖性。 第三，我们正在研究与细胞质丝 (CF) 相关的核孔蛋白，其中包括 RanBP2（也称为 Nup358）。 Ran GTP/RanGDP 细胞梯度对于核质运输、核膜 (NE) 组装和有丝分裂染色体分离至关重要。该梯度是由不对称定位的 Rans GTP 交换因子（与染色质结合）的活性和 Rans GTP 酶激活蛋白 RanGAP 的胞质定位建立的。哺乳动物 RanBP2 与 RanGAP 的 SUMO1 修饰形式 (RanGAP1-SUMO1) 以及稳定复合物（RRSU 复合物）中的 SUMO 结合酶 Ubc9 结合。在有丝分裂期间，RRSU 复合体以 Crm1 和 Ran 依赖性方式与有丝分裂着丝粒结合，这种募集对于纺锤体-着丝粒附着的形成非常重要。虽然 RanGAP 在多细胞生物中被束缚在 NE 的细胞质侧，但其定位的功能后果仍然未知。为了研究这个问题，我们使用了人类组织培养细胞和果蝇。令人惊讶的是，RanGAP1 NE 定位的破坏既不会对组织培养细胞的活力产生明显影响，也不会导致模型底物的核细胞质运输缺陷。然后，我们关注果蝇并确定了核孔蛋白 dmRanBP2 内的一个区域，该区域是 dmRanGAP 与 NPC 直接、不依赖 SUMO 的束缚所必需的。我们分析了这种相互作用被破坏的突变体的发育表型。总的来说，我们的结果表明，虽然 dmRanGAP 的 NE 定位在多细胞生物中广泛保守，但靶向机制却并非如此。此外，我们发现这种定位的要求在组织发育过程中至关重要。 这些实验共同表明，我们现在能够评估单个核孔蛋白在间期和有丝分裂期间重要细胞过程中的功能，并在分子水平上剖析这些过程。这提供了一个极好的机会来评估细胞功能的新机制以及它们如何导致与核孔蛋白基因突变相关的多样化发育表型。