Mitotic roles of the Nuclear Transport Machinery

核运输机械的有丝分裂作用

• 批准号: 10691097
• 负责人: MARY C. DASSO
• 金额: $ 220.01万
• 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/10691097
• 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; 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是通过称为核孔复合物（NPC）的导管发生的，该导管由大约30种不同的蛋白质（核孔）组成，形成了一个中心通道，其细丝延伸到核和细胞质中。除了大分子运输外，核孔蛋白还通过与基因组以及染色质维持和有丝分裂进展来控制基因表达。它们在这些不同的过程中的作用为生物学调节和细胞功能之间的协调提供了许多可能的机制。最近的发现记录了许多由核孔扰动引起的发育阶段或组织特异性表型，与复杂的作用相一致，这些作用超出了简单的管家功能。此外，核孔功能受到损害的人类疾病表现出非常明显的组织特异性表型，例如在神经退行性疾病中，如肌萎缩性侧面硬化症（ALS）或肾脏疾病（如诸如类固醇抗性肾病综合征（SRNS））中的肾脏疾病。但是，了解单个核孔蛋白在脊椎动物细胞中的作用是有限的，因为它们通过标准方法（例如RNAi）进行操作由于它们的丰度及其在细胞活力中的多个基本作用而存在问题：脊椎动物核核蛋白耗竭会导致高度的菌落现象，其中许多可能会导致替代效果。为了解决这个问题，我们使用CRISPR/CAS9基因编辑来系统地靶向核孔蛋白基因，以创建细胞系，其中内源性核苷具有生长素可诱导的Degron（AID）标签，从而可以快速且受调节的方式降解。我们正在使用这种方法来分析各种情况下各个核孔蛋白的功能。这项工作的主要目的是破译基于基于核孔蛋白的发育表型和组织特异性病理的特定机制和细胞过程。目前，我们专注于NPC的三个领域。 首先，NUP153，TPR和NUP50定位于核质细丝，它们被统称为篮子核苷。已经提出了核质细丝作为RNA修饰和导出的平台，以及染色质重塑。辅助标记的篮子核孔正确定位，在NPC中起作用，并在加入生长素（<2小时）上迅速降解。为了评估每个核孔蛋白的作用，我们在不存在和存在生长素以及核运输以及基因表达谱（RNA-Semecting）中的直接反应下跟随细胞生长。此外，我们评估了篮子成分的相互依存关系，并与篮子蛋白（SENP1，SENP2，MAD1）相关，这是根据组装核孔的稳定性以及改革有丝分裂后改革核孔的能力。我们的数据表明，单个篮子核孔在核功能和基因表达中起着不同的作用，并且该系统为我们提供了在分子水平上剖析这些作用的能力。 TPR的急性耗竭尤其引起转录组轮廓的快速变化。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的耗竭或转录 - export-Export-2（TREX-2）mRNA MRNA导出复合物引起的变化密切相关。此外，TPR耗竭破坏了TREX-2亚基（GANP，PCID2，ENY2）与NPC的关联，并在RNA异常的RNA转录和导出中结果。我们的发现表明，通过TREX-2-和/或NXF1依赖性mRNA转换，TPR在基因表达中的独特而关键的作用。 其次，NPC的中央结构域由三个同轴环组成，每个环分别显示出晶格样的排列，分别称为细胞质环，内环和核质环。 NUP107-160复合物包含9个核心核苷（NUP37，NUP85，SEH1，SEC13，NUP96，NUP96，NUP107，NUP133和NUP133和NUP160），其中第十个亚基，称为Elys所需的ELYS所需的Chomatin募集所需。 NUP107-160络合物形成了细胞质和核环的基础支架。 NUP107-160复合物还将其与动力学有丝分裂中的动力学相关联，在该分裂中，它在纺锤体组装和染色体隔离中起着独立于运输的作用。在相间和有丝分裂过程中对个体脊椎动物NUP107-160复合构件进行体内分析的早期努力是有问题的，因为它们的丰度和稳定性使得它们难以被RNAi耗尽：耗尽所需的延长时间会导致相互作用和有丝分裂功能的逐渐缺陷，从而产生不利的次要后果。此外，在延长的RNAi耗竭期间，非靶向亚基的水平降低，可能表明当不存在较大的复合物时它们变得不稳定。辅助标签的NUP107-160复合核丁物组装成功能性NPC，加强生长素后，它们迅速降解（<4小时），对其他NUP107-160复合构件的稳定性影响很小。我们已经通过比较了生长素的存在和存在，评估了NUP107-160复合亚基在核贩运中的作用。现在，我们正在研究单个复合构件如何促进NPC的结构稳定性，以及NUP107-160复杂持久性的亚基之间的相互依赖性，以及纺锤体功能和丝质后NPC组装。 第三，我们正在研究与细胞质丝（CFS）相关的核苷，其中包括RANBP2（也称为NUP358）。 RAN GTP/RANGDP细胞梯度对于核胞质转运，核包膜（NE）组装和有丝分裂染色体分离至关重要。该梯度是由染色质结合的不对称局部rans GTP交换因子的活性以及RANS rans GTPase激活蛋白Rangap的胞质定位的活性来确定的。哺乳动物RANBP2结合了Rangap（Rangap1-Sumo1）的SUMO1修饰形式，并在稳定的复合物（RRSU复合物）中结合了SUMO共轭酶UBC9。在有丝分裂过程中，RRSU复合物以CRM1和RAN依赖性方式与有丝分裂运动学相关，并且该募集对于形成纺锤体 - 金底附件很重要。虽然在多细胞生物的NE中，Rangap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，我们使用了人体组织培养细胞和果蝇。 Rangap1 NE定位的破坏令人惊讶地对组织培养细胞活力产生明显影响，也没有引起模型底物的核质转运的缺陷。然后，我们专注于果蝇，并鉴定出核孔蛋白DMranBP2中的一个区域，该区域是直接，独立于SUMO独立于DMRANGAP到NPC的区域。我们已经分析了这种相互作用中断的突变体的发育表型。总的来说，我们的结果表明，尽管DMrangap在NE中的定位在多细胞生物中广泛保守，但靶向机制却没有。此外，我们发现在组织发育过程中，这种定位至关重要。 这些实验共同表明，我们现在能够评估单个核孔蛋白在相间和有丝分裂过程中的重要细胞过程中的功能，并在分子水平上剖析这些过程。这为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种发育表型提供了绝佳的机会。