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Mitotic roles of the Nuclear Transport Machinery

核运输机械的有丝分裂作用

基本信息

批准号：
10915324
负责人：
MARY C. DASSO
金额：
$ 254.65万
依托单位：
EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
依托单位国家：
美国
项目类别：
财政年份：
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/10915324
关键词：
Acute Amyotrophic Lateral Sclerosis Auxins Binding Biological CRISPR/Cas technology Cell Cycle Cell Line Cell Nucleus Cell Survival Cell physiology Cells Chromatin Chromosome Segregation Complex Cytoplasm Cytoplasmic Filaments Defect 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 Hour Housekeeping Human Human Genome Incubated Individual Interphase Interphase Cell Kidney Diseases Kinetochores MXD1 gene Maintenance 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 Nucleoplasm Organism Pathology Phenotype Physiological Play Process Proteins RNA RNA Interference Regulation Role Running SUMO1 gene Side Steroid-resistant idiopathic nephrotic syndrome System Time Tissues Work cell growth chromatin remodeling 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 postmitotic receptor recruit response scaffold tissue/cell culture trafficking transcriptome sequencing transcriptomics

项目摘要

Exchange of molecules between the cytoplasm and the nucleus in all human cells 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 have systematically targeted 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 proteins associated with the basket (SENP1, SENP2, MAD1) on each other, on the stability of the assembled nuclear pore, and ability to reform the nuclear pore post mitosis. We find 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 the asymmetrically localized Ran GTP exchange factor, which is chromatin-bound, and cytosolic Ran GTPase activating protein, RanGAP. Mammalian RanBP2 binds the SUMO1-modified form of 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 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.

在所有人类细胞中，细胞质和细胞核之间的分子交换都是通过称为核孔复合物（npc）的管道进行的，核孔复合物由大约30种不同的蛋白质（核孔蛋白）组成，形成一个中心通道，其细丝延伸到细胞核和细胞质中。除了大分子运输外，核孔蛋白还通过与基因组的相互作用参与基因表达的控制，以及染色质维持和有丝分裂过程。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。