Mitotic roles of the Nuclear Transport Machinery

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

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.