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 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 wide range of possible mechanisms for biological regulation and coordination amongst cellular functions. Nucleoporin perturbation can cause a variety of developmental stage- or tissue-specific phenotypes, 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 or in renal diseases like steroid-resistant nephrotic syndromes. 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 comprise the 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. 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 transcriptomic profiles. We further assessed the interdependence of the basket nucleoporins and other proteins associated with the basket for localization at the NPC. We find that individual basket nucleoporins play distinct roles in nuclear function and gene expression. 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, revealing a key role of TPR in TREX-2-mediated mRNA export. We are currently utilizing the AID degron system to elucidate the function of individual TREX-2 subunits (GANP, PCID2, ENY2, DSS1 and CETN2/3) and how they interface with the NPC during RNA export. We are also examining how mitotic regulatory proteins (Mad1, Mad2, p31Comet) associate to the NPC basket and how this association impacts mitotic progression. 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. 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 Nup107-160 complex subunits for 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 mammalian 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. We find that although the localization of RanGAP to the NE is widely conserved in multicellular organisms, the targeting mechanisms are not. Further, disruption of RanGAP association to the NE did not cause obvious disruption of either bulk nuclear trafficking or mitotic progression in flies (D. melanogaster), yet this localization was critical during tissue developmental processes. We are currently examining the importance of RanGAP localization for differentiation, particularly in the context of spermatogenesis using the fly testis as a model system. 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.