The Ran GTPase is required for many cellular functions, including nucleocytoplasmic trafficking, spindle assembly, nuclear assembly and cell cycle control. The sole nucleotide exchange factor for Ran, RCC1, binds chromatin throughout the cell cycle. The GTPase activating protein for Ran, RanGAP1, localizes to the cytosolic face of the nuclear pore complex (NPC) during interphase through association with RanBP2, a large nucleoporin. The interphase distribution of Ran regulators leads to a high concentration of Ran-GTP in nuclei, and low Ran-GTP in cytosol. The major effectors for Ran are a family of Ran-GTP binding proteins that were discovered as nuclear transport receptors. These receptors are collectively called Karyopherins;those that mediate import are called Importins, and those that mediate export are called Exportins. Karyopherins transit the NPC in a Ran- and cargo-independent fashion. Their cargo loading is governed by Ran-GTP levels: Importins bind to their cargo in the cytoplasm. Import complexes traverse the NPC and dissociate upon Ran-GTP-Importin binding. Exportins bind their cargo inside nuclei in complexes that contain Ran-GTP. After passage through the NPC, export complexes dissociate upon Ran-GTP hydrolysis. Notably, the capacity of Ran-GTP to bind some Importins and promote their dissociation from their cargos is conditional upon events other than nuclear import, such as the assembly of cargo into macromolecular complexes, suggesting that karyopherins can act as chaperones that assist in the targeting of cargos molecules beyond the NPC. To date, two karyopherins have been shown to act as Ran effectors during mitosis, Importin-beta and the exportin Crm1. Importin-beta binds and imports cargo with classical nuclear localization sequences (cNLSs) through an adaptor subunit, Importin-alpha. In mitotic metazoan cells, Importin-alpha/beta bind and inhibit spindle assembly factors. Elevation of diffusible Ran-GTP concentrations near mitotic chromatin releases inhibition by Importin-alpha/beta, allowing localized activation of such factors. Our studies have been particularly concerned with Ran functions at kinetochores. Kinetochores are proteinaceous structures that assemble at the centromere of each sister chromatid during mitosis, and that serve as sites of spindle microtubule (MT) attachment. The kinetochore fibers (k-fibers) that link mammalian kinetochores to spindle poles contain both MTs that are directly attached to the kinetochores at their plus ends (kMTs) and MTs that are not. Kinetochore attachment is monitored through the spindle assembly checkpoint (SAC), which prevents mitotic exit by blocking anaphase promoting complex/cyclosome (APC/C) activation until all chromosomes are attached and aligned onto the metaphase plate. The APC/C is a ubiquitin ligase that regulates the destruction of key mitotic regulatory proteins. Components of the SAC include: Mad1, Mad2, Mps1, Bub1, Bub3, BubR1, and CENP-E. We have examined the dynamics of RCC1 association to chromatin within Xenopus egg extracts (XEEs). We observe that there is a dramatic increase in the chromatin-bound RCC1 at the metaphase-anaphase transition, and that the addition of exogenous RCC1 to XEEs can abrogate SAC arrest. This abrogation is correlated with disruption of the kinetochore localization of SAC components, suggesting that the SAC is directly responsive to RCC1 dynamics. We find that the capacity of RCC1 to mediate this effect does not require karyopherins Importin beta or Crm1, and we are currently investigating the molecular mechanism behind the function of RCC1 in this context. At the same time, we are elucidating the upstream pathway that controls RCC1 dynamics on chromatin at the time of this transition. Crm1 is the exportin for proteins with classical export signals (NESs). We have found that Crm1 localizes to kintochores, and inhibition of Crm1 by Leptomycin B (LMB) in mitosis results in abnormal kinetochore attachment, decreased microtubule (MT) stability and reduced spindle size. These defects are correlated with a failure to recruit the RanGAP1/RanBP2 complex onto kinetochores after MT attachment is established. We examined proteins that bind Crm1, in order to determine whether Crm1 might regulate targets proteins through sequestration as has been reported for Importin-beta, and to ascertain whether any targets bind Crm1 in a mitosis-specific fashion. Specifically, we examined the protein binding profile of Crm1 within interphase and mitotic HeLa cell extacts: Many nuclear export cargos bound transiently to Crm1, with only a very small fraction of these proteins bound to Crm1 at any given time. However, we also found a small set of sequestered cargos (SCs) that bind quantitatively to Crm1 in a Ran-GTP-dependent manner. This unusual property of SCs suggests that they may be regulated by Crm1 beyond simple control of their nuclear localization. A putative histone H3 lysine demethylase (KDM3B) was identified as a SC and studied further. We examined the localization of KDM3B, and found that it co-localizes with Crm1 in the nucleoplasm and in intranucleolar bodies (INBs). Strikingly, treatment with LMB caused rapid and complete loss of KDM3B from INBs, indicating that Crm1 is required for KDM3B localization at that site. Cells treated with Actinomycin D, an RNA polymerase I inhibitor, also lost KDM3B from INBs. Chromatin IP (ChIP) assays suggests that KDM3B binds to rRNA genes and qPCR data reveal that treatment with LMB causes decreased transcription of rRNA genes. Collectively, these data suggest that interaction with Crm1-RanGTP regulates KDM3B-mediated histone demethylation and rRNA gene transcription in a novel, transport independent manner.
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