Insights into Dynamic Mitotic Chromatin Organization Through the NIMA Kinase Suppressor SonC,a Chromatin-Associated Protein Involved in the DNA Damage Response

The nuclear pore complex proteins SonA and SonB, the orthologs of mammalian RAE1 and NUP98, respectively, were identified in Aspergillus nidulans as cold-sensitive suppressors of a temperature-sensitive allele of the essential mitotic NIMA kinase (nimA1). Subsequent analyses found that sonB1 mutants exhibit temperature-dependent DNA damage sensitivity. To understand this pathway further, we performed a genetic screen to isolate additional conditional DNA damage-sensitive suppressors of nimA1. We identified two new alleles of SonA and four intragenic nimA mutations that suppress the temperature sensitivity of the nimA1 mutant. In addition, we identified SonC, a previously unstudied binuclear zinc cluster protein involved with NIMA and the DNA damage response. Like sonA and sonB, sonC is an essential gene. SonC localizes to nuclei and partially disperses during mitosis. When the nucleolar organizer region (NOR) undergoes mitotic condensation and removal from the nucleolus, nuclear SonC and histone H1 localize in a mutually exclusive manner with H1 being removed from the NOR region and SonC being absent from the end of the chromosome beyond the NOR. This region of chromatin is adjacent to a cluster of nuclear pore complexes to which NIMA localizes last during its progression around the nuclear envelope during initiation of mitosis. The results genetically extend the NIMA regulatory system to include a protein with selective large-scale chromatin location observed during mitosis. The data suggest a model in which NIMA and SonC, its new chromatin-associated suppressor, might help to orchestrate global chromatin states during mitosis and the DNA damage response.     

The mitotic checkpoint protein hBUB3 and the mRNA export factor hRAE1 interact with GLE2p-binding sequence (GLEBS)-containing proteins

The mRNA export factor RAE1 (also called GLE2) and the mitotic checkpoint protein BUB3 share extensive sequence homology in yeast as well as higher eukaryotes, although the biological relevance of their similarity is unclear. Previous work in HeLa cells has shown that human (h)RAE1 binds the nuclear pore complex protein hNUP98 via a short NUP98 motif called GLEBS (for GLE2p-binding sequence). Here we report that the two known binding partners of hBUB3, the mitotic checkpoint proteins hBUB1 and hBUBR1, both carry a region with remarkable similarity to the GLEBS motif of hNUP98. We show that the GLEBS-like motifs of mouse (m)BUB1 and mBUBR1 are sufficient for mBUB3 binding. mBUB3 lacks affinity for the hNUP98 GLEBS, demonstrating its binding specificity for GLEBS motifs of mitotic checkpoint proteins. Interestingly, mRAE1 does not exclusively bind to the GLEBS motif of hNUP98 and can cross-interact with the mBUB1 GLEBS. We show that full-length RAE1 and BUB1 proteins interact in mammalian cells and accumulate both at the kinetochores of prometaphase chromosomes. Our findings demonstrate that GLEBS motifs reside in mammalian nucleoporins and mitotic checkpoint proteins and apparently serve as specific binding sites for either BUB3, RAE1, or both.     

RAE1 is a shuttling mRNA export factor that binds to a GLEBS-like NUP98 motif at the nuclear pore complex through multiple domains

Gle2p is implicated in nuclear export of poly(A)+ RNA and nuclear pore complex (NPC) structure and distribution in Saccharomyces cerevisiae. Gle2p is anchored at the nuclear envelope (NE) via a short Gle2p-binding motif within Nup116p called GLEBS. The molecular mechanism by which Gle2p and the Gle2p-Nup116p interaction function in mRNA export is unknown. Here we show that RAE1, the mammalian homologue of Gle2p, binds to a GLEBS-like NUP98 motif at the NPC through multiple domains that include WD-repeats and a COOH-terminal non-WD-repeat extension. This interaction is direct, as evidenced by in vitro binding studies and chemical cross-linking. Microinjection experiments performed in Xenopus laevis oocytes demonstrate that RAE1 shuttles between the nucleus and the cytoplasm and is exported from the nucleus in a temperature-dependent and RanGTP-independent manner. Docking of RAE1 to the NE is highly dependent on new mRNA synthesis. Overexpression of the GLEBS-like motif also inhibits NE binding of RAE1 and induces nuclear accumulation of poly(A)+ RNA. Both effects are abrogated either by the introduction of point mutations in the GLEBS-like motif or by overexpression of RAE1, indicating a direct role for RAE1 and the NUP98-RAE1 interaction in mRNA export. Together, our data suggest that RAE1 is a shuttling transport factor that directly contributes to nuclear export of mRNAs through its ability to anchor to a specific NUP98 motif at the NPC.     

A point mutation in the Aspergillus nidulans sonBNup98 nuclear pore complex gene causes conditional DNA damage sensitivity

The nuclear pore complex (NPC) is embedded in the nuclear envelope where it mediates transport between the cytoplasm and nucleus and helps to organize nuclear architecture. We previously isolated sonB1, a mutation encoding a single amino acid substitution within the Aspergillus nidulans SONBnNup98 NPC protein (nucleoporin). Here we demonstrate that this mutation causes marked DNA damage sensitivity at 42 degrees . Although SONBnNup98 has roles in the G2 transition, we demonstrate that the G2 DNA damage checkpoint is functional in the sonB1 mutant at 42 degrees . The MRN complex is composed of MRE11, RAD50, and NBS1 and functions in checkpoint signaling, DNA repair, and telomere maintenance. At 42 degrees we find that the DNA damage response defect of sonB1 mutants causes synthetic lethality when combined with mutations in scaANBS1, the A. nidulans homolog of NBS1. We provide evidence that this synthetic lethality is independent of MRN cell cycle checkpoint functions or MREAMRE11-mediated DNA repair functions. We also demonstrate that the single A. nidulans histone H2A gene contains the C-terminal SQE motif of histone H2AX isoforms and that this motif is required for the DNA damage response. We propose that the sonB1 nucleoporin mutation causes a defect in a novel part of the DNA damage response.     

A Role for NIMA in the Nuclear Localization of Cyclin B in Aspergillus nidulans

NIMA promotes entry into mitosis in late G2 by some mechanism that is after activation of the Aspergillus nidulans G2 cyclin-dependent kinase, NIMX^CDC2/NIME^{Cyclin B}. Here we present two independent lines of evidence which indicate that this mechanism involves control of NIMX^CDC2/NIME^{Cyclin B} localization. First, we found that NIME^{Cyclin B} localized to the nucleus and the nucleus-associated organelle, the spindle pole body, in a NIMA-dependent manner. Analysis of cells from asynchronous cultures, synchronous cultures, and cultures arrested in S or G2 showed that NIME^{Cyclin B} was predominantly nuclear during interphase, with maximal nuclear accumulation in late G2. NIMX^CDC2 colocalized with NIME^{Cyclin B} in G2 cells. Although inactivation of NIMA using either the nimA1 or nimA5 temperature-sensitive mutations blocked cells in G2, NIMX^CDC2/NIME^{Cyclin B} localization was predominantly cytoplasmic rather than nuclear. Second, we found that nimA interacts genetically with sonA, which is a homologue of the yeast nucleocytoplasmic transporter GLE2/RAE1. Mutations in sonA were identified as allele-specific suppressors of nimA1. The sonA1 suppressor alleviated the nuclear division and NIME^{Cyclin B} localization defects of nimA1 cells without markedly increasing NIMX^CDC2 or NIMA kinase activity. These results indicate that NIMA promotes the nuclear localization of the NIMX^CDC2/ NIME^{Cyclin B} complex, by a process involving SONA. This mechanism may be involved in coordinating the functions of NIMX^CDC2 and NIMA in the regulation of mitosis.     

Expression of the noncatalytic domain of the NIMA kinase causes a G2 arrest in Aspergillus nidulans.

Temperature-sensitive mutation of the nimA gene of Aspergillus nidulans causes a reversible G2 arrest, whereas overexpression of nimA causes premature entry into mitosis from which the cells cannot exit. The nimA gene encodes a Ser/Thr-specific protein kinase (NIMA) which contains an extended COOH-terminal noncatalytic domain. To evaluate the role of this enzyme in nuclear division control, we introduced various mutant nimA cDNAs under the control of the inducible alcohol dehydrogenase gene promoter into a strain of Aspergillus nidulans containing a temperature-sensitive nimA mutation (nimA5). While expression of the wild type NIMA complemented the nimA5 mutation and induced a premature mitotic arrest when overexpressed, expression of a kinase-negative NIMA containing a single amino acid mutation in the putative ATP-binding site could not rescue the nimA5 mutation but resulted in a specific G2 arrest when overexpressed. An identical phenotype was observed with cells expressing only the noncatalytic COOH-terminal domain of NIMA, whereas overexpression of the inactive kinase domain was without effect. The G2 arrest produced by overexpression of the full-length inactive or COOH-terminal NIMA molecules did not prevent activation of the endogenous NIMA or H1 kinase activity precipitable by p13 beads. We suggest that this dominant-negative phenotype results from competitive inhibition of the association of active NIMA with a cellular target(s) and that appropriate targeting is essential for the mitotic function of the NIMA kinase.     

Activation of the nimA protein kinase plays a unique role during mitosis that cannot be bypassed by absence of the bimE checkpoint.

Mutation of nimA reversibly arrests cells in late G2 and nimA overexpression promotes premature mitosis. Here we demonstrate that the product of nimA (designated NIMA) has protein kinase activity that can phosphorylate beta-casein but not histone proteins. NIMA kinase activity is cell cycle regulated being 20-fold higher at mitosis when compared to S-phase arrested cells. NIMA activation is normally required in G2 to initiate chromosome condensation, to nucleate spindle pole body microtubules, and to allow an MPM-2 specific mitotic phosphorylation. All three of these mitotic events can occur in the absence of activated NIMA when the bimE gene is mutated (bimE7). However, the bimE7 mutation cannot completely bypass the requirement for nimA during mitosis as entry into mitosis in the absence of NIMA activation results in major mitotic defects that affect both the organization of the nuclear envelope and mitotic spindle. Thus, although nimA plays an essential but limited role during mitosis, mutation of nimA arrests all of mitosis. We therefore propose that mutation of nimA prevents mitotic initiation due to a checkpoint arrest that is negatively mediated by bimE. The checkpoint ensures that mitosis is not initiated until NIMA is mitotically activated.     

Nup116p and nup100p are interchangeable through a conserved motif which constitutes a docking site for the mRNA transport factor gle2p.

Nup116p and Nup100p are highly related yeast GLFG nucleoporins, but only Nup116p is stoichiometrically bound to Gle2p, a previously identified mRNA export factor. A short Gle2p-binding sequence within Nup116p (GLEBS; residues 110-166) is sufficient and necessary to anchor Gle2p at the nuclear pores, whereas the carboxy-terminal domain of Nup116p mediates its own nuclear pore complex (NPC) association. The GLEBS is evolutionarily conserved and found in rat/Xenopus Nup98 and an uncharacterized Caenorhabditis elegans ORF, but is absent from Nup100p. When the GLEBS is deleted from Nup116p, Gle2p dissociates from the nuclear envelope and clusters of herniated nuclear pores form. When the GLEBS is inserted into Nup100p, Nup100p-GLEBS complements both the thermosensitive and NPC-herniated phenotype of nup116- cells, and Gle2p is retargeted concomitantly to the NPCs. Thus, the in vivo function of Gle2p is strictly coupled to the short GLEBS within Nup116p which links this putative mRNA transport factor to the nuclear pores.     

GLE2, a Saccharomyces cerevisiae homologue of the Schizosaccharomyces pombe export factor RAE1, is required for nuclear pore complex structure and function.

To identify and characterize novel factors required for nuclear transport, a genetic screen was conducted in the yeast Saccharomyces cerevisiae. Mutations that were lethal in combination with a null allele of the gene encoding the nucleoporin Nup100p were isolated using a colony-sectoring assay. Three complementation groups of gle (for GLFG lethal) mutants were identified. In this report, the characterization of GLE2 is detailed. GLE2 encodes a 40.5-kDa polypeptide with striking similarity to that of Schizosaccharomyces pombe RAE1. In indirect immunofluorescence and nuclear pore complex fractionation experiments, Gle2p was associated with nuclear pore complexes. Mutated alleles of GLE2 displayed blockage of polyadenylated RNA export; however, nuclear protein import was not apparently diminished. Immunofluorescence and thin-section electron microscopic analysis revealed that the nuclear pore complex and nuclear envelope structure was grossly perturbed in gle2 mutants. Because the clusters of herniated pore complexes appeared subsequent to the export block, the structural perturbations were likely indirect consequences of the export phenotype. Interestingly, a two-hybrid interaction was detected between Gle2p and Srp1p, the nuclear localization signal receptor, as well as Rip1p, a nuclear export signal-interacting protein. We propose that Gle2p has a novel role in mediating nuclear transport.     

PINA is essential for growth and positively influences NIMA function in Aspergillus nidulans

The phospho-Ser/Thr-directed prolyl-isomerase Pin1 was originally identified in vertebrate systems as a negative regulator of NIMA, a Ser/Thr protein kinase that regulates the G(2)/M transition in Aspergillus nidulans. Here we explore the physiological roles of the Pin1 orthologue, PINA, in A. nidulans and evaluate the relevance of the interaction of PINA with NIMA in this fungus. We find pinA to be an essential gene in A. nidulans. In addition, when PINA levels are reduced 50-fold the cells grow at a reduced rate. Upon germination under conditions that repress PINA expression, the cells are delayed in the interphase activation of NIMX(cdc2), whereas they traverse the other phases of the cell cycle at a similar rate to controls. These results indicate that a marked reduction of PINA results in a lengthening of G(1). Additionally, PINA repression increases the rate at which the cells enter mitosis following release from a hydroxyurea arrest without altering the sensitivity of the fungus to agents that activate the replication or DNA damage checkpoints. In contrast to predictions based on Pin1, the physical interaction between PINA and NIMA is primarily dependent upon the prolylisomerase domain of PINA and the C-terminal 303 amino acids of NIMA. Finally, reduction of PINA levels exacerbates the nimA5 temperature-sensitive mutant, whereas overexpression of PINA decreases the severity of this mutation, results that are consistent with a positive genetic interaction between PINA and NIMA. Thus, although PINA is essential and positively regulates NIMA function, A. nidulans is most sensitive to a reduction in PINA concentration in G(1) rather than in G(2)/M.     

Nup116p associates with the Nup82p-Nsp1p-Nup159p nucleoporin complex

Nup116p is a GLFG nucleoporin involved in RNA export processes. We show here that Nup116p physically interacts with the Nup82p-Nsp1p-Nup159p nuclear pore subcomplex, which plays a central role in nuclear mRNA export. For this association, a sequence within the C-terminal domain of Nup116p that includes the conserved nucleoporin RNA-binding motif was sufficient and necessary. Consistent with this biochemical interaction, protein A-Nup116p and the protein A-tagged Nup116p C-terminal domain, like the members of the Nup82p complex, localized to the cytoplasmic side of the nuclear pore complex, as revealed by immunogold labeling. Finally, synthetic lethal interactions were found between mutant alleles of NUP116 and all members of the Nup82p complex. Thus, Nup116p consists of three independent functional domains: 1) the C-terminal part interacts with the Nup82p complex; 2) the Gle2p-binding sequence interacts with Gle2p/Rae1p; and 3) the GLFG domain interacts with shuttling transport receptors such as karyopherin-beta family members.     

Complex formation among the RNA export proteins Nup98, Rae1/Gle2, and TAP

Most nucleocytoplasmic traffic through the nuclear pore complex is mediated by soluble receptors of the importin/exportin or karyopherin family. mRNA export is unique in that no receptor of this family has been implicated in trafficking of the bulk of mRNAs. Instead, many diverse proteins have been linked to mRNA export, but an all-encompassing model remains elusive. Understanding how these proteins interact with each other is central to the development of such a model. Here, we have focused on the interactions between three proteins implicated in mRNA export, Nup98, Rae1/Gle2, and TAP. We have defined the binary complexes that form among these proteins. We find that Gle2 requires two sites within TAP for stable interaction. Strikingly, rather than a general affinity for all nucleoporin FG repeats, TAP has highest affinity for a specific region within the GLFG domain of Nup98, indicating that not all repeats are identical in function. We have established that the ternary complex can form through simultaneous binding of both Gle2 and TAP to adjacent sites on Nup98. In contrast, Nup98 competes with TAP for Gle2 binding; when bound to Nup98, Gle2 no longer interacts directly with TAP. From these interactions, we propose that Gle2 may act to deliver TAP to Nup98 and that this may represent the first in a series of interactions between an export complex and a nucleoporin.     

Mitotic destruction of the cell cycle regulated NIMA protein kinase of Aspergillus nidulans is required for mitotic exit.

NIMA is a cell cycle regulated protein kinase required, in addition to p34cdc2/cyclin B, for initiation of mitosis in Aspergillus nidulans. Like cyclin B, NIMA accumulates when cells are arrested in G2 and is degraded as cells traverse mitosis. However, it is stable in cells arrested in mitosis. NIMA, and related kinases, have an N-terminal kinase domain and a C-terminal extension. Deletion of the C-terminus does not completely inactivate NIMA kinase activity but does prevent functional complementation of a temperature sensitive mutation of nimA, showing it to be essential for function. Partial C-terminal deletion of NIMA generates a highly toxic kinase although the kinase domain alone is not toxic. Transient induction experiments demonstrate that the partially truncated NIMA is far more stable than the full length NIMA protein which likely accounts for its toxicity. Unlike full length NIMA, the truncated NIMA is not degraded during mitosis and this affects normal mitotic progression. Cells arrested in mitosis with non-degradable NIMA are able to destroy cyclin B, demonstrating that the arrest is not due to stabilization of p34cdc2/cyclin B activity. The data establish that NIMA degradation during mitosis is required for correct mitotic progression in A. nidulans.     

Schizosaccharomyces pombe NIMA-related kinase,Fin1, regulates spindle formation and an affinity of Polo for the SPB

The Aspergillus nidulans protein kinase NIMA regulates mitotic commitment, while the human and Xenopus equivalents influence centrosome function. Two recessive, temperature-sensitive mutations in the Schizosaccharomyces pombe NIMA homologue, Fin1, blocked spindle formation at 37 degrees C. One of the two spindle pole bodies (SPBs) failed to nucleate microtubules. This phenotype was reduced by accelerating mitotic commitment through genetic inhibition of Wee1 or activation of either Cdc25 or Cdc2. Polo kinase (Plo1) normally associates with the SPB of mitotic, but not interphase cells. cut12.s11 is a dominant mutation in an SPB component that both suppresses cdc25 mutants and promotes Plo1 association with the interphase SPB. Both cut12.s11 phenotypes were abolished by removing Fin1 function. Elevating Fin1 levels promoted Plo1 recruitment to the interphase SPB of wild-type cells and reduced the severity of the cdc25.22 phenotype. These data are consistent with Fin1 regulating Plo1 function during mitotic commitment. The fin1 mitotic commitment and spindle phenotypes resemble distinct nimA phenotypes in different systems and suggest that the function of this family of kinases may be conserved across species.     

A zebrafish model of lethal congenital contracture syndrome 1 reveals Gle1 function in spinal neural precursor survival and motor axon arborization

In humans, GLE1 is mutated in lethal congenital contracture syndrome 1 (LCCS1) leading to prenatal death of all affected fetuses. Although the molecular roles of Gle1 in nuclear mRNA export and translation have been documented, no animal models for this disease have been reported. To elucidate the function of Gle1 in vertebrate development, we used the zebrafish (Danio rerio) model system. gle1 mRNA is maternally deposited and widely expressed. Altering Gle1 using an insertional mutant or antisense morpholinos results in multiple defects, including immobility, small eyes, diminished pharyngeal arches, curved body axis, edema, underdeveloped intestine and cell death in the central nervous system. These phenotypes parallel those observed in LCCS1 human fetuses. Gle1 depletion also results in reduction of motoneurons and aberrant arborization of motor axons. Unexpectedly, the motoneuron deficiency results from apoptosis of neural precursors, not of differentiated motoneurons. Mosaic analyses further indicate that Gle1 activity is required extrinsically in the environment for normal motor axon arborization. Importantly, the zebrafish phenotypes caused by Gle1 deficiency are only rescued by expressing wild-type human GLE1 and not by the disease-linked Fin(Major) mutant form of GLE1. Together, our studies provide the first functional characterization of Gle1 in vertebrate development and reveal its essential role in actively dividing cells. We propose that defective GLE1 function in human LCCS1 results in both neurogenic and non-neurogenic defects linked to the apoptosis of proliferative organ precursors.     

NUP98 fusion oncoproteins interact with the APC/CCdc20 as a pseudosubstrate and prevent mitotic checkpoint complex binding

NUP98 is a recurrent partner gene in translocations causing acute myeloid leukemias and myelodisplastic syndrome. The expression of NUP98 fusion oncoproteins has been shown to induce mitotic spindle defects and chromosome missegregation, which correlate with the capability of NUP98 fusions to cause mitotic checkpoint attenuation. We show that NUP98 oncoproteins physically interact with the APC/C^Cdc20 in the absence of the NUP98 partner protein RAE1, and prevent the binding of the mitotic checkpoint complex to the APC/C^Cdc20. NUP98 oncoproteins require the GLEBS-like domain present in their NUP98 moiety to bind the APC/C^Cdc20. We found that NUP98 wild-type is a substrate of APC/C^Cdc20 prior to mitotic entry, and that its binding to APC/C^Cdc20 is controlled via phosphorylation of a PEST sequence located within its C-terminal portion. We identify S606, within the PEST sequence, as a key target site, whose phosphorylation modulates the capability of NUP98 to interact with APC/C^Cdc20. We finally provide evidence for an involvement of the peptidyl-prolyl isomerase PIN1 in modulating the possible conformational changes within NUP98 that lead to its dissociation from the APC/C^Cdc20 during mitosis. Our results provide novel insight into the mechanisms underlying the aberrant capability of NUP98 oncoproteins to interact with APC/C^Cdc20 and to interfere with its function.