The Integral Membrane Protein Snl1p Is Genetically Linked to Yeast Nuclear Pore Complex Function

Integral membrane proteins are predicted to play key roles in the biogenesis and function of nuclear pore complexes (NPCs). Revealing how the transport apparatus is assembled will be critical for understanding the mechanism of nucleocytoplasmic transport. We observed that expression of the carboxyl-terminal 200 amino acids of the nucleoporin Nup116p had no effect on wild-type yeast cells, but it rendered the nup116 null strain inviable at all temperatures and coincidentally resulted in the formation of nuclear membrane herniations at 23°C. To identify factors related to NPC function, a genetic screen for high-copy suppressors of this lethal nup116-C phenotype was conducted. One gene (designated SNL1 for suppressor of nup116-C lethal) was identified whose expression was necessary and sufficient for rescuing growth. Snl1p has a predicted molecular mass of 18.3 kDa, a putative transmembrane domain, and limited sequence similarity to Pom152p, the only previously identified yeast NPC-associated integral membrane protein. By both indirect immunofluorescence microscopy and subcellular fractionation studies, Snl1p was localized to both the nuclear envelope and the endoplasmic reticulum. Membrane extraction and topology assays suggested that Snl1p was an integral membrane protein, with its carboxyl-terminal region exposed to the cytosol. With regard to genetic specificity, the nup116-C lethality was also suppressed by high-copy GLE2 and NIC96. Moreover, high-copy SNL1 suppressed the temperature sensitivity of gle2–1 and nic96-G3 mutant cells. The nic96-G3 allele was identified in a synthetic lethal genetic screen with a null allele of the closely related nucleoporin nup100. Gle2p physically associated with Nup116p in vitro, and the interaction required the N-terminal region of Nup116p. Therefore, genetic links between the role of Snl1p and at least three NPC-associated proteins were established. We suggest that Snl1p plays a stabilizing role in NPC structure and function.

Phosphorothioate Antisense Oligonucleotides Induce the Formation of Nuclear Bodies

Antisense oligonucleotides are powerful tools for the in vivo regulation of gene expression. We have characterized the intracellular distribution of fluorescently tagged phosphorothioate oligodeoxynucleotides (PS-ONs) at high resolution under conditions in which PS-ONs have the potential to display antisense activity. Under these conditions PS-ONs predominantly localized to the cell nucleus where they accumulated in 20–30 bright spherical foci designated phosphorothioate bodies (PS bodies), which were set against a diffuse nucleoplasmic population excluding nucleoli. PS bodies are nuclear structures that formed in cells after PS-ON delivery by transfection agents or microinjection but were observed irrespectively of antisense activity or sequence. Ultrastructurally, PS bodies corresponded to electron-dense structures of 150–300 nm diameter and resembled nuclear bodies that were found with lower frequency in cells lacking PS-ONs. The environment of a living cell was required for the de novo formation of PS bodies, which occurred within minutes after the introduction of PS-ONs. PS bodies were stable entities that underwent noticeable reorganization only during mitosis. Upon exit from mitosis, PS bodies were assembled de novo from diffuse PS-ON pools in the daughter nuclei. In situ fractionation demonstrated an association of PS-ONs with the nuclear matrix. Taken together, our data provide evidence for the formation of a nuclear body in cells after introduction of phosphorothioate oligodeoxynucleotides.

Role of the Box C/D Motif in Localization of Small Nucleolar RNAs to Coiled Bodies and Nucleoli

Small nucleolar RNAs (snoRNAs) are a large family of eukaryotic RNAs that function within the nucleolus in the biogenesis of ribosomes. One major class of snoRNAs is the box C/D snoRNAs named for their conserved box C and box D sequence elements. We have investigated the involvement of cis-acting sequences and intranuclear structures in the localization of box C/D snoRNAs to the nucleolus by assaying the intranuclear distribution of fluorescently labeled U3, U8, and U14 snoRNAs injected into Xenopus oocyte nuclei. Analysis of an extensive panel of U3 RNA variants showed that the box C/D motif, comprised of box C′, box D, and the 3′ terminal stem of U3, is necessary and sufficient for the nucleolar localization of U3 snoRNA. Disruption of the elements of the box C/D motif of U8 and U14 snoRNAs also prevented nucleolar localization, indicating that all box C/D snoRNAs use a common nucleolar-targeting mechanism. Finally, we found that wild-type box C/D snoRNAs transiently associate with coiled bodies before they localize to nucleoli and that variant RNAs that lack an intact box C/D motif are detained within coiled bodies. These results suggest that coiled bodies play a role in the biogenesis and/or intranuclear transport of box C/D snoRNAs.

The Kinesin-related Proteins, Kip2p and Kip3p, Function Differently in Nuclear Migration in Yeast

The roles of two kinesin-related proteins, Kip2p and Kip3p, in microtubule function and nuclear migration were investigated. Deletion of either gene resulted in nuclear migration defects similar to those described for dynein and kar9 mutants. By indirect immunofluorescence, the cytoplasmic microtubules in kip2Δwere consistently short or absent throughout the cell cycle. In contrast, in kip3Δ strains, the cytoplasmic microtubules were significantly longer than wild type at telophase. Furthermore, in the kip3Δ cells with nuclear positioning defects, the cytoplasmic microtubules were misoriented and failed to extend into the bud. Localization studies found Kip2p exclusively on cytoplasmic microtubules throughout the cell cycle, whereas GFP-Kip3p localized to both spindle and cytoplasmic microtubules. Genetic analysis demonstrated that the kip2Δ kar9Δ double mutants were synthetically lethal, whereas kip3Δ kar9Δ double mutants were viable. Conversely, kip3Δ dhc1Δ double mutants were synthetically lethal, whereas kip2Δ dhc1Δ double mutants were viable. We suggest that the kinesin-related proteins, Kip2p and Kip3p, function in nuclear migration and that they do so by different mechanisms. We propose that Kip2p stabilizes microtubules and is required as part of the dynein-mediated pathway in nuclear migration. Furthermore, we propose that Kip3p functions, in part, by depolymerizing microtubules and is required for the Kar9p-dependent orientation of the cytoplasmic microtubules.

Characterization of ATM Expression, Localization, and Associated DNA-dependent Protein Kinase Activity

Ataxia telangiectasia–mutated gene (ATM) is a 350-kDa protein whose function is defective in the autosomal recessive disorder ataxia telangiectasia (AT). Affinity-purified polyclonal antibodies were used to characterize ATM. Steady-state levels of ATM protein varied from undetectable in most AT cell lines to highly expressed in HeLa, U2OS, and normal human fibroblasts. Subcellular fractionation showed that ATM is predominantly a nuclear protein associated with the chromatin and nuclear matrix. ATM protein levels remained constant throughout the cell cycle and did not change in response to serum stimulation. Ionizing radiation had no significant effect on either the expression or distribution of ATM. ATM immunoprecipitates from HeLa cells and the human DNA-dependent protein kinase null cell line MO59J, but not from AT cells, phosphorylated the 34-kDa subunit of replication protein A (RPA) complex in a single-stranded and linear double-stranded DNA–dependent manner. Phosphorylation of p34 RPA occurred on threonine and serine residues. Phosphopeptide analysis demonstrates that the ATM-associated protein kinase phosphorylates p34 RPA on similar residues observed in vivo. The DNA-dependent protein kinase activity observed for ATM immunocomplexes, along with the association of ATM with chromatin, suggests that DNA damage can induce ATM or a stably associated protein kinase to phosphorylate proteins in the DNA damage response pathway.

Organization of Highly Acetylated Chromatin around Sites of Heterogeneous Nuclear RNA Accumulation

Histones found within transcriptionally competent and active regions of the genome are highly acetylated. Moreover, these highly acetylated histones have very short half-lives. Thus, both histone acetyltransferases and histone deacetylases must enrich within or near these euchromatic regions of the interphase chromatids. Using an antibody specific for highly acetylated histone H3, we have investigated the organization of transcriptionally active and competent chromatin as well as nuclear histone acetyltransferase and deacetylase activities. We observe an exclusion of highly acetylated chromatin around the periphery of the nucleus and an enrichment near interchromatin granule clusters (IGCs). The highly acetylated chromatin is found in foci that may reflect the organization of highly acetylated chromatin into “chromonema” fibers. Transmission electron microscopy of Indian muntjac fibroblast cell nuclei indicates that the chromatin associated with the periphery of IGCs remains relatively condensed, most commonly found in domains containing chromatin folded beyond 30 nm. Using electron spectroscopic imaging, we demonstrate that IGCs are clusters of ribonucleoprotein particles. The individual granules comprise RNA-rich fibrils or globular regions that fold into individual granules. Quantitative analysis of individual granules indicates that they contain variable amounts of RNA estimated between 1.5 and >10 kb. We propose that interchromatin granules are heterogeneous nuclear RNA-containing particles, some of which may be pre-mRNA generated by nearby transcribed chromatin. An intermediary zone between the IGC and surrounding chromatin is described that contains factors with the potential to provide specificity to the localization of sequences near IGCs.

Activation of Androgen Receptor Function by a Novel Nuclear Protein Kinase

Androgen receptor (AR) belongs to the nuclear receptor superfamily and mediates the biological actions of male sex steroids. In this work, we have characterized a novel 130-kDa Ser/Thr protein kinase ANPK that interacts with the zinc finger region of AR in vivo and in vitro. The catalytic kinase domain of ANPK shares considerable sequence similarity with the minibrain gene product, a protein kinase suggested to contribute to learning defects associated with Down syndrome. However, the rest of ANPK sequence, including the AR-interacting interface, exhibits no apparent homology with other proteins. ANPK is a nuclear protein that is widely expressed in mammalian tissues. Its overexpression enhances AR-dependent transcription in various cell lines. In addition to the zinc finger region, ligand-binding domain and activation function AF1 of AR are needed, as the activity of AR mutants devoid of these domains was not influenced by ANPK. The receptor protein does not appear to be a substrate for ANPK in vitro, and overexpression of ANPK does not increase the extent of AR phosphorylation in vivo. In view of this, it is likely that ANPK-mediated activation of AR function is exerted through modification of AR-associated proteins, such as coregulatory factors, and/or through stabilization of the receptor protein against degradation.

A Nuclear Protein Involved in Apoptotic-like DNA Degradation in Stylonychia: Implications for Similar Mechanisms in Differentiating and Starved Cells

Ciliates are unicellular eukaryotic organisms containing two types of nuclei: macronuclei and micronuclei. After the sexual pathway takes place, a new macronucleus is formed from a zygote nucleus, whereas the old macronucleus is degraded and resorbed. In the course of macronuclear differentiation, polytene chromosomes are synthesized that become degraded again after some hours. Most of the DNA is eliminated, and the remaining DNA is fragmented into small DNA molecules that are amplified to a high copy number in the new macronucleus. The protein Pdd1p (programmed DNA degradation protein 1) from Tetrahymena has been shown to be present in macronuclear anlagen in the DNA degradation stage and also in the old macronuclei, which are resorbed during the formation of the new macronucleus. In this study the identification and localization of a Pdd1p homologous protein in Stylonychia (Spdd1p) is described. Spdd1p is localized in the precursor nuclei in the DNA elimination stage and in the old macronuclei during their degradation, but also in macronuclei and micronuclei of starved cells. In all of these nuclei, apoptotic-like DNA breakdown was detected. These data suggest that Spdd1p is a general factor involved in programmed DNA degradation in Stylonychia.

A Role for the GSG Domain in Localizing Sam68 to Novel Nuclear Structures in Cancer Cell Lines

The GSG (GRP33, Sam68, GLD-1) domain is a protein module found in an expanding family of RNA-binding proteins. The numerous missense mutations identified genetically in the GSG domain support its physiological role. Although the exact function of the GSG domain is not known, it has been shown to be required for RNA binding and oligomerization. Here it is shown that the Sam68 GSG domain plays a role in protein localization. We show that Sam68 concentrates into novel nuclear structures that are predominantly found in transformed cells. These Sam68 nuclear bodies (SNBs) are distinct from coiled bodies, gems, and promyelocytic nuclear bodies. Electron microscopic studies show that SNBs are distinct structures that are enriched in phosphorus and nitrogen, indicating the presence of nucleic acids. A GFP-Sam68 fusion protein had a similar localization as endogenous Sam68 in HeLa cells, diffusely nuclear with two to five SNBs. Two other GSG proteins, the Sam68-like mammalian proteins SLM-1 and SLM-2, colocalized with endogenous Sam68 in SNBs. Different GSG domain missense mutations were investigated for Sam68 protein localization. Six separate classes of cellular patterns were obtained, including exclusive SNB localization and association with microtubules. These findings demonstrate that the GSG domain is involved in protein localization and define a new compartment for Sam68, SLM-1, and SLM-2 in cancer cell lines.

cut11+: A Gene Required for Cell Cycle-dependent Spindle Pole Body Anchoring in the Nuclear Envelope and Bipolar Spindle Formation in Schizosaccharomyces pombe

The “cut” mutants of Schizosaccharomyces pombe are defective in spindle formation and/or chromosome segregation, but they proceed through the cell cycle, resulting in lethality. Analysis of temperature-sensitive alleles of cut11+ suggests that this gene is required for the formation of a functional bipolar spindle. Defective spindle structure was revealed with fluorescent probes for tubulin and DNA. Three-dimensional reconstruction of mutant spindles by serial sectioning and electron microscopy showed that the spindle pole bodies (SPBs) either failed to complete normal duplication or were free floating in the nucleoplasm. Localization of Cut11p tagged with the green fluorescent protein showed punctate nuclear envelope staining throughout the cell cycle and SPBs staining from early prophase to mid anaphase. This SPB localization correlates with the time in the cell cycle when SPBs are inserted into the nuclear envelope. Immunoelectron microscopy confirmed the localization of Cut11p to mitotic SPBs and nuclear pore complexes. Cloning and sequencing showed that cut11+ encodes a novel protein with seven putative membrane-spanning domains and homology to the Saccharomyces cerevisiae gene NDC1. These data suggest that Cut11p associates with nuclear pore complexes and mitotic SPBs as an anchor in the nuclear envelope; this role is essential for mitosis.

Activation and Cellular Localization of the Cyclosporine A-sensitive Transcription Factor NF-AT in Skeletal Muscle Cells

The widely used immunosuppressant cyclosporine A (CSA) blocks nuclear translocation of the transcription factor, NF-AT (nuclear factor of activated T cells), preventing its activity. mRNA for several NF-AT isoforms has been shown to exist in cells outside of the immune system, suggesting a possible mechanism for side effects associated with CSA treatment. In this study, we demonstrate that CSA inhibits biochemical and morphological differentiation of skeletal muscle cells while having a minimal effect on proliferation. Furthermore, in vivo treatment with CSA inhibits muscle regeneration after induced trauma in mice. These results suggest a role for NF-AT–mediated transcription outside of the immune system. In subsequent experiments, we examined the activation and cellular localization of NF-AT in skeletal muscle cells in vitro. Known pharmacological inducers of NF-AT in lymphoid cells also stimulate transcription from an NF-AT–responsive reporter gene in muscle cells. Three isoforms of NF-AT (NF-ATp, c, and 4/x/c3) are present in the cytoplasm of muscle cells at all stages of myogenesis tested. However, each isoform undergoes calcium-induced nuclear translocation from the cytoplasm at specific stages of muscle differentiation, suggesting specificity among NF-AT isoforms in gene regulation. Strikingly, one isoform (NF-ATc) can preferentially translocate to a subset of nuclei within a single multinucleated myotube. These results demonstrate that skeletal muscle cells express functionally active NF-AT proteins and that the nuclear translocation of individual NF-AT isoforms, which is essential for the ability to coordinate gene expression, is influenced markedly by the differentiation state of the muscle cell.

A Mutation in a Novel Yeast Proteasomal Gene, RPN11/MPR1, Produces a Cell Cycle Arrest, Overreplication of Nuclear and Mitochondrial DNA, and an Altered Mitochondrial Morphology

We report here the functional characterization of an essential Saccharomyces cerevisiae gene, MPR1, coding for a regulatory proteasomal subunit for which the name Rpn11p has been proposed. For this study we made use of the mpr1-1 mutation that causes the following pleiotropic defects. At 24°C growth is delayed on glucose and impaired on glycerol, whereas no growth is seen at 36°C on either carbon source. Microscopic observation of cells growing on glucose at 24°C shows that most of them bear a large bud, whereas mitochondrial morphology is profoundly altered. A shift to the nonpermissive temperature produces aberrant elongated cell morphologies, whereas the nucleus fails to divide. Flow cytometry profiles after the shift to the nonpermissive temperature indicate overreplication of both nuclear and mitochondrial DNA. Consistently with the identification of Mpr1p with a proteasomal subunit, the mutation is complemented by the human POH1 proteasomal gene. Moreover, the mpr1-1 mutant grown to stationary phase accumulates ubiquitinated proteins. Localization of the Rpn11p/Mpr1p protein has been studied by green fluorescent protein fusion, and the fusion protein has been found to be mainly associated to cytoplasmic structures. For the first time, a proteasomal mutation has also revealed an associated mitochondrial phenotype. We actually showed, by the use of [rho°] cells derived from the mutant, that the increase in DNA content per cell is due in part to an increase in the amount of mitochondrial DNA. Moreover, microscopy of mpr1-1 cells grown on glucose showed that multiple punctate mitochondrial structures were present in place of the tubular network found in the wild-type strain. These data strongly suggest that mpr1-1 is a valuable tool with which to study the possible roles of proteasomal function in mitochondrial biogenesis.

Apoptosis Induced by the Nuclear Death Domain Protein p84N5 Is Inhibited by Association with Rb Protein

Rb protein inhibits both cell cycle progression and apoptosis. Interaction of specific cellular proteins, including E2F1, with Rb C-terminal domains mediates cell cycle regulation. In contrast, the nuclear N5 protein associates with an Rb N-terminal domain with unknown function. The N5 protein contains a region of sequence similarity to the death domain of proteins involved in apoptotic signaling. We demonstrate here that forced N5 expression potently induces apoptosis in several tumor cell lines. Mutation of conserved residues within the death domain homology compromise N5-induced apoptosis, suggesting that it is required for normal function. Endogenous N5 protein is specifically altered in apoptotic cells treated with ionizing radiation. Furthermore, dominant interfering death domain mutants compromise cellular responses to ionizing radiation. Finally, physical association with Rb protein inhibits N5-induced apoptosis. We propose that N5 protein plays a role in the regulation of apoptosis and that Rb directly coordinates cell proliferation and apoptosis by binding specific proteins involved in each process through distinct protein binding domains.

Mechanisms of Human Mitochondrial DNA Maintenance: The Determining Role of Primary Sequence and Length over Function

Although the regulation of mitochondrial DNA (mtDNA) copy number is performed by nuclear-coded factors, very little is known about the mechanisms controlling this process. We attempted to introduce nonhuman ape mtDNA into human cells harboring either no mtDNA or mutated mtDNAs (partial deletion and tRNA gene point mutation). Unexpectedly, only cells containing no mtDNA could be repopulated with nonhuman ape mtDNA. Cells containing a defective human mtDNA did not incorporate or maintain ape mtDNA and therefore died under selection for oxidative phosphorylation function. On the other hand, foreign human mtDNA was readily incorporated and maintained in these cells. The suicidal preference for self-mtDNA showed that functional parameters associated with oxidative phosphorylation are less relevant to mtDNA maintenance and copy number control than recognition of mtDNA self-determinants. Non–self-mtDNA could not be maintained into cells with mtDNA even if no selection for oxidative phosphorylation was applied. The repopulation kinetics of several mtDNA forms after severe depletion by ethidium bromide treatment showed that replication and maintenance of mtDNA in human cells are highly dependent on molecular features, because partially deleted mtDNA molecules repopulated cells significantly faster than full-length mtDNA. Taken together, our results suggest that mtDNA copy number may be controlled by competition for limiting levels of trans-acting factors that recognize primarily mtDNA molecular features. In agreement with this hypothesis, marked variations in mtDNA levels did not affect the transcription of nuclear-coded factors involved in mtDNA replication.

Nup93, a Vertebrate Homologue of Yeast Nic96p, Forms a Complex with a Novel 205-kDa Protein and Is Required for Correct Nuclear Pore Assembly

Yeast and vertebrate nuclear pores display significant morphological similarity by electron microscopy, but sequence similarity between the respective proteins has been more difficult to observe. Herein we have identified a vertebrate nucleoporin, Nup93, in both human and Xenopus that has proved to be an evolutionarily related homologue of the yeast nucleoporin Nic96p. Polyclonal antiserum to human Nup93 detects corresponding proteins in human, rat, and Xenopus cells. Immunofluorescence and immunoelectron microscopy localize vertebrate Nup93 at the nuclear basket and at or near the nuclear entry to the gated channel of the pore. Immunoprecipitation from both mammalian and Xenopus cell extracts indicates that a small fraction of Nup93 physically interacts with the nucleoporin p62, just as yeast Nic96p interacts with the yeast p62 homologue. However, a large fraction of vertebrate Nup93 is extracted from pores and is also present in Xenopus egg extracts in complex with a newly discovered 205-kDa protein. Mass spectrometric sequencing of the human 205-kDa protein reveals that this protein is encoded by an open reading frame, KIAAO225, present in the human database. The putative human nucleoporin of 205 kDa has related sequence homologues in Caenorhabditis elegans and Saccharomyces cerevisiae. To analyze the role of the Nup93 complex in the pore, nuclei were assembled that lack the Nup93 complex after immunodepletion of a Xenopus nuclear reconstitution extract. The Nup93-complex–depleted nuclei are clearly defective for correct nuclear pore assembly. From these experiments, we conclude that the vertebrate and yeast pore have significant homology in their functionally important cores and that, with the identification of Nup93 and the 205-kDa protein, we have extended the knowledge of the nearest-neighbor interactions of this core in both yeast and vertebrates.