Participation of the nuclear cap binding complex in pre-mRNA 3′ processing

Communication between the 5′ and 3′ ends is a common feature of several aspects of eukaryotic mRNA metabolism. In the nucleus, the pre-mRNA 5′ end is bound by the nuclear cap binding complex (CBC). This RNA–protein complex plays an active role in both splicing and RNA export. We provide evidence for participation of CBC in the processing of the 3′ end of the message. Depletion of CBC from HeLa cell nuclear extract strongly reduced the endonucleolytic cleavage step of the cleavage and polyadenylation process. Cleavage was restored by addition of recombinant CBC. CBC depletion was found to reduce the stability of poly(A) site cleavage complexes formed in nuclear extract. We also provide evidence that the communication between the 5′ and 3′ ends of the pre-mRNA during processing is mediated by the physical association of the CBC/cap complex with 3′ processing factors bound at the poly(A) site. These observations, along with previous data on the function of CBC in splicing, illustrate the key role played by CBC in pre-mRNA recognition and processing. The data provides further support for the hypothesis that pre-mRNAs and mRNAs may exist and be functional in the form of “closed-loops,” due to interactions between factors bound at their 5′ and 3′ ends.

Nuclear translocation of NF-κB is increased in dopaminergic neurons of patients with Parkinson disease

Evidence from postmortem studies suggest an involvement of oxidative stress in the degeneration of dopaminergic neurons in Parkinson disease (PD) that have recently been shown to die by apoptosis, but the relationship between oxidative stress and apoptosis has not yet been elucidated. Activation of the transcription factor NF-κB is associated with oxidative stress-induced apoptosis in several nonneuronal in vitro models. To investigate whether it may play a role in PD, we looked for the translocation of NF-κB from the cytoplasm to the nucleus, evidence of its activation, in melanized neurons in the mesencephalon of postmortem human brain from five patients with idiopathic PD and seven matched control subjects. In PD patients, the proportion of dopaminergic neurons with immunoreactive NF-κB in their nuclei was more than 70-fold that in control subjects. A possible relationship between the nuclear localization of NF-κB in mesencephalic neurons of PD patients and oxidative stress in such neurons has been shown in vitro with primary cultures of rat mesencephalon, where translocation of NF-κB is preceded by a transient production of free radicals during apoptosis induced by activation of the sphingomyelin-dependent signaling pathway with C2-ceramide. The data suggest that this oxidant-mediated apoptogenic transduction pathway may play a role in the mechanism of neuronal death in PD.

Feedback-inducible nuclear-receptor-driven reporter gene expression in transgenic mice

Understanding nuclear receptor signaling in vivo would be facilitated by an efficient methodology to determine where a nuclear receptor is active. Herein, we present a feedback-inducible expression system in transgenic mice to detect activated nuclear receptor effector proteins by using an inducible reporter gene. With this approach, reporter gene induction is not limited to a particular tissue, and, thus, this approach provides the opportunity for whole-animal screens. Furthermore, the effector and reporter genes are combined to generate a single strain of transgenic mice, which enables direct and rapid analysis of the offspring. The system was applied to localize sites where the retinoic acid receptor ligand-binding domain is activated in vivo. The results identify previously discovered sources of retinoids in the embryo and indicate the existence of previously undiscovered regions of retinoic acid receptor signaling in vivo. Notably, the feedback-inducible nuclear-receptor-driven assay, combined with an independent in vitro assay, provides evidence for a site of retinoid synthesis in the isthmic mesenchyme. These data illustrate the potential of feedback-inducible nuclear-receptor-driven analyses for assessing in vivo activation patterns of nuclear receptors and for analyzing pharmacological properties of natural and synthetic ligands of potential therapeutic value.

Multiple protein domains determine the cell type-specific nuclear distribution of the catalytic subunit required for apolipoprotein B mRNA editing

Apolipoprotein B (apoB) mRNA editing catalyzed by apoB mRNA editing catalytic subunit 1 (APOBEC-1) has been proposed to be a nuclear process. To test this hypothesis, the subcellular distribution of hemagglutinin-(HA) tagged APOBEC-1 expressed in transiently transfected hepatoma cells was determined by indirect immunofluorescence microscopy. HA-APOBEC-1 was detected in both the nucleus and cytoplasm of rat and human hepatoma cells. Mutagenesis of APOBEC-1 demonstrated that the N-terminal 56 amino acids (1–56) were necessary for the nuclear distribution of APOBEC-1, but this region did not contain a functional nuclear localization signal (NLS). However, we identified a 24-amino acid domain in the C terminus of APOBEC-1 with characteristics of a cytoplasmic retention signal (CRS) or a nuclear export signal (NES). These data suggest, therefore, that the nuclear import of APOBEC-1 may not be mediated by a positive NLS; rather, it may be achieved by overcoming the effect of a CRS/NES. We also demonstrated that the nuclear distribution of APOBEC-1 occurred only in cell lines that were capable of editing apoB RNA. We propose that the cellular distribution of APOBEC-1 is determined by multiple domains within this protein, and a nuclear localization of the enzyme may be regulated by cell type-specific factors that render these cells uniquely editing competent.

A cytosolic activity distinct from Crm1 mediates nuclear export of protein kinase inhibitor in permeabilized cells

The leucine-rich nuclear export signal (NES) is used by a variety of proteins to facilitate their delivery from the nucleus to the cytoplasm. One of the best-studied examples, protein kinase inhibitor (PKI), binds to the catalytic subunit of protein kinase A in the nucleus and mediates its rapid export to the cytoplasm. We developed a permeabilized cell assay that reconstitutes nuclear export mediated by PKI, and we used it to characterize the cytosolic factors required for this process. The two-step assay involves an import phase and an export phase, and quantitation is achieved by digital fluorescence microscopy. During the import phase, a fluorescent derivative of streptavidin is imported into the nuclei of digitonin-permeabilized HeLa cells. During the export phase, biotinylated PKI diffuses into the nucleus, binds to fluorescent streptavidin, and mediates export of the complex to the cytoplasm. Nuclear export of the PKI complex is cytosol dependent and can be stimulated by addition of the purified NES receptor, Crm1. HeLa cell cytosol treated with N-ethylmaleimide (NEM) or phenyl-Sepharose to inactivate or deplete Crm1, respectively, is still fully active in the PKI export assay. Significantly, the export activity can be depleted from cytosol by preadsorption with a protein conjugate that contains a functional NES. These data indicate that cytosol contains an export activity that is distinct from Crm1 and is likely to correspond to an NES receptor.

Retina-specific nuclear receptor: A potential regulator of cellular retinaldehyde-binding protein expressed in retinal pigment epithelium and Müller glial cells

In an effort to identify nuclear receptors important in retinal disease, we screened a retina cDNA library for nuclear receptors. Here we describe the identification of a retina-specific nuclear receptor (RNR) from both human and mouse. Human RNR is a splice variant of the recently published photoreceptor cell-specific nuclear receptor [Kobayashi, M., Takezawa, S., Hara, K., Yu, R. T., Umesono, Y., Agata, K., Taniwaki, M., Yasuda, K. & Umesono, K. (1999) Proc. Natl. Acad. Sci. USA 96, 4814–4819] whereas the mouse RNR is a mouse ortholog. Northern blot and reverse transcription–PCR analyses of human mRNA samples demonstrate that RNR is expressed exclusively in the retina, with transcripts of ≈7.5 kb, ≈3.0 kb, and ≈2.3 kb by Northern blot analysis. In situ hybridization with multiple probes on both primate and mouse eye sections demonstrates that RNR is expressed in the retinal pigment epithelium and in Müller glial cells. By using the Gal4 chimeric receptor/reporter cotransfection system, the ligand binding domain of RNR was found to repress transcriptional activity in the absence of exogenous ligand. Gel mobility shift assays revealed that RNR can interact with the promoter of the cellular retinaldehyde binding protein gene in the presence of retinoic acid receptor (RAR) and/or retinoid X receptor (RXR). These data raise the possibility that RNR acts to regulate the visual cycle through its interaction with cellular retinaldehyde binding protein and therefore may be a target for retinal diseases such as retinitis pigmentosa and age-related macular degeneration.

Cholesterol feeding reduces nuclear forms of sterol regulatory element binding proteins in hamster liver

Cholesterol feeding reduces the mRNAs encoding multiple enzymes in the cholesterol biosynthetic pathway and the low density lipoprotein receptor in livers of hamsters. Here we show that cholesterol feeding also reduces the levels of the nuclear NH2-terminal domains of sterol regulatory element binding proteins (SREBPs), which activate transcription of sterol-regulated genes. We show that livers of hamsters, like those of mice and humans, predominantly produce SREBP-2 and the 1c isoform of SREBP-1. Both are produced as membrane-bound precursors that must be proteolyzed to release the transcriptionally active NH2-terminal domains. Diets containing 0.1% to 1.0% cholesterol decreased the amount of nuclear SREBP-1c without affecting the amount of the membrane precursor or its mRNA, suggesting that cholesterol inhibits the proteolytic processing of SREBP-1 in liver as it does in cultured cells. Cholesterol also appeared to reduce the proteolytic processing of SREBP-2. In addition, at high levels of dietary cholesterol the mRNA encoding SREBP-2 declined and the amount of the precursor also fell, suggesting that cholesterol accumulation also may inhibit transcription of the SREBP-2 gene. The high-cholesterol diets reduced the amount of low density lipoprotein receptor mRNA by 30% and produced a more profound 70–90% reduction in mRNAs encoding 3-hydroxy-3-methylglutaryl CoA synthase and reductase. Treatment with lovastatin and Colestipol, which increases hepatic demands for cholesterol, increased the amount of SREBP-2 mRNA as well as the precursor and nuclear forms of the protein. This treatment caused a reciprocal decline in SREBP-1c mRNA and protein. Considered together, these data suggest that SREBPs play important roles in controlling transcription of sterol-regulated genes in liver, as they do in cultured cells.

Ancient mtDNA sequences in the human nuclear genome: A potential source of errors in identifying pathogenic mutations

Nuclear-localized mtDNA pseudogenes might explain a recent report describing a heteroplasmic mtDNA molecule containing five linked missense mutations dispersed over the contiguous mtDNA CO1 and CO2 genes in Alzheimer’s disease (AD) patients. To test this hypothesis, we have used the PCR primers utilized in the original report to amplify CO1 and CO2 sequences from two independent ρ° (mtDNA-less) cell lines. CO1 and CO2 sequences amplified from both of the ρ° cells, demonstrating that these sequences are also present in the human nuclear DNA. The nuclear pseudogene CO1 and CO2 sequences were then tested for each of the five “AD” missense mutations by restriction endonuclease site variant assays. All five mutations were found in the nuclear CO1 and CO2 PCR products from ρ° cells, but none were found in the PCR products obtained from cells with normal mtDNA. Moreover, when the overlapping nuclear CO1 and CO2 PCR products were cloned and sequenced, all five missense mutations were found, as well as a linked synonymous mutation. Unlike the findings in the original report, an additional 32 base substitutions were found, including two in adjacent tRNAs and a two base pair deletion in the CO2 gene. Phylogenetic analysis of the nuclear CO1 and CO2 sequences revealed that they diverged from modern human mtDNAs early in hominid evolution about 770,000 years before present. These data would be consistent with the interpretation that the missense mutations proposed to cause AD may be the product of ancient mtDNA variants preserved as nuclear pseudogenes.

Nuclear Activities of Basic Fibroblast Growth Factor: Potentiation of Low-Serum Growth Mediated by Natural or Chimeric Nuclear Localization Signals

Human basic fibroblast growth factor (FGF-2) occurs in four isoforms: a low molecular weight (LMW FGF-2, 18 kDa) and three high molecular weight (HMW FGF-2, 22, 22.5, and 24 kDa) forms. LMW FGF-2 is primarily cytoplasmic and functions in an autocrine manner, whereas HMW FGF-2s are nuclear and exert activities through an intracrine, perhaps nuclear, pathway. Selective overexpression of HMW FGF-2 forms in fibroblasts promotes growth in low serum, whereas overexpression of LMW FGF-2 does not. The HMW FGF-2 forms have two functional domains: an amino-terminal extension and a common 18-kDa amino acid sequence. To investigate the role of these regions in the intracrine signaling of HMW FGF-2, we produced stable transfectants of NIH 3T3 fibroblasts overexpressing either individual HMW FGF-2 forms or artificially nuclear-targeted LMW FGF-2. All of these forms of FGF-2 localize to the nucleus/nucleolus and induce growth in low serum. The nuclear forms of FGF-2 trigger a mitogenic stimulus under serum starvation conditions and do not specifically protect the cells from apoptosis. These data indicate the existence of a specific role for nuclear FGF-2 and suggest that LMW FGF-2 represents the biological messenger in both the autocrine/paracrine and intracrine FGF-2 pathways.

Saccharomyces cerevisiae Cells with Defective Spindle Pole Body Outer Plaques Accomplish Nuclear Migration via Half-Bridge–organized Microtubules

Cnm67p, a novel yeast protein, localizes to the microtubule organizing center, the spindle pole body (SPB). Deletion of CNM67 (YNL225c) frequently results in spindle misorientation and impaired nuclear migration, leading to the generation of bi- and multinucleated cells (40%). Electron microscopy indicated that CNM67 is required for proper formation of the SPB outer plaque, a structure that nucleates cytoplasmic (astral) microtubules. Interestingly, cytoplasmic microtubules that are essential for spindle orientation and nuclear migration are still present in cnm67Δ1 cells that lack a detectable outer plaque. These microtubules are attached to the SPB half- bridge throughout the cell cycle. This interaction presumably allows for low-efficiency nuclear migration and thus provides a rescue mechanism in the absence of a functional outer plaque. Although CNM67 is not strictly required for mitosis, it is essential for sporulation. Time-lapse microscopy of cnm67Δ1 cells with green fluorescent protein (GFP)-labeled nuclei indicated that CNM67 is dispensable for nuclear migration (congression) and nuclear fusion during conjugation. This is in agreement with previous data, indicating that cytoplasmic microtubules are organized by the half-bridge during mating.

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.

Nuclear Import of Sterol Regulatory Element–binding Protein-2, a Basic Helix-Loop-Helix–Leucine Zipper (bHLH-Zip)–containing Transcription Factor, Occurs through the Direct Interaction of Importin β with HLH-Zip

The sterol regulatory element–binding protein-2 (SREBP-2) is produced as a large precursor molecule attached to the endoplasmic reticulum membrane. In response to the sterol depletion, the N-terminal segment of the precursor, which contains a basic helix-loop-helix–leucine zipper domain, is released by two sequential cleavages and is translocated to the nucleus, where it activates the transcription of target genes. The data herein show that released SREBP-2 uses a distinct nuclear transport pathway, which is mediated by importin β. The mature form of SREBP-2 is actively transported into the nucleus when injected into the cell cytoplasm. SREBP-2 binds directly to importin β in the absence of importin α. Ran-GTP but not Ran-GDP causes the dissociation of the SREBP-2–importin β complex. G19VRan-GTP inhibits the nuclear import of SREBP-2 in living cells. In the permeabilized cell in vitro transport system, nuclear import of SREBP-2 is reconstituted only by importin β in conjunction with Ran and its interacting protein p10/NTF2. We further demonstrate that the helix-loop-helix–leucine zipper motif of SREBP-2 contains a novel type of nuclear localization signal, which binds directly to importin β.

The Nuclear Actin-related Protein of Saccharomyces cerevisiae, Act3p/Arp4, Interacts with Core Histones

Act3p/Arp4, an essential actin-related protein of Saccharomyces cerevisiae located within the nucleus, is, according to genetic data, involved in transcriptional regulation. In addition to the basal core structure of the actin family members, which is responsible for ATPase activity, Act3p possesses two insertions, insertions I and II, the latter of which is predicted to form a loop-like structure protruding from beyond the surface of the molecule. Because Act3p is a constituent of chromatin but itself does not bind to DNA, we hypothesized that insertion II might be responsible for an Act3p-specific function through its interaction with some other chromatin protein. Far Western blot and two-hybrid analyses revealed the ability of insertion II to bind to each of the core histones, although with somewhat different affinities. Together with our finding of coimmunoprecipitation of Act3p with histone H2A, this suggests the in vivo existence of a protein complex required for correct expression of particular genes. We also show that a conditional act3 mutation affects chromatin structure of an episomal DNA molecule, indicating that the putative Act3p complex may be involved in the establishment, remodeling, or maintenance of chromatin structures.

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.

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.