Interchromatin Granules Research Articles

P300/CBP-associated Factor Drives DEK into Interchromatin Granule Clusters

DEK is a mammalian protein that has been implicated in the pathogenesis of autoimmune diseases and cancer, including acute myeloid leukemia, melanoma, glioblastoma, hepatocellular carcinoma, and bladder cancer. In addition, DEK appears to participate in multiple cellular processes, including transcriptional repression, mRNA processing, and chromatin remodeling. Sub-nuclear distribution of this protein, with the attendant functional ramifications, has remained a controversial topic. Here we report that DEK undergoes acetylation in vivo at lysine residues within the first 70 N-terminal amino acids. Acetylation of DEK decreases its affinity for DNA elements within the promoter, which is consistent with the involvement of DEK in transcriptional repression. Furthermore, deacetylase inhibition results in accumulation of DEK within interchromatin granule clusters (IGCs), sub-nuclear structures that contain RNA processing factors. Overexpression of P/CAF acetylase drives DEK into IGCs, and addition of a newly developed, synthetic, cell-permeable P/CAF inhibitor blocks this movement. To our knowledge, this is the first reported example of acetylation playing a direct role in relocation of a protein to IGCs, and this may explain how DEK can function in multiple pathways that take place in distinct sub-nuclear compartments. These findings also suggest that DEK-associated malignancies and autoimmune diseases might be amenable to treatment with agents that alter acetylation.

Secretion of the Human T Cell Leukemia Virus Type I Transactivator Protein Tax

Human T cell leukemia virus type I (HTLV-I) is the etiologic agent of adult T cell leukemia and HTLV-I-associated myelopathy/tropical spastic paraparesis. The HTLV-I protein Tax is well known as a transcriptional transactivator and inducer of cellular transformation. However, it is also known that extracellular Tax induces the production and release of cytokines, such as tumor necrosis factor-alpha and interleukin-6, which have adverse effects on cells of the central nervous system. The cellular process by which Tax exits the cell into the extracellular environment is currently unknown. In most cell types, Tax has been shown to localize primarily to the nucleus. However, Tax has also been found to accumulate in the cytoplasm. The results contained herein begin to characterize the process of Tax secretion from the cell. Specifically, cytoplasmic Tax was demonstrated to localize to organelles associated with the cellular secretory process including the endoplasmic reticulum and Golgi complex. Additionally, it was demonstrated that full-length Tax was secreted from both baby hamster kidney cells and a human kidney tumor cell line, suggesting that Tax enters the secretory pathway in a leaderless manner. Tax secretion was partially inhibited by brefeldin A, suggesting that Tax migrated from the endoplasmic reticulum to the Golgi complex. In addition, combined treatment of Tax-transfected BHK-21 cells with phorbol myristate acetate and ionomycin resulted in a small increase in the amount of Tax secreted, suggesting that a fraction of cytoplasmic Tax was present in the regulated secretory pathway. These studies begin to provide a link between Tax localization to the cytoplasm, the detection of Tax in the extracellular environment, its possible role as an extracellular effector molecule, and a potential role in neurodegenerative disease associated with HTLV-I infection.

Higher order chromatin architecture in the cell nucleus: on the way from structure to function

The cell nucleus is a highly compartmentalized structure. In this review we describe controversial views on higher order chromatin organization from the level of higher order chromatin domains built up from folded chromatin fibers to the level of chromosome territories and the interchromatin compartment (IC), which harbors non-chromatin nuclear domains, such as interchromatin granule clusters (IGCs visualized in the electron microscope) or splicing factor-containing speckles (visualized by fluorescence microscopy). Emphasis is laid on the definition and functional importance of a nuclear compartment located at the periphery of chromatin domains in direct contact with the IC, termed the perichromatin region (PR). Ongoing experiments to elucidate the topological relationships between PR and IC have provided new insights into the functional interplay between transcription and splicing. As an example, we discuss the structure and nuclear topology of perichromatin fibrils (FPs) contained in the PR and their functional interplay with IGCs/speckles. In addition we discuss the advantages and drawbacks of experimental approaches currently used to study nuclear architecture and function in fixed and living cells.

Rearrangement of nuclear ribonucleoprotein (RNP)-containing structures during apoptosis and transcriptional arrest

The aim of this paper is to review the data in the literature concerning ribonucleoprotein components during apoptosis, where a major rearrangement of RNPs takes place. In parallel with chromatin changes, the nucleoplasmic constituents (perichromatin fibrils; perichromatin granules; interchromatin granules and nuclear bodies) as well as the nucleoli aggregate into heterogeneous clusters called HERDS, in the interchromatin space. Later, these RNP-containing structures are extruded from the nucleus and leave the cell within cytoplasmic blebs. We propose also a role for HERDS as markers of irreversible transcriptional arrest.

Proteomic analysis of interchromatin granule clusters.

A variety of proteins involved in gene expression have been localized within mammalian cell nuclei in a speckled distribution that predominantly corresponds to interchromatin granule clusters (IGCs). We have applied a mass spectrometry strategy to identify the protein composition of this nuclear organelle purified from mouse liver nuclei. Using this approach, we have identified 146 proteins, many of which had already been shown to be localized to IGCs, or their functions are common to other already identified IGC proteins. In addition, we identified 32 proteins for which only sequence information is available and thus these represent novel IGC protein candidates. We find that 54% of the identified IGC proteins have known functions in pre-mRNA splicing. In combination with proteins involved in other steps of pre-mRNA processing, 81% of the identified IGC proteins are associated with RNA metabolism. In addition, proteins involved in transcription, as well as several other cellular functions, have been identified in the IGC fraction. However, the predominance of pre-mRNA processing factors supports the proposed role of IGCs as assembly, modification, and/or storage sites for proteins involved in pre-mRNA processing.

The Human Nuclear SRcyp Is a Cell Cycle-regulated Cyclophilin

Cyclophilins of the Moca family (Cavarec, L., Kamphausen, T., Dubourg, B., Callebaut, I., Lemeunier, F., Metivier, D., Feunteun, J., Fischer, G., and Modjtahedi, N. (2002) J. Biol. Chem. 277, 41171-41182) are found only in organisms of the animal kingdom and share several structural and enzymatic features. The presence of serine/arginine (S/R) dipeptide repeats in their C-terminal tail suggests that these enzymes belong to the SR protein family involved in the regulation of gene expression. The function of this group of cyclophilins is currently unknown. However, their C-terminal tails contain a highly conserved polypeptide signature segment (the moca domain), which may well be involved in the functional regulation of these proteins. We report here the identification of five Cdc2-type phosphorylation sites gathered in and around the moca domain of SRcyp, a human cyclophilin belonging to the Moca family. The segment of SRcyp containing the identified sites is specifically phosphorylated in mitotic cells. This mitosis-specific phosphorylation was inhibited by treatment of the cells with roscovitine, a specific inhibitor of cyclin-dependent kinases, suggesting that the unknown activity of the moca domain of SRcyp requires mitotic regulation by the Cdc2-cyclin B kinase complex. The Cdc2-cyclin B complex was found to phosphorylate four of the five identified phosphorylation sites in vitro, providing further support for this possibility. Like many factors stored in nuclear speckles and involved in the regulation of gene expression, this nuclear cyclophilin displays a predominantly diffuse cytoplasmic distribution at the onset of mitosis. Only in late telophase is SRcyp recruited to the newly formed nuclei. The transit of SRcyp through mitotic interchromatin granule clusters, before re-entering the nucleus, suggests that the timing of the appearance of this cyclophilin in the telophasic nuclei is tightly coordinated with post-mitotic events. Human SRcyp is the first cell cycle-regulated cyclophilin to be described.

Tissue-specific Expression and Dynamic Organization of SR Splicing Factors inArabidopsis

The organization of the pre-mRNA splicing machinery has been extensively studied in mammalian and yeast cells and far less is known in living plant cells and different cell types of an intact organism. Here, we report on the expression, organization, and dynamics of pre-mRNA splicing factors (SR33, SR1/atSRp34, and atSRp30) under control of their endogenous promoters in Arabidopsis. Distinct tissue-specific expression patterns were observed, and differences in the distribution of these proteins within nuclei of different cell types were identified. These factors localized in a cell type-dependent speckled pattern as well as being diffusely distributed throughout the nucleoplasm. Electron microscopic analysis has revealed that these speckles correspond to interchromatin granule clusters. Time-lapse microscopy revealed that speckles move within a constrained nuclear space, and their organization is altered during the cell cycle. Fluorescence recovery after photobleaching analysis revealed a rapid exchange rate of splicing factors in nuclear speckles. The dynamic organization of plant speckles is closely related to the transcriptional activity of the cells. The organization and dynamic behavior of speckles in Arabidopsis cell nuclei provides significant insight into understanding the functional compartmentalization of the nucleus and its relationship to chromatin organization within various cell types of a single organism.

Nuclear bodies and compartmentalization of pre-mRNA splicing factors in higher plants.

We studied the fine structural organization of nuclear bodies in the root meristem during germination of maize and Arabidopsis thaliana using electron microscopy (EM). Cajal bodies (CBs) were observed in quiescent embryos and germinating cells in both species. The number and distribution of CBs were investigated. To characterize the nuclear splicing domains, immunofluorescence labelling with antibodies against splicing factors (U2B" and m3G-snRNAs) and in situ hybridisation (with U1/U6 antisense probes) were performed combined with confocal microscopy. Antibodies specific to the Arabidopsis SR splicing factor atRSp31 were produced. AtRSp31 was detected in quiescent nuclei and in germinating cells. This study revealed an unexpected speckled nuclear organization of atRSp31 in root epidermal cells where micro-clusters of interchromatin granules were also observed by EM. Therefore, we examined the distribution of green fluorescent protein (GFP)-tagged atRSp31 in living cells after Agrobacterium -mediated transient expression. When expressed transiently, atRSp31-GFP exhibited a speckled distribution in leaf cells. Treatments with alpha-amanitin, okadaic acid, staurosporine or heat shock induced the speckles to reorganize. Furthermore, we generated stable Arabidopsis transgenics expressing atRSp31-GFP. The distribution of the fusion protein was identical to that of endogenous atRSp31. Three-dimensional time-lapse confocal microscopy showed that speckles were highly dynamic domains over time.

Nuclear substructure and dynamics

Many aspects of nuclear organization exhibit dynamic properties. Both nuclear bodies and individual chromatin regions can move through the nucleoplasm. Certain nuclear bodies are able to assemble and disassemble from soluble components in response to the metabolic demands of the cell. The cycling behaviour of nuclear factors and the rapid turnover of nuclear body components also underlines the importance of measuring the dynamic properties of proteins and RNPs when interpreting both localization patterns and their possible functional roles. Static fluorescence or electron microscopy images usually indicate steady state accumulations of nuclear factors, but can fail to reveal sites where factors localize transiently. Recent work has also shown that some nuclear proteins and RNPs participate in directional trafficking pathways, moving in a temporal sequence between distinct classes of nuclear bodies. For example, the nucleolar snoRNP protein NHPX/p15.5kDa transiently accumulates in splicing speckles before adopting a steady state concentration in nucleoli and Cajal bodies.Our understanding of the dynamic organization of the cell nucleus is expanding rapidly and this is facilitated by the increasing range of new methods to study the composition, structure and movement of nuclear bodies and their components. In the case of nucleoli, NPCs, speckles and Cajal bodies, they can be purified and their protein components can be identified using mass spectrometry. The ability to label nuclear structures in vivo through the expression of fluorescent protein-tagged factors also now allows many dynamic parameters to be measured directly in living cells using photobleaching techniques. Further developments in quantitative fluorescence microscopy and quantitative proteomics will help to develop a more robust model of how the nucleus is organized and should inform our view as to how nuclear structure influences cellular function.

Nuclear speckles: a model for nuclear organelles

Speckles are subnuclear structures that are enriched in pre-messenger RNA splicing factors and are located in the interchromatin regions of the nucleoplasm of mammalian cells. At the fluorescence-microscope level they appear as irregular, punctate structures, which vary in size and shape, and when examined by electron microscopy they are seen as clusters of interchromatin granules. Speckles are dynamic structures, and both their protein and RNA-protein components can cycle continuously between speckles and other nuclear locations, including active transcription sites. Studies on the composition, structure and behaviour of speckles have provided a model for understanding the functional compartmentalization of the nucleus and the organization of the gene-expression machinery.

Nuclear distribution of Oct-4 transcription factor in transcriptionally active and inactive mouse oocytes and its relation to RNA polymerase II and splicing factors.

The intranuclear distribution of the transcription factor Oct-4, which is specifically expressed in totipotent mice stem and germ line cells, was studied in mouse oocytes using immunogold labeling/electron microscopy and immunofluorescence/confocal laser scanning microcopy. The localization of Oct-4 was studied in transcriptionally active (uni/bilaminar follicles) and inactive (antral follicles) oocytes. Additionally, the Oct-4 distribution was examined relative to that of the unphosphorylated form of RNA polymerase II (Pol II) and splicing factor (SC 35) in the intranuclear entities such as perichromatin fibrils (PFs), perichromatin granules (PGs), interchromatin granule clusters (IGCs), Cajal bodies (CBs), and nucleolus-like bodies (NLBs). It was shown that: (i) Oct-4 is localized in PFs, IGCs, and in the dense fibrillar component (DFC) of the nucleolus at the transcriptionally active stage of the oocyte nucleus; (ii) Oct-4 present in PFs and IGCs colocalizes with Pol II and SC 35 at the transcriptionally active stage; (iii) Oct-4 accumulates in NLBs, CBs, and PGs at the inert stage of the oocyte. The results confirm the previous suggestion that PFs represent the major nucleoplasmic structural domain involved in active pre-mRNA transcription/processing. The colocalization of Oct-4 with Pol II in both IGCs and PFs in active oocytes (uni/bilaminar follicles) suggests that Oct-4 is intimately associated with the Pol II holoenzyme before and during transcription. The colocalization of Oct-4, Pol II, and SC 35 with coilin-containing structures such as NLBs and CBs at the inert stage (antral follicles) suggests that the latter may represent storage sites for the transcription/splicing machinery during the decline of transcription.

Adenovirus early region 4 promotes the localization of splicing factors and viral RNA in late-phase interchromatin granule clusters

Adenovirus early region 4 (E4) mutants are defective for late gene expression and show reduced levels of late RNA in both the cytoplasm and the nucleus. These reductions reflect a posttranscriptional defect in the production of viral late RNA. We find that E4 mutants form replication centers during the initial stages of infection and are able to redistribute splicing factors to transcription sites that surround viral replication centers. However, E4 mutant infected cultures have reduced numbers of cells with splicing factors localized in enlarged interchromatin granule clusters during the late phase. Although the late-phase interchromatin granule clusters that formed in wild-type and E4 mutant infected cells had similar levels of poly(A) RNA, they contained reduced levels of viral RNA. These results suggest that E4 mutants do not efficiently accumulate viral late RNA in late-phase interchromatin granule clusters following the onset of late RNA transcription.

Nuclear speckle localisation of the small heat shock protein αB-crystallin and its inhibition by the R120G cardiomyopathy-linked mutation

In this study, the small heat shock protein (sHSP) chaperones, αB-crystallin and HSP27, are identified as nuclear speckle components in unstressed cells in tissue culture. This new finding suggests a constitutive function for these sHSP chaperones in the nucleus and suggests a new perspective on the cardiomyopathy-causing mutation for αB-crystallin that could involve transcriptional splicing effects. Both αB-crystallin and HSP27 were immunolocalised to nuclear speckles (interchromatin granule clusters). While αB-crystallin was preferentially localised to speckles as shown by colocalisation with non-snRNP, SC35, as well as the snRNP components Sm and U1A, HSP27 was also seen associated with the nucleolar compartment, indicating a subtle difference between these closely related sHSPs. Actinomycin D treatment caused the relocalisation of αB-crystallin along with Sm and SC35 to a smaller number of more distinct spots, suggesting a link between speckle localisation and the transcriptional status of the cells. We then examined several transformed, immortalised, and primary cells expressing endogenous αB-crystallin as well as some cells with ectopic αB-crystallin expression. All consistently showed αB-crystallin in nuclear speckles. The nuclear localisation of the sHSPs was also confirmed biochemically and 2D gel electrophoresis revealed that there was only one major nuclear αB-crystallin isoform. This suggested that phosphorylation was not required for nuclear localisation of αB-crystallin. This was confirmed by the transient transfection of HeLa cells with a phosphorylation-defective αB-crystallin. In contrast, the transfection of R120G αB-crystallin, the mutation that causes cardiomyopathy, inhibited the nuclear speckle localisation of αB-crystallin. These data suggest that the cardiomyopathy-causing mutation for αB-crystallin has nuclear as well as cytoplasmic consequences, suggesting an explanation for the difference in severity of the desmin and αB-crystallin transgenic models of their respective cardiomyopathies.

Diacylglycerol kinase-θ is localized in the speckle domains of the nucleus

It is well established that the nucleus is endowed with enzymes that are involved in lipid-dependent signal transduction pathways. Diacylglycerol (DAG) is a fundamental lipid second messenger that is produced in the nucleus. Previous reports have shown that the nucleus contains diacylglycerol kinases (DGKs), i.e., the enzymes that, by converting DAG into phosphatidic acid (PA), terminate DAG-dependent events. Here, we show, by immunofluorescence staining and confocal analysis, that DGK-θ localizes mainly to the nucleus of various cell lines, such as MDA-MB-453, MCF-7, PC12, and HeLa. Nuclear DGK-θ co-localizes with phosphatidylinositol 4,5-bisphosphate (PIP 2) in domains that correspond to nuclear speckles, as revealed by the use of an antibody to the splicing factor SC-35, a well-established marker for these structures. The spatial distribution of nuclear DGK-θ was dynamic in that it was affected by inhibition of mRNA transcription with α-amanitin. Immuno-electron microscopy analysis demonstrated that DGK-θ, PIP 2, and phosphoinositide-specific phospholipase Cβ1 (PLCβ1) associated with electron-dense particles within the nucleus that correspond to interchromatin granule clusters. Cell fractionation experiments performed in MDA-MB-453, HeLa, and PC12 cells showed a preferential association of DGK-θ with the nucleus. Western blots demonstrated that DGK-θ was enriched in the nuclear matrix fraction prepared from MDA-MB-453 cells. Immunoprecipitation experiments with an antibody to PLCβ1 revealed in MDA-MB-453 cells an association between this enzyme and both DGK-θ and phosphatidylinositol phosphate kinase Iα (PIPKIα). Our findings strengthen the contention that speckles represent a crucial site for the nuclear-based inositol lipid cycle. We may speculate that nuclear speckle-located DGK-θ, on cell stimulation with an agonist, converts to PA the DAG derived from PLCβ1-dependent PIP 2 hydrolysis.

Distinct nuclear body components, PML and SMRT, regulate the trans-acting function of HTLV-1 Tax oncoprotein

Several viruses target cellular promyelocytic leukemia (PML)-nuclear bodies (PML-NBs) to induce their disruption, marked morphological changes in these structures or the relocation to PML-NB components to the cytoplasm of infected cells. PML conversely interferes with viral replication. We demonstrate that PML acts as a coactivator for the human T-cell leukemia virus type 1 (HTLV-1) Tax oncoprotein without direct binding. Tax was identified within interchromatin granule clusters (IGCs)/RNA splicing bodies (SBs), not PML-NBs; Tax expression did not affect PML-NB formation. Moreover, PML and CBP/p300 cooperatively activated Tax-mediated HTLV-1-LTR-dependent gene expression. Interestingly, two PML mutants, PML-RAR and PMLDelta216-331, which fail to form PML-NBs, could also coactivate Tax-mediated trans-acting function but had no effect on retinoic acid receptor (RAR)- or p53-dependent gene expression. In contrast, SMRT (silencing mediator for retinoic acid and thyroid hormone receptors), a nuclear corepressor found within the matrix-associated deacetylase (MAD) nuclear body, relocalized into Tax-associated nuclear bodies upon coexpression with Tax. SMRT coactivated the trans-acting function of Tax through direct binding. Coexpression of SMRT and PML resulted in an additive activation of Tax trans-acting function. Thus, crosstalk between distinct nuclear bodies may control Tax function.

The prodomain of interleukin 1alpha interacts with elements of the RNA processing apparatus and induces apoptosis in malignant cells.

Interleukin 1alpha (IL-1alpha), a 33 kDa precursor, is cleaved releasing the 17 kDa carboxyl-terminal cytokine IL-1alpha to which all of the biological properties of IL-1alpha have been attributed. We investigated the potential independent properties of the remaining 16 kDa IL-1alpha amino-terminal propiece by expression in human tumor and primary human cell lines. The IL-1alpha propiece produced apoptosis in malignant but not normal cell lines. A minimal fragment comprised of amino acids 55-108 was required for apoptosis. Deletion and mutation studies identified an extended nuclear localization sequence required for nuclear localization, induction of apoptosis and concentration of the IL-1alpha propiece in interchromatin granule clusters, concentrations of proteins in the RNA splicing and processing pathways. The IL-1alpha propiece interacted with five known components of the RNA splicing/processing pathway, suggesting that the mechanism of action may involve changes in RNA splicing or processing. Expression of the IL-1alpha propiece caused a shift in the ratio of Bcl-Xl/Bcl-Xs toward the apoptotic direction. Our findings indicate that the IL-1alpha propiece induces apoptosis in a range of tumor cells and likely operates through a mechanism involving the RNA processing apparatus and the alternate splicing of apoptosis regulatory proteins.

Sm and U2B" proteins redistribute to different nuclear domains in dormant and proliferating onion cells.

Monoclonal antibodies against the spliceosomal proteins Sm and U2B", and against p105, a protein component of interchromatin granules, were used to investigate the nuclear distribution of the splicing factors in Allium cepa L. meristematic cells. Confocal microscopy showed that in steady-state proliferating cells, the spliceosomal components were distributed into two nuclear domains: (i) a diffuse nucleoplasmic network similar to that formed by interchromatin granules and (ii) numerous Cajal bodies. These domains were the counterpart of the perichromatin fibrils and granules, interchromatin granules and Cajal bodies observed by electron microscopy after EDTA and bismuth oxynitrate stainings. Dormant cells showed a nuclear distribution of the proteins in small Cajal bodies and numerous micro-speckles, correlated with the distribution of ribonucleoproteins (RNPs) observed by electron microscopy. The spliceosomal proteins relocated to the diffuse nucleoplasmic network and Cajal bodies when the cells were released from dormancy by water soaking and they re-started their proliferative activity. Inhibition of RNA synthesis by 5,6-dichloro-1-beta- d-ribofuranosylbenzimidazole (DRB) treatment in proliferating cells demonstrated that the micro-speckles were not the morphological expression of a transcription block. Fractionation and confocal microscopy studies showed a differential association of the splicing factors with the nuclear matrix depending not only on the protein, but also on nuclear activity. Our results suggest a reversible relocation of the spliceosomal proteins between different sub-nuclear domains in physiological conditions. We report here an unusual nuclear domain in dormant nuclei, the micro-speckles, corresponding to storage sites for RNPs, which were rapidly mobilised after water imbibition.

Rev inhibition strongly affects intracellular distribution of human immunodeficiency virus type 1 RNAs.

To define the human immunodeficiency virus type 1 (HIV-1) RNA maturation pathways, we analyzed the intracellular distribution of HIV-1 RNA and the viral regulatory proteins Rev and Tat in transfected COS cells and HIV-1-infected lymphoid C8166 cells by means of ultrastructural in situ hybridization using antisense RNA probes and immunoelectron microscopy. The intranuclear viral RNA occurs in ribonucleoprotein fibrils in the perichromatin and interchromatin regions. The simultaneous demonstration of Rev, Tat, Br-labeled RNA, and cellular proteins SC35 and CRM1 in such fibrils reveals the potential of Rev to associate with nascent HIV pre-mRNA and its splicing complex and transport machinery. In a rev-minus system, the env intron-containing, incompletely spliced viral RNAs are revealed only in the nucleus, indicating that Rev is required to initiate the transport to the cytoplasm. Moreover, env intron sequences frequently occur in the periphery of interchromatin granule clusters, while the probe containing the rev exon sequence does not associate with this nucleoplasmic domain. When cells were treated with the CRM1 inhibitor leptomycin B in the presence of Rev protein, the env intron containing HIV RNAs formed clusters throughout the nucleoplasm and accumulated at the nuclear pores. This suggests that Rev is necessary and probably also sufficient for the accumulation of incompletely spliced HIV RNAs at the nuclear pores while CRM1 is needed for translocation across the nuclear pore complex.

MAPKs entry into the nucleus at specific interchromatin domains in plant differentiation and proliferation processes

Mitogen-activated protein kinases (MAPKs) are involved in the signaling of extracellular stimuli in eukaryotes, including plants. Different MAPKs have recently been shown to be expressed during plant cell proliferation and developmental processes such as pollen development and embryogenesis, but the structural subdomain where these MAPKs are targeted in the nucleus has not yet been characterized. We have determined the changes in the expression and subcellular localization of ERK homologues, proteins belonging to the MAPK family, and MAPK-active forms in two plant developmental processes which involved differentiation (pollen maturation) and proliferation (the initials of pollen embryogenesis). Immunofluorescence and immunogold labeling in the species studied showed that the progression of differentiation and proliferation was accompanied by an increase in the expression of ERKs and MAPK activation together with a translocation to the nucleus. Combining ultrastructural cytochemistry and immunogold for RNA and phosphorylated proteins we have identified the nuclear sites housing these MAPKs in areas of the interchromatin region enriched in RNA and phosphoproteins that include clusters of interchromatin granules. This could suggest a role of these MAPKs in the early events of activation of the transcription and processing machinery, via phosphorylation, which subsequently would be recruited to the transcription sites. The association of the nuclear localization of MAPKs with the progression through the cell cycle and the commitment toward differentiation in the two plant developmental processes can be correlated.

Walleye dermal sarcoma virus cyclin interacts with components of the mediator complex and the RNA polymerase II holoenzyme.

Walleye dermal sarcoma virus (WDSV) encodes an accessory protein, OrfA, with sequence homology to cyclins (retrovirus cyclin). In cells transfected with an expression construct, OrfA was localized to the nucleus and was concentrated in interchromatin granule clusters (IGCs), sites where splicing factors are concentrated. Other proteins identified in IGCs include transcription factors, the large subunit of RNA polymerase II (Pol II), and cyclin-dependent kinase 8 (cdk8). cdk8 is the kinase partner of cyclin C and a component of the mediator complex, associated with the Pol II holoenzyme. cdk8 and cyclin C can regulate transcription via phosphorylation of cyclin H and the carboxy-terminal domain of Pol II. OrfA in transfected HeLa cells was found to colocalize and copurify with hyperphosphorylated forms of Pol II (Pol IIO) in IGCs, and OrfA was coimmunoprecipitated from lysates of transfected cells with an antibody against Pol IIO. Likewise, Pol IIO could be coprecipitated with an antibody against OrfA. A survey with antibodies against several different cdks resulted in coimmunoprecipitation of OrfA with anti-cdk8, and antiserum against OrfA was able to coprecipitate cdk8 from lysates of cells that express OrfA. Coprecipitation of OrfA with anti-cyclin C demonstrated that it was included in complexes with OrfA and cdk8. OrfA has sequence and structural similarities to cyclin C, and, functionally, OrfA appears to have the capacity to both enhance and inhibit the activity of promoters in a cell-specific manner, similar to functions of the mediator complex. These data suggest that WDSV OrfA functions through its interactions with these large, transcription complexes. Further investigations will clarify the role of the retrovirus cyclin in control of virus expression and transformation.