Nuclear matrix-like filaments and fibrogranular complexes form through the rearrangement of specific nuclear ribonucleoproteins

The behavior of nuclear pre-mRNA-binding proteins after their nuclease and/or salt-induced release from RNA was investigated. After RNase digestion or salt extraction, two proteins that initially exist as tetramers (A2)(3)B1 in isolated heterogeneous nuclear ribonucleoprotein (hnRNP) complexes quantitatively reassociated to form regular helical filaments ranging in length from 100 nm to >10 microm. In highly magnified preparations prepared for scanning transmission electron microscopy, single filaments have diameters near 18 nm. In conventional negatively stained preparations viewed at low magnification, the diameters of the thinnest filaments range from 7 to 10 nm. At protein concentrations of >0.1 mg/ml, the filaments rapidly aggregated to form thicker filamentous networks that look like the fibrogranular structures termed the "nuclear matrix." Like the residual material seen in nuclear matrix preparations, the hnRNP filaments were insoluble in 2 M NaCl. Filament formation is associated with, and may be dependent on, disulfide bridge formation between the hnRNP proteins. The reducing agent 2-mercaptoethanol significantly attenuates filament assembly, and the residual material that forms is ultrastructurally distinct from the 7- to 10-nm fibers. In addition to the protein rearrangement leading to filament formation, nearly one-third of the protein present in chromatin-clarified nuclear extracts was converted to salt-insoluble material within 1 min of digestion with RNase. These observations are consistent with the possibility that the residual material termed the nuclear matrix may be enriched in, if not formed by, denatured proteins that function in pre-mRNA packaging, processing, and transport.     

Considerations in the isolation of rat liver nuclear matrix,nuclear envelope,and pore complex lamina

A number of recent studies have demonstrated a salt-, nuclease, and detergent-resistant subnuclear structure termed the nuclear protein matrix which consists of a fibrogranular intranuclear network, residual components of the nucleolus, and a peripheral lamina. Other workers, however, have shown that somewhat similar methods result in the isolation of the peripheral lamina devoid of the intranuclear components. In this report we demonstrate that seemingly slight changes in the isolation procedure cause major changes in the morphology of the residual structures obtained. When freshly purified rat liver nuclei were digested with DNase I and RNase A and then extracted with buffers of low magnesium ion concentration (LS buffer) and high ionic strength (HS buffer), the resulting structures isolated prior to or after Triton X-100 extraction lacked the extensive intranuclear network and the easily identifiable residual nucleoli present in the nuclear protein matrix. Systematic modification of this extraction procedure revealed that morphologically identifiable residual nucleoli were present when digestion with RNase A followed extraction with HS buffer but were absent when the order of these steps was reversed. The removal of the nucleolus by RNase A and HS buffer correlated with the removal of nuclear RNA by the same treatments. These coordinate events could not be prevented by treatment with protease inhibitors but were prevented by treatment of the RNase A with diethylpyrocarbonate, an RNase inhibitor. The extensive intranuclear network seen in the nuclear protein matrix was sparse or absent when residual structures were prepared from DNase- and RNase-treated nuclei under conditions which minimized the oxidation of protein sulfhydryl groups. In contrast, an extensive non-chromatin intranuclear network was seen if the formation of intermolecular protein disulfide bonds was promoted by extraction of nuclei with cationic detergents, by overnight incubation, or by treatment with oxidizing agents like sodium tetrathionate prior to nuclease digestion and subsequent extraction. By varying the order of extraction steps and the extent of disulfide cross-linking, it is possible to isolate from a single batch of nuclei residual structures with a wide range of morphologies and compositions.     

The nuclear matrix of duck erythroblasts is associated with globin mRNA coding sequences but not with the major proteins of 40S nuclear RNP

A residual protein matrix has been prepared from avian erythroblast nuclei by extensive extraction with salines and detergent and subsequent digestion with high concentrations of RNase and DNase. Ultrastructural examination reveals considerable internal structure, the most prominent feature being the remains of the nucleoli embedded in a network of fibres of fairly uniform diameter of 50 Å. The proteins which make up this structure have been examined by two-dimensional electrophoresis and are shown to consist of a characteristic set of about 30, mainly acidic components, including four prominent species of 43 000, 52 000, 66 000 and 68 000 molecular weight (MW). In parallel preparations of the nuclear matrix digested with DNase alone, much of the nuclear RNA is found associated with the residual structure, including globin-coding sequences. These results correlate well with the ultrastructural appearance of DNase-digested matrix preparations which show that superimposed on the 50 Å fibrous network is a 200–300 Å granular component, the combined fibrillo-granular structure resembling the interchromatin RNP previously identified in situ. However, the proteins of the DNase-digested matrix seen by two-dimensional electrophoresis are indistinguishable from the proteins of matrix preparations digested with both DNase and RNase. Furthermore, two-dimensional comparison between the proteins of the DNase-digested matrix and purified 40S nuclear RNP particles shows that the bulk of the proteins found associated with nuclear RNA in vitro are extracted during matrix preparation, and only two, with MWs of 43 000 and 73 000, remain. The latter species co-migrates with the poly(A)-binding protein.     

Characterization of the 2A7 Antigen as a 85-kDa Human Nucleocytoplasmic Shuttling Protein

The murine monoclonal antibody 2A7 was found to react specifically with a 85-kDa human protein which is distributed throughout the nuclear interior in interphase and becomes associated with condensed chromosomes during mitosis. The 2A7 epitope was not detected in cells from other species. Two-dimensional immunoblotting analysis of HeLa cell homogenates further indicated that the 85-kDa polypeptide species recognized by the 2A7 antibody corresponds to an acidic protein which may be complexed in vivo within high-molecular-weight protein structures. Immunofluorescence monitoring of the 2A7 staining pattern during in situ preparation of nuclear matrices from HeLa cells demonstrated that the nucleoplasmic fraction of the antigen is readily solubilized by detergent and salts, whereas the nucleolar fraction resists detergent/salt extraction and DNase digestion, to be released only upon RNase activity. Mobility assays in human-mouse heterokaryons provided evidence that the 2A7 antigen is a nucleocytoplasmic shuttling protein. The nuclear distribution of this antigen remained unchanged upon drug-induced inhibition of RNA synthesis but was markedly altered by heat shock stress. All together, the data presented here suggest that the 2A7 antigen may have a function in RNA metabolism.     

Phospholipid interactions in rat liver nuclear matrix

Rat liver nuclear matrix has been isolated by salt extraction and nuclease digestion of nuclei. Under the electron microscope, the matrix appears as a spongelike network joined by thinner fibrils. Biochemical analysis shows a high protein content and low amounts of nucleic acid and phospholipid. Treatment of the matrix with phospholipase C results in a release of most of the nucleic acid, and a disappearance of the fibrils, however the appearance of the matrix is largely unaffected. It seems likely that phospholipids are responsible for the hydrophobic interactions between nucleic acids and matrix fibrils. From $\text{in vitro}$ labelling studies the released DNA is more recently synthesised than the bulk material, however the matrix bound RNA appears to label less rapidly than total nuclear RNA.     

SV40 virions and viral RNA metabolism are associated with cellular substructures

Nuclear matrices were prepared by DNase and high salt extraction of SV40-infected epithelial monkey cells. The matrices retain the majority of SV40 virions. This conclusion is based on electron microscopic observations of the occurrence of encapsidated viral DNA that is resistant to DNase digestion and on the analysis of viral proteins by gel electrophoresis. Pulse labeled SV40 RNA is also associated with the nuclear matrix (less than 15% of the viral RNA is removed by DNase and high salt). Pulse-chase experiments revealed that processing of SV40 RNA takes place on the nuclear matrix and the processed molecules are directly transported to the cytoplasm where they are associated with the cytoskeleton. These results suggest a central role for the nuclear and cytoplasmic substructures in virus assembly and in the biogenesis of viral RNA.     

Core filaments of the nuclear matrix

The nuclear matrix is concealed by a much larger mass of chromatin, which can be removed selectively by digesting nuclei with DNase I followed by elution of chromatin with 0.25 M ammonium sulfate. This mild procedure removes chromatin almost completely and preserves nuclear matrix morphology. The complete nuclear matrix consists of a nuclear lamina with an interior matrix composed of thick, polymorphic fibers and large masses that resemble remnant nucleoli. Further extraction of the nuclear matrices of HeLa or MCF-7 cells with 2 M sodium chloride uncovered a network of core filaments. A few dark masses remained enmeshed in the filament network and may be remnants of the nuclear matrix thick fibers and nucleoli. The highly branched core filaments had diameters of 9 and 13 nm measured relative to the intermediate filaments. They may serve as the core structure around which the matrix is constructed. The core filaments retained 70% of nuclear RNA. This RNA consisted both of ribosomal RNA precursors and of very high molecular weight hnRNA with a modal size of 20 kb. Treatment with RNase A removed the core filaments. When 2 M sodium chloride was used directly to remove chromatin after DNase I digestion without a preceding 0.25 M ammonium sulfate extraction, the core filaments were not revealed. Instead, the nuclear interior was filled with amorphous masses that may cover the filaments. This reflected a requirement for a stepwise increase in ionic strength because gradual addition of sodium chloride to a final concentration of 2 M without an 0.25 M ammonium sulfate extraction uncovered core filaments.     

Alterations of neuronal nuclear matrix and chromatin structure after irradiation under aerobic and anoxic conditions

This study was undertaken to determine if structural alterations of the bulk chromatin and the amount of protein associated with the nuclear matrix in cerebellar neurons depend on radiation dose and a cell's state of oxygenation. After irradiation with 2.5 to 25.0 Gy under both aerobic and anoxic conditions, the sensitivity of the neuronal chromatin to m. nuclease digestion increase linearly with dose up to about 5 Gy, beyond which there was no further increase. The same increase in accessibility of chromatin to micrococcal nuclease digestion was observed when neuronal nuclei were irradiated at 4 degrees C. Neuronal nuclei were stained with propidium iodide (PI) for DNA and with fluorescein isothiocyanate (FITC) for protein, both before and after complete digestion with DNase I, and analyzed by flow cytometry. There was no change in either the PI (P greater than 0.4) or the FITC (P greater than 0.9) fluorescence of undigested nuclei after irradiation. For the DNase I digested nuclei, the PI fluorescence was unchanged after irradiation (P greater than 0.4), but the FITC fluorescence increased significantly (P less than 0.02). This increase in the FITC fluorescence was linear with dose up to about 5 Gy, beyond which there was no further increase. The flow cytometry results from DNase I digested nuclei were identical for neurons irradiated under aerobic or anoxic conditions, indicating that this phenomenon is oxygen independent. This increase in FITC fluorescence after irradiation was inhibited at ice-cold temperatures and probably reflects an increase in protein content at the nuclear matrix that requires metabolism. This may explain our previously observed resistance of nuclear matrix-associated DNA to digestion by DNase I. This protein increase at the nuclear matrix appears to follow "saturation" kinetics identical to that previously reported for repair of DNA strand breaks in cerebellar neurons. However, the exact molecular nature of this process and its role in DNA repair or cell survival remains to be determined.     

Residual structure and constituent proteins of the peripheral framework of the cell nucleus in somatic embryos from Daucus carota L.

Nuclei were isolated from somatic embryos of carrot (Daucus carota L.) using a buffer system containing non-ionic detergent. To prepare nuclear matrices, the purified membrane-depleted nuclei were digested with DNase I in combination with RNase A, followed by extraction with 1 M NaCl. The DNA residue in the final insoluble fraction was less than 4% of that in isolated nuclei, and most of the residual nuclei retained their sphericity. Electron microscopy revealed that the nuclear matrix was composed of a distinct peripheral layer, an internal matrix structure and some fibrils; residual nucleoli were observed when exogeneous RNase was not incorporated. The proteins extracted from the nuclei and nuclear subfractions were compared by gel electrophoresis, which showed that the residual fraction contained many minor proteins. To identify proteins showing specific localization at the nuclear periphery, we prepared monoclonal antibodies (MAbs) against an ion-exchange chromatography fraction extracted from carrot nuclear matrices. Immunofluorescence microscopy with one of the MAbs, CML-1, showed exclusive staining of the nuclear periphery. The MAb recognized several spots showing microheterogeneity, with a narrow range of pI and molecular mass upon immunoblotting. A complete set of these spots was shown to be conserved in nuclear matrices. On the other hand, MAb CML-13 appeared to react with the nuclear interior as well as the periphery, recognizing a 96-kDa polypeptide of the nuclear matrix. These proteins were thus demonstrated to lie at the nuclear periphery, and to constitute the nuclear matrices in carrot. The 96-kDa polypeptide is suggested to be similar to the 92-kDa nuclear protein reported by Beven et al. in carrot (Beven et al., 1991, J. Cell Sci. 98, 293–302).Abbreviations DEAE diethylaminoethyl - MAb monoclonal antibody - NEPHGE nonequilibrium pH gradient electrophoresis We wish to thank Ms. Akiko Itoh for excellent technical assistance. This work was supported by a Grant-in-Aid (05640738) from the Ministry of Education of Japan.     

Nuclear protein following heat shock: protein removal kinetics and cell cycle rearrangements

Nuclear protein and DNA content of HeLa cells was determined as a function of time following hyperthermia by staining isolated nuclei with two fluorescent dyes: fluorescein isothiocyanate (FITC) for protein content and propidium iodide (PI) for DNA content. Bivariate FITC and PI histograms were obtained by flow cytometry. Univariate flow cytometric analysis was shown to be inadequate for this study, because some of the nuclear protein changes were due to cell cycle redistribution. Posthyperthermia cell kinetics could be divided into two distinct phases: an early phase characterized by the removal of heat-induced excess nuclear proteins with little or no cell progression through the cell cycle; and a late phase characterized by a redistribution of cells in the cell cycle resulting in an accumulation of cells in G2. The duration of these phases was dependent upon the hyperthermia dose. In the early phase, the rate of removal of excess nuclear protein was found to vary with heating time and temperature for time-temperature combinations which resulted in the same amount of excess nuclear protein. In the late phase, the cells blocked in G2 did not reduce their nuclear protein levels back to control values.     

Use of CpG island microarrays to identify colorectal tumors with a high degree of concurrent methylation

Oligonucleotide-targeted RNase H protection assays are powerful means to analyze protein binding domains in ribonucleoprotein particles (RNPs). In such an assay, the RNA component of a RNP and, in an essential control reaction, the corresponding deproteinized RNA are targeted with an antisense DNA oligonucleotide and RNase H. If the oligonucleotide is able to anneal to the complementary sequence of the RNA, RNase H will cleave the RNA within the double-stranded DNA/RNA region. However, protein binding to a specific RNA sequence may prevent hybridization of the DNA oligonucleotide, thereby protecting the RNA molecule from endonucleolytic cleavage. An RNase H protection analysis can usually be carried out with crude cell extract and does not require further RNP purification. On the other hand, purified RNP fractions are preferable when a crude extract contains RNase activity or a heterogenous RNP population of a specific RNA. The cleavage pattern of RNase H digestion can be analyzed by Northern blotting or primer-extension assays. In addition, the investigation of RNP fragments, for example, by native gel electrophoresis, may reveal important structural information about a RNP. In this article, we describe procedures for RNP and RNA preparation, the oligonucleotide-targeted RNase H protection assay, and methods for the analysis of RNA and RNP cleavage products. As an example, we show oligonucleotide-targeted RNase H protection of the Trypanosoma brucei U1 small nuclear RNP.     

Nuclear matrix-associated NMN adenylyltransferase activity in human placenta.

This paper presents data about the presence of the NMN adenylyltransferase at the nuclear matrix level of human placenta nuclei. It was found that 40-45% of the activity (depending on the extraction procedure) referred to the total nuclear NMN adenylyltransferase was tightly associated with this subnuclear compartment. The matrices purified by two different procedures exhibited DNA, RNA and protein contents comparable with those described in literature. Extensive digestion of human placenta nuclei with DNase I was not able to solubilize the NMN adenylyltransferase activity. Therefore, the data we present are consistent with the conclusion that a part of the total nuclear NMN adenylyltransferase is associated with the nuclear matrix.     

Synaptonemal complexes are integral components of the isolated mouse spermatocyte nuclear matrix

Synaptonemal complexes (SCs) have been isolated as integral components of the nuclear matrix from purified mouse pachytene spermatocytes. These nuclear synaptonemal complex-matrices are prepared by extracting Triton X-100-treated nuclei with low (0.2 M) and high (1.0 or 2.0 M) NaCl, DNase I, and RNase A to remove 85% of the nuclear proteins, 97% of the RNA, and 99% of the DNA. Studies with the light and electron microscopes indicate that these matrices, while lacking a distinct lamina, contain nuclear pores interconnected by a fiber network, residual nucleoli, and interchromatin fibers. In addition, the pachytene spermatocyte matrices contain residual XY heterochromatin and the principal components of the SCs, including two lateral elements, a central element, a presumptive centromere, and attachment plaques. These SCs are preserved within the matrix and retain their structural association with the pore-fiber complex, even when subjected to strong dissociating conditions. Nuclear matrices from pachytene spermatocytes and spermatids (steps 1-8), when analyzed by SDS PAGE, contain an array of polypeptides distinct from those of mouse liver nuclear matrices. Proteins of spermatogenic matrices range in Mr from 8,000 to approximately 150,000. The prominent lamina proteins (Mr approximately 60,000-70,000) of somatic nuclear matrices are either absent or represent only a minor part of the spermatogenic matrix. The polypeptide composition of the pachytene spermatocyte and spermatid matrices are similar, although minor quantitative and qualitative differences are evident. These observations suggest that the SC constituents may consist of a heterogeneous group of proteins present in low proportion relative to total matrix proteins, or they may be retained, but in a different form, within the spermatid matrix.     

Correspondence of two nuclear networks observed in situ with the nuclear matrix

In resting lymphocytes, three well defined networks are observed and attempts were made to superimpose them upon the networks described in isolated nuclear matrices. These three nuclear structures are seen after DNase and RNase treatment. They are digested by pepsin but their sensitivity to this enzyme is different. The more resistant network corresponds to the outer lamina of the isolated nuclear matrix. The second network is located in the inter-chromatin area. It is more sensitive to pepsin than the lamina and this sensitivity is increased ten-fold when digestion with pepsin is preceded by RNase digestion. This network corresponds to the internal network of isolated nuclear matrices. The third network is located in the intrachromatin area and is the most sensitive to pepsin action.     

Unraveling the organization of the internal nuclear matrix: RNA-dependent anchoring of NuMA to a lamin scaffold

Using quantitative immunoelectron microscopy we show here that when the nuclear matrix is isolated from rat hepatocytes in the presence of an inhibitor of RNase activity both lamins and the nuclear mitotic apparatus protein (NuMA) preferentially localize within the electron-dense domains of the internal nuclear matrix (INM). After RNA digestion NuMA undergoes a sharp depletion, while labeling by an antibody against lamins A and C within the electron-transparent regions increases, suggesting that a subset of lamin epitopes is masked by the interaction with RNA. We were able to explain this result by visualizing for the first time a thin web of lamin protofibrils which connects the electron-dense regions. Confirmation of these changes has been obtained by immunoblot analysis and confocal microscopy. As RNA digestion results both in the release of NuMA and in the collapse of the INM, we propose that a fraction of nuclear RNA brings about the association of NuMA islands with a lamin scaffold and that this interaction is required to maintain the latter in a state of high molecular dispersion.     

Quantitative immunofluorescence and immunoelectron microscopy of the topoisomerase IIα associated with nuclear matrices from wild-type and drug-resistant Chinese hamster ovary cell lines

Topo IIα is considered an important constituent of the nuclear matrix, serving as a fastener of DNA loops to the underlying filamentous scaffolding network. To further define a mechanism of drug resistance to topo II poisons, we studied the quantity of topo IIα associated with the nuclear matrix in drug-resistant SMR₁₆ and parental cells in the presence and absence of VP-16. Nuclear matrices were prepared from nuclei isolated in EDTA buffer, followed by nuclease digestion with DNase II in the absence of RNase treatment and extraction with 2 M NaCl. Whole-mount spreading of residual structures permits, by means of isoform-specific antibody and colloidal-gold secondary antibodies, an estimate of the amount of topo IIα in individual nuclear matrices. There are significant variations in topo IIα amounts between individual nuclear matrices due to the cell cycle distribution. The parental cell line contained eight to ten times more nuclear matrix–associated topo IIα than the resistant cell line matrices. Nuclear matrix–associated topo IIα from wild-type and resistant cell lines correlated well with the immunofluorescent staining of the enzyme in nuclei of intact cells. The amount of DNA associated with residual nuclear structures was five times greater in the resistant cell line. This quantity of DNA was not proportional to the quantity of topo IIα in the same matrix; in fact they were inversely related. In situ whole-mount nuclear matrix preparations were obtained from cells grown on grids and confirmed the results from labeling of isolated residual structures. J. Cell. Biochem. 67:112–130, 1997. © 1997 Wiley-Liss, Inc.     

Differential nuclear localization and nuclear matrix association of the splicing factors PSF and PTB

A monoclonal antibody raised against nuclear matrix proteins detected a protein of basic pI in human nuclear matrix protein samples of various cellular origin. The ubiquitously occurring (common) nuclear matrix protein was identified as splicing factor PSF (PTB associated splicing factor). The interaction between the splicing factors PSF and PTB/hnRNP I was confirmed by co-immunoprecipitation from nuclear salt extracts. However, the nuclear localization of PSF and PTB and their distribution in subnuclear fractions differed markedly. Isolated nuclear matrices contained the bulk of PSF, but only minor amounts of PTB. In confocal microscopy both proteins appeared in speckles, the majority of which did not co-localize. Removing a large fraction of the soluble PTB structures by salt extraction revealed some colocalization of the more stable PTB fraction with PSF. These PTB/PSF complexes as well as the observed PSF-PTB interaction may reflect the previously reported presence of PTB and PSF in spliceosomal complexes during RNA processing. The present data, however, point to different cellular distribution and nuclear matrix association of the majority of PSF and PTB.