Localization of nuclear matrix core filament proteins at interphase and mitosis.

The gentle removal of chromatin uncovers a nuclear matrix consisting of two parts: a nuclear lamina connected to the intermediate filaments of the cytoskeleton and an internal matrix of thick, polymorphic fibers connecting the lamina to masses in the nuclear interior. This internal nuclear matrix can be further fractionated to uncover a highly branched network of 9 nm and 13 nm core filaments retaining some enmeshed bodies. The core filament network retains most of the nuclear RNA, as well as the fA12RNP antigen, and may be the most basic or core element of internal nuclear structure. One high molecular weight protein component of the core filament network, the H1B2 antigen, is normally masked in the interphase nucleus and is uncovered as the chromatin condenses at mitosis. This protein is associated with a fibrogranular network surrounding and connected to the chromosomes. The core filament-associated fA12 antigen also becomes associated with this perichromosomal network. We propose that the core filament nuclear matrix structure may not completely disassemble at mitosis but, rather, that parts remain as a structural network connected to chromosomes and other mitotic structures. These mitotic networks may, in turn, serve as the core structures on which the nuclear matrices of daughter cells are built.     

Nuclear matrix: isolation and characterization of a framework structure from rat liver nuclei

A nuclear framework structure termed the nuclear matrix has been isolated and characterized. This matrix forms the major residual structure of isolated nuclei and consists largely of protein with smaller amounts of RNA, DNA, carbohydrate, and phospholipid. The nuclear matrix can be further resolved by combined treatment with DNase and RNase. The remaining nuclear protein structure, after extraction of 90 percent of the nuclear protein, 99.9 percent of the DNA, and 98 percent of the RNA and phospholipid, is termed the nuclear protein matrix. Electron microscopy of this final nuclear protein matrix reveals an interior framework structure composed of residual nucleolar structures associated with a granular and fibrous internal matrix structure. The internal matrix framework is derived from the interchromatinic structures of the nucleus, and is connected to a surrounding residual nuclear envelope layer containing residual nuclear pore complex structures. Sodium dodecyl sulfate-acrylamide gel electrophoresis of the nuclear matrix proteins demonstrates three major polypeptide fractions, P-1, P-2, and P-3, with average molecular weights of approximately 69,000, 66,000 and 62,000, as well as several minor polypeptides which migrate at approximately 50,000 and at higher molecular weights (>100,000). Polypeptides with molecular weights identical to those of P-1, P-2 and P-3 are also components of isolated nuclear envelopes and nucleoli, whereas isolated chromatin contains no detectable matrix polypeptides. This suggests that the major matrix polypeptides are localized in specific structural regions of the nucleus, i.e., nuclear envelope, nucleoli, and interchromatinic structures. The presence of cytochrome oxidase activity in the isolated nuclear matrix indicates that at least some integral proteins of the nuclear membrane are associated with the matrix.     

Nuclear development in locust fat body: the influence of juvenile hormone on inclusion bodies and the nuclear matrix

The hormonal induction of vitellogenesis in insects and in oviparous vertebrates are prime models of gene regulation in eukaryotes. In vertebrates the process is under estrogenic control and normally confined to females, although males can be artificially induced. In locust in contrast, juvenile hormone (JH) is central to fat body development in both males and females, yet the response is strongly sex limited not only for vitellogenin production but also in terms of total protein, DNA and RNA synthesis and nuclear ploidy levels. To differentiate further possible sex and/or JH related developmental aspects in locusts, large-scale nuclear events were examined during normal adult maturation and in animals treated with antiallatropins and JH analogs. Fat body nuclei undergo extensive restructuring during normal development in both sexes. This included progressive nuclear enlargement, accompanied by extensive proliferation of nuclear matrix components and elaboration of complex inclusion bodies (NB). The isolated protein matrix was unusually complex relative to similar structures from vertebrates and the NB were firmly anchored to it. Although matrix proteins were qualitatively similar to those from other sources, as assessed by SDS polyacrylamide gel electrophoresis, several major matrix polypeptides, including lamins A and B, and components greater than 150 kD, fluctuated quantitatively during development and in concert with nuclear enlargement. The number and morphology of the NB were unrelated to sex, but increased in direct proportion to absolute nuclear volumes. All changes were more pronounced in females, where higher ploidy levels, larger nuclei and correspondingly more internal matrix elements occurred. Suppression of JH production by precocene prevented all foregoing nuclear changes, but re-exposure to methoprene rapidly induced normal development. The results are compared to analogous nuclear changes in steroid responsive vertebrate tissues.     

Nuclear dreams: The malignant alteration of nuclear architecture

Cancer is diagnosed by examining the architectural alterations to cells and tissues. Changes in nuclear structure are among the most universal of these and include increases in nuclear size, deformities in nuclear shape, and changes in the internal organization of the nucleus. These may all reflect changes in the nuclear matrix, a non-chromatin nuclear scaffolding determining nuclear form, higher order chromatin folding, and the spatial organization of nucleic acid metabolism. Malignancy-induced changes in this structure may have profound effects on chromatin folding, on the fidelity of genome replication, and on gene expression. Elucidating the mechanisms and the biological consequences of nuclear changes will require the identification of the major structural molecules of the internal nuclear matrix and an understanding of their assembly into structural elements. If biochemical correlates to malignant alterations in nuclear structure can be identified then nuclear matrix proteins and, perhaps nuclear matrix-associated structural RNAs, may be an attractive set of diagnostic markers and therapeutic targets. J. Cell. Biochem. 70:172–180, 1998. © 1998 Wiley-Liss, Inc.     

Characterization of heterogeneous nuclear RNA-protein complexes in vivo with monoclonal antibodies.

Exposure of cells to UV light of sufficient intensity brings about cross-linking of RNA to proteins which are in direct contact with it in vivo. The major [35S]methionine-labeled proteins which become cross-linked to polyadenylated heterogeneous nuclear RNA in HeLa cells have molecular weights of 120,000 (120K), 68K, 53K, 43K, 41K, 38K, and 36K. Purified complexes of polyadenylated RNA with proteins obtained by UV cross-linking in intact cells were used to immunize mice and generate monoclonal antibodies to several of these proteins. Some properties of three of the proteins, 41K, 43K, and 120K, were characterized with these antibodies. The 41K and 43K polypeptides are highly related. They were recognized by the same antibody (2B12) and have identical isoelectric points (pl = 6.0 +/- 0.2) but different partial peptide maps. The 41K and 43K polypeptides were part of the 40S heterogeneous nuclear ribonucleoprotein particle and appear to correspond to the previously described C proteins (Beyer et al., Cell II:127-138, 1977). A different monoclonal antibody (3G6) defined a new major heterogeneous ribonucleoprotein of 120K. The 41K, 43K, and 120K polypeptides were associated in vivo with both polyadenylated and non-polyadenylated nuclear RNA, and all three proteins were phosphorylated. The monoclonal antibodies recognized similar proteins in human and monkey cells but not in several other vertebrates. Immunofluorescence microscopy demonstrated that these proteins are segregated to the nucleus, where they are part of a fine particulate nonnucleolar structure. In cells extracted in situ with nonionic detergent, all of the 41K and 43K polypeptides were associated with the nucleus at salt concentrations up to 0.5 M NaCl, whereas the 120K polypeptide was completely extracted at this NaCl concentration. A substantial fraction of the 41K and 43K polypeptides (up to 40%) was retained with a nuclear matrix--a structure which is resistant to digestion with DNase I and to extraction by 2 M NaCl, but the 41K and 43K polypeptides were quantitatively removed at 0.5 M NaCl after digestion with RNase.     

Characterization of lamin proteins in BHK cells

Lamins are structural proteins found in rat liver nuclear envelope and are major constituents of the nuclear matrix. 2-D gel electrophoresis indicates that BHK cell nuclear matrix is composed of four major proteins (62 kD, 68 kD, 70 kD and 72 kD). Three of these proteins are very similar to lamins A, B and C of rat liver nuclear envelope according to their molecular mass and isoelectric points. An anti-serum specific to BHK matrix proteins has been raised. On 2-D immunoblot, this serum detects all the 62, 68 and 72 kD polypeptide isovariants but only one of the two isovariants of the 70 kD polypeptide. Rat lamins A, B and C react with the anti-BHK matrix serum. However, when a monoclonal antibody to rat liver lamins A, B and C is used (Burke, B, Tooze, J & Warren, G, EMBO j 2 (1983) 361 [23]), only the 72 kD (lamin A-like) and the 62 kD (lamin C-like) BHK polypeptides are detected. Our results suggest that although a strong similarity exists between BHK and rat lamins, there is no identical cross-reactivity between the two species.     

Organization of the lamin scaffold in the internal nuclear matrix of normal and transformed hepatocytes

Nuclear lamins are among the more abundant proteins making up the internal nuclear matrix, but very little is known about their structure in the nucleoplasm. Using immunoelectron microscopy, we demonstrate the organization of lamins in the nuclear matrix isolated from rat hepatocytes for the first time. Lamin epitopes are arrayed both in locally ordered clusters and in quasi-regular rows. Fourier filtering of the images demonstrates that the epitopes are placed at the nodes and halfway between the nodes of square or rhombic lattices that are about 50 nm on each side, as well as along rows at regular ∼25-nm intervals. In addition, we have compared this structure with that of the internal nuclear matrix isolated from persistent hepatocyte nodules. In transformed hepatocytes, the islands of lamin lattice are lost, and only a partial regularity in the rows of gold particles remains. We suggest that orthogonal lattice assembly might be an intrinsic property of lamin molecules, and that the disassembly may be triggered by simple molecular events such as phosphorylation.     

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.     

Adenoviral heterogeneous nuclear RNA is associated with the host nuclear matrix during splicing

After infection of HeLa cells with adenovirus type 2, virus-specific heterogeneous nuclear RNA is quantitatively associated with a higher ordered structure, the nuclear matrix. Analysis of this matrix-associated RNA by S₁ nuclease mapping showed that precursors as well as processed messenger RNAs from the late region L4 were present. By irradiation of intact cells with ultraviolet light, proteins tightly associated with heterogeneous nuclear RNA can be induced to cross-link with the RNA. Characterization of the cross-linked RNA-protein complexes showed that all viral polyadenylated RNAs (precursors, products and processing intermediates) could be cross-linked to two host proteins, earlier found to be involved in the association of host-specific heterogeneous nuclear RNA to the nuclear matrix (van Eekelen &; van Venrooij, 1981). Our results thus further support the concept that the nuclear matrix may function in the localization and the structural organization of (viral) heterogeneous nuclear RNA during its processing.     

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.     

The nuclear matrix from cells of different origin. Evidence for a common set of matrix proteins

We compared the protein composition of the nuclear matrix isolated from several murine embryonal carcinoma cells and mature tissues by two-dimensional gel electrophoresis. Two nuclear matrix fractions were investigated: the "peripheral" nuclear matrix (matrix proteins that remain insoluble after reduction), and the "internal" nuclear matrix (matrix proteins released by reduction). The two subfractions have completely different protein compositions. Although numerous differences in nuclear matrix protein composition among different cell types were observed, a limited set of polypeptides common to all mouse cell types was identified. A majority of these common proteins was also present in cells from other mammalian species (i.e. rat and human). For this set of proteins, we coin the term "minimal matrix." As expected, lamin B, known to be expressed throughout differentiation, is part of the common set of peripheral nuclear matrix proteins. Lamins A and C are not because these proteins were absent from undifferentiated embryonal carcinoma cells. Since these common nuclear matrix proteins occur in all mammalian nuclear matrices analyzed so far, it is likely that they have a basic role in nuclear organization and function.     

A monoclonal antibody recognizes an epitope common to an avian-specific nuclear antigen and to cytokeratins

X3, a monoclonal antibody of unusual specificity, is described. This antibody reacts with one or more cytokeratin polypeptides and also reacts with an avian (chicken, quail) nuclear antigen that appears to be present in all cell types (chicken) tested, although with variable staining pattern and intensity. This antigen is distinct from the cytokeratins but does have an epitope in common with this class of proteins. It disappears from the nucleus during the early stages of cell division and reappears during anaphase as a granular cytoplasmic structure. In late telophase the antigen is relocated in the nucleus. This antigen, which we have designated as avian-specific nuclear antigen (AVNA), is not associated with chromatin or ribonucleoproteins. From immunoblotting experiments on chicken fibroblast nuclei, AVNA is probably a complex composed of one or several polypeptides, one of which has a molecular weight of approximately 60 kD. The proteins were identified as nuclear matrix proteins rather than pore complex-lamina proteins by immunoblotting experiments on the purified nuclear matrix of chicken erythrocytes. The major polypeptide had a molecular weight of 60 kD and the minor polypeptide a molecular weight of 69 kD.     

Association of actin with the nuclear matrix from bovine lymphocytes

Nuclear matrix prepared from bovine lymphocytes contained a significant amount of actin. Both nuclear matrix actin and rabbit muscle actin showed the same electrophoretic mobility on SDS-gel. The matrix-associated actin could be separated into three isoproteins which may correspond to alpha-, beta- and gamma-actin. The most acidic spot of these isoproteins co-migrated with rabbit muscle actin (alpha-actin) on two-dimensional electrophoresis. The amino-acid composition of the nuclear matrix actin was closely related to that of rabbit muscle and to that of porcine brain actin. Moreover, the actin filaments, treated with 0.75 M guanidine hydrochloride, changed from the polymerized form of the nuclear matrix actin into a monomeric form (G-actin), which had strong inhibitor activity against pancreatic DNase I. From this inhibition, the actin content of the nuclear matrix was estimated to be about 12% of total matrix protein. When the nuclear matrix was digested with trypsin, the bulk of matrix protein was hydrolyzed, but about 80% of the actin remained associated with sphere structures (trypsin-treated nuclear matrix) precipitable by low speed centrifugation. SDS-gel analysis revealed that actin was one of the major components of the trypsin-treated nuclear matrix, which had a similar size and structure as the untreated nuclear matrix. The fibrogranular structure and residual nucleoli of the original nuclear matrix were well preserved against trypsin digestion; however, the peripheral lamina was removed. These results indicate that the matrix-associated actin is localized predominantly in the matrix interior, where it presumably interacts closely with the fibrogranular structure and/or the residual nucleoli.     

DNA binding properties of the nuclear matrix and individual nuclear matrix proteins. Evidence for salt-resistant DNA binding sites

The DNA binding characteristics of the rat nuclear matrix were investigated. A saturable and temperature-dependent, salt-resistant DNA binding to the nuclear matrix was discovered, with 70-80% of total bound DNA resistant to extraction with high concentrations of salt at 37 degrees C, compared to less than 5% at 0 degrees C. The initial binding of DNA to nuclear matrix is sensitive to salt concentration, indicating a transition to a salt-resistant binding state. The nuclear matrix shows a preference for single-stranded DNA, both in saturation and competition assays, with little binding of RNA or double-stranded DNA. Further competition studies show a preference for matrix-attached DNA probably involving predominantly AT-rich sequences, while a specific sequence defined previously as a matrix-attached region (MAR; Cockerill, P. N., and Garrard, W. T. (1986) Cell 46, 273-282) only showed preference for a limited number of the total matrix binding sites. These results and estimates from saturation data of approximately 150,000 single-stranded DNA binding sites per matrix lead us to propose that the nuclear matrix contains different classes of DNA binding sites, each with a separate sequence specificity. Binding of DNA to individual matrix polypeptides separated on sodium dodecyl sulfate-polyacrylamide gels and transferred to nitrocellulose blots was also temperature-dependent, salt-resistant, and showed a preference for binding DNA over RNA and nuclear matrix DNA over total genomic DNA. Subnuclear fractionation experiments further demonstrated that the nuclear matrix is enriched in the subset of higher molecular weight (greater than 50,000) DNA binding proteins of isolated nuclei and correspondingly depleted of the lower molecular weight ones. Of the approximately 12 major proteins separated on nonequilibrium two-dimensional gels, 7 were identified as specific DNA binding proteins including lamins A and C (but not B), and the internal nuclear matrix proteins, matrins D, E, F, G, and 4.     

hnRNA and its attachment to a nuclear protein matrix

In this study, DNA-depleted nuclear protein matrices are isolated from HeLa S3 cells. These nuclear matrices consist of peripheral laminae, residual nucleoli, and internal fibrillar structures. High molecular weight, heterogeneous nuclear RNA (hnRNA) is quantitatively associated with these structures and can be released intact only by affecting the integrity of the matrices. It is, therefore, concluded that hnRNA is part of a highly organized nuclear structure. By irradiation of intact cells or isolated nuclear matrices with ultraviolet light, proteins tightly associated with hnRNA can be induced to cross-link with the RNA. Performing the cross-linking in vivo is an extra guarantee that only hnRNA-protein (hnRNP) complexes existing in the intact cell are covalently linked. Such hnRNP complexes were isolated and purified under conditions that completely dissociate nonspecific RNA-protein complexes. By comparison of the hnRNP found in nuclear matrices and the published data on the composition of hnRNP particles, it was found that the so-called hnRNP "packaging" proteins (32,000-38,000 mol wt) were not efficiently cross-linked to hnRNA by UV irradiation. They were, however, present in the matrix preparations, bound to hnRNA, because they were released from nuclear matrices after ribonuclease treatment of these structures. On the other hand, two major hnRNPs (41,500 and 43,000 mol wt) were efficiently cross-linked to hnRNA. These proteins were not released by ribonuclease treatment, which suggests that they are involved in the binding of hnRNA to the nuclear matrix.