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PubMed2004

NuMA and nuclear lamins are cleaved during viral infection--inhibition of caspase activity prevents cleavage and rescues HeLa cells from measles virus-induced but not from rhinovirus 1B-induced cell death.

Nuclear matrix is a structural framework of important nuclear processes. We studied the effect of two different types of viral infections on nuclear matrix. HeLa cells were infected with human rhinovirus 1B (HRV 1B) or measles virus (MV), and Nuclear Mitotic Apparatus protein (NuMA) and lamins A/C and B were used as markers for internal nuclear matrix and peripheral nuclear lamina, respectively. We show that NuMA, lamins, and poly(ADP-ribose) polymerase-1 are cleaved during viral infection in a virus family-specific manner suggesting that these viruses activate different sets of proteases. Morphologically, NuMA was excluded from the condensed chromatin, lamins showed a folded distribution, and both proteins finally remained around the nuclear fragments. A general caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD-FMK) prevented the nuclear disintegration and the cleavage of the proteins studied. Interestingly, z-VAD-FMK rescued MV-infected but not HRV 1B-infected cells from cell death. These results show for the first time that NuMA and lamins are specific target proteins during virus-induced programmed cell death.

Amino Acid Chloromethyl Ketones↗
PubMed1993

[Nuclear matrix-intermediate filament scaffold of cybrid cells crossed between rabbit reticulocytes and K562 cells].

To elucidate the role of the mammalian erythroid cytoplasmic factor on erythroid cell denucleation, cybridization of neo gene transferred rabbit reticulocytes with human K562 erythroleukemia cells was performed, the structure and composition of the nuclear matrix-intermediate filaments (NM-IF) system of the reticulocytes, K562 cells and the cybrids between them, the K-RRneo Cells, were studied by employing the techniques of selective extraction and whole mount electron microscopy. It was shown that regional condensation of nuclear matrix with a thinned nuclear lamina were seen in the cybrids, while the intermediate filaments exhibited a network pattern reorganization comparatively similar with those of reticulocytes but different from the radiation pattern of the K562 cells. SDS-PAGE and western blot analysis also revealed that the proteins affinity to vimentin antibody in the cybrids migrate in a pattern similar with that of the rabbit reticulocytes, both of them with only residual depolymerized vimentin complex but not the polymerized vimentin band of 55 kd. These results suggested that the factors in the cytoplasm of the rabbit reticulocytes might function through a mechanism of vimentin depolymerization or blockage of gene expression, which finally resulted in destruction of intermediate filaments that facilitates the expellation of nucleus from cytoplasm.

Animals↗
PubMed1986

Selective repair of methylated purines in regions of chromatin DNA.

The distribution of methylated purines in different regions of liver chromatin DNA has been examined after treating rats with [14C]dimethylnitrosamine (2 mg/kg). At different times after administration of the carcinogen, liver nuclei were isolated and fractionated by micrococcal nuclease digestion and low and high salt extractions into an active chromatin fraction, two fractions comprising the bulk of the genome, and a nuclear matrix fraction. Regions of active chromatin and nuclear matrix tended to be methylated more readily than bulk chromatin, with respect to formation of both O6-methylguanine and N-methyl purines. Removal of both 7-methylguanine and 3-methyladenine (by repair and depurination reactions) occurred at a relatively uniform rate in all chromatin fractions. In contrast, repair of O6-methylguanine proceeded more rapidly from active chromatin than from bulk chromatin, whereas repair of this lesion from nuclear matrix DNA was much slower than for bulk DNA. Pretreatment of rats for 4 weeks with non-radioactive dimethylnitrosamine before the administration of [14C]dimethylnitrosamine enhanced the rate of repair of radioactive O6-methylguanine from all chromatin fractions. Nevertheless the rate of loss of the adduct was still faster from active chromatin and slower from matrix DNA than from the bulk of the genome. Since pretreatment also elevated the rate of liver DNA synthesis especially in the nuclear matrix fraction, there is an increased probability of the fixation of mutations due to the presence of O6-methylguanine in this selected region of the genome. The implications of this persistent O-alkylation of matrix DNA, and rapid repair of O6-alkylguanine in active chromatin for the toxicity and carcinogenicity of alkylating agents are discussed.

Animals↗
PubMed2001

Cloning and expression of a novel nuclear matrix-associated protein that is regulated during the retinoic acid-induced neuronal differentiation.

Retinoic acid (RA), a derivative of vitamin A, is essential for the normal patterning and neurogenesis during development. RA treatment induces growth arrest and terminal differentiation of a human embryonal carcinoma cell line (NT2) into postmitotic central nervous system neurons. Using RNA fingerprinting by arbitrarily primed polymerase chain reaction, we identified a novel serine/threonine-rich protein, RA-regulated nuclear matrix-associated protein (Ramp), that was down-regulated during the RA-induced differentiation of NT2 cells. Prominent mRNA expression of ramp could be detected in adult placenta and testis as well as in all human fetal tissues examined. The genomic clone of ramp has been mapped to the telomere of chromosome arm 1q, corresponding to band 1q32.1-32.2. Associated with the nuclear matrix of NT2 cells, Ramp translocates from the interphase nucleus to the metaphase cytoplasm during mitosis. During the late stage of cytokinesis, Ramp concentrates at the midzone of the dividing daughter cells. The transcript expression of ramp is closely correlated with the cell proliferation rate of NT2 cells. Moreover, overexpression of Ramp induces a transient increase in the proliferation rate of NT2 cells. Taken together, our data suggest that Ramp plays a role in the proliferation of the human embryonal carcinoma cells.

Adult↗
PubMed1996

DNA supercoiling changes and nuclear matrix-associated proteins: possible role in oncogene-mediated radioresistance.

PURPOSE: Transfection with either H-ras or H-ras and c-myc has been shown to confer radioresistance in rat embryonal cells (REC). REC primary, transfected with either c-myc, H-ras or cotransfected with c-myc and H-ras (in ascending order of radioresistance and tumorigenicity), were used as an in vitro model system to determine if nuclear matrix-mediated higher order DNA organization contributes to oncogene-mediated radioresistance. METHODS AND MATERIALS: DNA damage induction and repair were measured by the alkaline and neutral filter elution assays. Analysis of the ability of DNA loop domains to undergo supercoiling changes in the presence of radiation-induced damage was determined by the fluorescent halo assay (FHA). Because DNA loops are organized by the nuclear matrix (NM), a study of NM-associated proteins by high resolution two-dimensional gel electrophoresis was performed. RESULTS: Induction and repair rates of DNA single- and double-strand breaks were similar for the relatively radiosensitive c-myc transfected and the radioresistant c-myc + H-ras transfected cells. However, the degree of inhibition of DNA supercoil rewinding in the presence of radiation-induced damage was less in the radioresistant cells and was inversely correlated with survival. A progressive loss of NM-associated proteins was observed, which correlated with increasing radioresistance and tumorigenicity in these cell lines. In addition, some protein changes were consistent with the possibility that these changes could be involved in DNA anchoring. CONCLUSIONS: Increased radioresistance associated with increasing tumorigencity in these oncogene-transfected cell lines could be due to changes in NM-mediated DNA organization, possibly via differences in NM protein composition that occur following oncogenic transfection.

Animals↗
PubMed2000

Topoisomerase II cleavable complex formation within DNA loop domains.

The distribution of VM-26 (Teniposide)-stabilized cleavable complexes within DNA loops bound to the nuclear matrix was determined to provide further insights into the mode of DNA synthesis inhibition by VM-26. Covalent binding of [(3)H]VM-26 was 9-fold greater per milligram of nuclear matrix protein compared with high salt-soluble nonmatrix protein of CEM cells. The ratio declined from 9-fold in CEM cells to 4-fold in drug-resistant VM-1/C2 cells, which have decreased nuclear matrix DNA topoisomerase IIalpha. VM-26 induced a concentration-dependent increase in the frequency of cleavable complex formation with actively replicating matrix DNA. At 25 microM VM-26, the frequency was 32 +/- 2 (SEM) complexes per 10(6) bp of replicating matrix DNA compared with 13 +/- 2 (SEM) complexes per 10(6) bp of nonreplicating DNA in the matrix fraction. VM-26 at concentrations as high as 25 microM stabilized less than 3 complexes per 10(6) bp in the various nonmatrix DNA domains, since the nonmatrix DNA comprises the DNA loop domains that are distal to the matrix-bound replication sites. A negligible frequency of cleavable complex formation was detected in both the matrix and nonmatrix DNA domains of drug-resistant VM-1/C2 cells. Compared with untreated control cells, VM-26 induced an accumulation of nascent DNA in the nuclear matrix fraction of CEM cells but decreased the amount of nascent DNA in the nonmatrix fraction. The extensive cleavable complex formation on matrix replicating DNA stalled most of the replication forks within 1 kb of the replication sites on the nuclear matrix. The results provide evidence that nascent DNA bound to the nuclear matrix is an important site of VM-26 cleavable complex formation, and that these complexes inhibit DNA synthesis by blocking the movement of nascent DNA away from replication sites on the nuclear matrix.

Antineoplastic Agents↗
PubMed1996

Interrelationships between nuclear structure and ligand-activated intracellular receptors.

The role of ligand-activated intracellular receptors in activation of gene expression most probably involves a multistep process that requires alteration in chromatin structure. Results of studies with members of the steroid hormone receptor superfamily as well as the ligand-activated member of the basic region/helix-loop-helix family of transcriptional activators suggests that two different nuclear structures and their associated nuclear proteins are important in the initial stages of gene activation: the nuclear matrix and nucleosomes. Cell- and tissue-specific nuclear matrix proteins and the variant and modified histones appear to be important for tissue and species specificity of ligand-induced responses. Because the function of a receptor may be limited in vivo by promoter and transcription factor accessibility, the various roles of nuclear ligand-receptor complexes may involve interaction with nuclear matrix proteins and/or nucleosomes. Tissue-specific structural nuclear proteins could control the conformation (looping through matrix attachment regions) of the DNA and unwinding or rearrangement of nucleosomes, thus providing specificity to the expression of certain genes. Modulation of cooperative elements required for gene activation may involve association of the gene promoter with the nuclear matrix together with the presence of nucleosomes. Thus, the series of events involved in ligand-receptor activation of genes requires alterations in chromatin structure, which allow access of the receptor complex to elements within the gene.

Animals↗
PubMed2002

Identification and mapping of nuclear matrix-attachment regions in a one megabase locus of human chromosome 19q13.12: long-range correlation of S/MARs and gene positions.

The first draft human genome sequence now available allowed the identification of an enormous number of gene coding areas of the genomic DNA. However, a great number of regulatory elements such as enhancers, promoters, transcription terminators, or replication origins can not be identified unequivocally by their nucleotide sequences in complex eukaryotic genomes. One important subclass of these type of sequences is scaffold/matrix attachment regions (S/MARs) that were hypothesized to anchor chromatin loops or domains to the nuclear matrix and/or chromosome scaffold. We developed an experimental selection procedure to identify S/MARs within a completely sequenced one megabase (1 Mb) long gene-rich D19S208-COX7A1 locus of human chromosome 19. A library of S/MAR elements from the locus was prepared and shown to contain -20 independent S/MARs. Sixteen of them were isolated, sequenced, and assigned to certain positions within the locus. A majority of the S/MARs identified (11 out of 16) lie in intergenic regions, suggesting their structural role, i.e., delimitation of chromatin domains. These 11 S/MARs subdivide the locus into 10 domains ranging from 6 to 272 kb with an average domain size of 88 kb. The remaining five S/MARs were found within intronic sequences of APLP1, HSPOX1, MAG, and NPHS1 genes, and can be tentatively characterized as regulatory S/MARs. The correspondence of the chromatin domains defined by the S/MARs to functional characteristics of the genes therein is discussed. The approach described can be a prototype of a similar search of long sequenced genomic stretches and/or whole chromosomes for various regulatory elements.

Chromosome Mapping↗
PubMed1986

The nonchromatin substructures of the nucleus: the ribonucleoprotein (RNP)-containing and RNP-depleted matrices analyzed by sequential fractionation and resinless section electron microscopy.

The nonchromatin structure or matrix of the nucleus has been studied using an improved fractionation in concert with resinless section electron microscopy. The resinless sections show the nucleus of the intact cell to be filled with a dense network or lattice composed of soluble proteins and chromatin in addition to the structural nuclear constituents. In the first fractionation step, soluble proteins are removed by extraction with Triton X-100, and the dense nuclear lattice largely disappears. Chromatin and nonchromatin nuclear fibers are now sharply imaged. Nuclear constituents are further separated into three well-defined, distinct protein fractions. Chromatin proteins are those that require intact DNA for their association with the nucleus and are released by 0.25 M ammonium sulfate after internucleosomal DNA is cut with DNAase I. The resulting structure retains most heterogeneous nuclear ribonucleoprotein (hnRNP) and is designated the RNP-containing nuclear matrix. The proteins of hnRNP are those associated with the nucleus only if RNA is intact. These are released when nuclear RNA is briefly digested with RNAase A. Ribonuclease digestion releases 97% of the hnRNA and its associated proteins. These proteins correspond to the hnRNP described by Pederson (Pederson, T., 1974, J. Mol. Biol., 83:163-184) and are distinct from the proteins that remain in the ribonucleoprotein (RNP)-depleted nuclear matrix. The RNP-depleted nuclear matrix is a core structure that retains lamins A and C, the intermediate filaments, and a unique set of nuclear matrix proteins (Fey, E. G., K. M. Wan, and S. Penman, 1984, J. Cell Biol. 98:1973-1984). This core had been previously designated the nuclear matrix-intermediate filament scaffold and its proteins are a third, distinct, and nonoverlapping subset of the nuclear nonhistone proteins. Visualizing the nuclear matrix using resinless sections shows that nuclear RNA plays an important role in matrix organization. Conventional Epon-embedded electron microscopy sections show comparatively little of the RNP-containing and RNP-depleted nuclear matrix structure. In contrast, resinless sections show matrix interior to be a three-dimensional network of thick filaments bounded by the nuclear lamina. The filaments are covered with 20-30-nm electron dense particles which may contain the hnRNA. The large electron dense bodies, enmeshed in the interior matrix fibers, have the characteristic morphology of nucleoli. Treatment of the nuclear matrix with RNAase results in the aggregation of the interior fibers and the extensive loss of the 20-30-nm particles.(ABSTRACT TRUNCATED AT 400 WORDS)

Cell Fractionation↗
PubMed1989

Ki-67 detects a nuclear matrix-associated proliferation-related antigen. II. Localization in mitotic cells and association with chromosomes.

In interphase cells the proliferation-associated antigen recognized by monoclonal antibody Ki-67 is almost exclusively located in the nucleoli. When cells at several stages of mitosis were examined for the localization of the Ki-67 antigen, a striking redistribution could be observed. During prophase the distinct nucleolar Ki-67 fluorescence changed to a bright irregular meshwork throughout the nucleoplasm. At metaphase the antigen appeared to be distributed in a reticulate structure surrounding the condensed chromosomes, while at late telophase a punctated staining of the entire nucleoplasm was observed, which preceded the typical nucleolar localization pattern in each of the two daughter cells. Immunolabelling with Ki-67 of metaphase chromosome spreads revealed a circumferential staining of the individual chromosomes. The Ki-67 antigen is preserved in nuclear matrix preparations obtained after in situ fractionation of interphase cells. When mitotic cells were exposed to such treatments, the obtained fluorescence data suggested that the antigen may be part of the chromosome scaffold. Quantification of the Ki-67 fluorescence signal using flow cytometry revealed the highest staining intensities in mitotic cells. Furthermore, it was shown that nutritionally deprived cells became negative for Ki-67.

Antibodies, Monoclonal↗
PubMed1989

[Study of nuclear poly(ADP-ribose)polymerase and DNA-topoisomerase II of brain cells during postnatal development of rats].

The nuclear poly(ADP-ribose)polymerase activity of neuronal and glial cells during postnatal development of rats was studied. It was shown that the poly(ADP-ribose)polymerase activity of nuclei and nuclear matrix of neuronal cells during postnatal development of rats is increased, whereas the polymerase activity of glial cell nuclei and nuclear matrix in newborn and adult rats is higher than in 14-day-old animals. The DNA-topoisomerase II activity of neuronal nuclear matrix during the postnatal development of rats does not change, whereas the topoisomerase activity of glial nuclear matrix decreases but is always higher than the DNA-topoisomerase II activity of neuronal cell matrix during the postnatal development of rats. It is suggested that ADP-ribosylation in the nuclear matrix of neuronal cells causes the inhibition of the DNA-topoisomerase II activity of nuclear matrix.

Animals↗
PubMed1996

Abnormal accumulation of copper in LEC rat liver induces expression of p53 and nuclear matrix-bound p21(waf 1/cip 1).

The LEC rat is an inbred mutant strain which spontaneously develops liver injury and subsequent liver cancer. Liver injury in LEC rats has recently been shown to be closely related to abnormal copper accumulation in the liver. Previously, we reported that LEC rat hepatocytes lose their growth potential, probably allowing selective growth of preneoplastic cells. In this study, to elucidate the effects of copper accumulation on the growth activity of LEC rat hepatocytes, we examined the growth activity and the expression of p53 and p21(waf 1/cip 1) in the livers of LEC rats fed on either a control or a low-copper diet. Potential for cell proliferation of hepatocytes obtained from normal diet fed LEC rats was almost comparable to that of the cells from age-matched Sprague-Dawley (SD) rats. Northern blot analysis showed that the expression of p53 and p21(waf 1/cip 1) was significantly high in the livers of LEC rats fed a control diet, while the expression of p53 and p21(waf 1/cip 1) in the LEC rats fed a low-copper diet was as low as that of SD rat livers. Western blot analysis consistently showed that the amount of p21(waf 1/cip 1) bound to the nuclear matrix scaffold of the LEC rat liver was reduced by feeding a low-copper diet. These findings suggest that abnormal accumulation of copper induced the expression of p53 and p21(waf 1/cip 1), resulting in the inhibition of cell proliferation of LEC rat hepatocytes.

Animals↗
PubMed1984

Epithelial cytoskeletal framework and nuclear matrix-intermediate filament scaffold: three-dimensional organization and protein composition.

Madin-Darby canine kidney (MDCK) cells grow as differentiated, epithelial colonies that display tissue-like organization. We examined the structural elements underlying the colony morphology in situ using three consecutive extractions that produce well-defined fractions for both microscopy and biochemical analysis. First, soluble proteins and phospholipid were removed with Triton X-100 in a physiological buffer. The resulting skeletal framework retained nuclei, dense cytoplasmic filament networks, intercellular junctional complexes, and apical microvillar structures. Scanning electron microscopy showed that the apical cell morphology is largely unaltered by detergent extraction. Residual desmosomes, as can be seen in thin sections, were also well-preserved. The skeletal framework was visualized in three dimensions as an unembedded whole mount that revealed the filament networks that were masked in Epon-embedded thin sections of the same preparation. The topography of cytoskeletal filaments was relatively constant throughout the epithelial sheet, particularly across intercellular borders. This ordering of epithelial skeletal filaments across contiguous cell boundaries was in sharp contrast to the more independent organization of networks in autonomous cells such as fibroblasts. Further extraction removed the proteins of the salt-labile cytoskeleton and the chromatin as separate fractions, and left the nuclear matrix-intermediate filament (NM-IF) scaffold. The NM-IF contained only 5% of total cellular protein, but whole mount transmission electron microscopy and immunofluorescence showed that this scaffold was organized as in the intact epithelium. Immunoblots demonstrate that vimentin, cytokeratins, desmosomal proteins, and a 52,000-mol-wt nuclear matrix protein were found almost exclusively in the NM-IF scaffold. Vimentin was largely perinuclear while the cytokeratins were localized at the cell borders. The 52,000-mol-wt nuclear matrix protein was confined to the chromatin-depleted matrix and the desmosomal proteins were observed in punctate polygonal arrays at intercellular junctions. The filaments of the NM-IF were seen to be interconnected, via the desmosomes, over the entire epithelial colony. The differentiated epithelial morphology was reflected in both the cytoskeletal framework and the NM-IF scaffold.

Animals↗
PubMed1991

Nuclear structure and the three-dimensional organization of DNA.

The organization of DNA within the nucleus has been demonstrated to be both cell and tissue specific and is arranged in a non-random fashion in both sperm and somatic cells. Nuclear structure has a pivotal role in this three-dimensional organization of DNA and RNA and contributes as well to forming fixed organizing sites for nuclear functions, such as DNA replication, transcription, and RNA processing. In sperm, DNA is also organized in a specific fashion by the nuclear matrix and DNA-protamine interactions. Within somatic cells, the nuclear matrix provides a three-dimensional framework for the tissue specific regulation of genes by directed interaction with transcriptional activators. This differential organization of the DNA by the nuclear matrix, in a tissue specific manner, contributes to tissue specific gene expression. The nuclear matrix is the first link from the DNA to the entire tissue matrix system and provides a direct structural linkage to the cytomatrix and extracellular matrix. In summary, the tissue matrix serves as a dynamic structural framework for the cell which interacts to organize and process spatial and temporal information to coordinate cellular functions and gene expression. The tissue matrix provides a structural system for integrating form and function.

Animals↗
PubMed1998

A nuclear matrix/scaffold attachment region co-localizes with the gypsy retrotransposon insulator sequence.

The 5'-untranslated region of the Drosophila gypsy retrotransposon contains an "insulator," which disrupts the interactions between enhancer and promoter elements located apart. The insulator effect is dependent on the suppressor of Hairy-wing (su(Hw)) protein, which binds to reiterated sites within the 350 base pairs of the gypsy insulator, whereby it additionally acts as a transcriptional activator of gypsy. Here, we show that the 350-base pair su(Hw) binding site-containing gypsy insulator behaves in addition as a matrix/scaffold attachment region (MAR/SAR), involved in interactions with the nuclear matrix. In vitro experiments using nuclear matrices from Drosophila, murine, and human cells demonstrate specific binding of the gypsy insulator, not observed with any other sequence within the retrotransposon. Moreover, we show that the gypsy insulator, like previously characterized MAR/SARs, specifically interacts with topoisomerase II and histone H1, i.e. with two essential components of the nuclear matrix. Finally, experiments within cells in culture demonstrate differential effects of the gypsy MAR sequence on reporter genes, namely no effect under conditions of transient transfection and a repressing effect in stable transformants, as expected for a sequence involved in chromatin structure and organization. A model for the gypsy insulator, which combines within a short "compacted" retroviral sequence three functional domains (insulator, enhancer, and the presently unraveled MAR/SAR) dispersed within more extended regions in other "boundary" domains, is discussed in relation to previously proposed models for insulation.

Animals↗
PubMed2003

[Influence of transfected EGFR-cDNA on bcl-2 and Bax in glioblastoma cells].

OBJECTIVE: To investigate the correlation of bcl-2 and Bax protein with nuclear matrix in glioblastoma cell line U87 as well as the effect of EGFR-cDNA transfection on the expression of bcl-2 and Bax in U87 cells. METHODS: The correlation of bcl-2 and Bax protein with nuclear matrix in glioblastoma cell line U87 was studied by confocal microscopy and Western blot. The expression of bcl-2 and Bax in EGFR-cDNA transfected and untransfected glioblastoma cell lines was studied by Western blot. RESULTS: Confocal microscopic images showed that bcl-2 protein was localized in the periphery of the nuclear matrix and Bax in the nuclear matrix. A 26 kDa bcl-2 band and a specific band of Bax at about 66 000 were detected in nuclear matrix proteins by western blot. The expression of bcl-2 was lower but that of Bax was higher in EGFR-cDNA transfected cells than the control. CONCLUSION: Bcl-2 and Bax, being nuclear matrix associated proteins, are probably involved in the EGFR-cDNA induced malignant conversion of glioblastoma cells by introducing EGFR cDNA into the tumor cells.

Apoptosis↗
PubMed1989

Ki-67 detects a nuclear matrix-associated proliferation-related antigen. I. Intracellular localization during interphase.

Ki-67 is a commercially available mouse monoclonal antibody, which reacts with a nuclear antigen in proliferating cells. The antibody can be used to determine the growth fraction of human tumours in situ and has been shown to be of prognostic importance. In this study it is shown that in interphase cells Ki-67 reacts with an antigen, mainly present in the nucleoli. Confocal scanning laser microscopy and immunoelectron microscopy on human MR65 monolayer cells revealed that this nucleolar antigen is predominantly localized in the nucleolar cortex and in the dense fibrillar components. The Ki-67 antigen appeared to be preserved in nuclear matrix preparations obtained after in situ fractionation of MR65 cells. Despite many efforts, we could not identify the antigen in immunoblotting or immunoprecipitation assays. Testing of cell cultures of different species by means of indirect immunofluorescence revealed that the antibody reacted with human cells and with the Rhesus monkey kidney-derived cell line LLC-MK2.

Antibodies, Monoclonal↗
PubMed2000

A nuclear matrix-associated factor, SAF-B, interacts with specific isoforms of AUF1/hnRNP D.

One class of heterogeneous nuclear ribonucleoproteins (hnRNPs), AUF1/hnRNP D, consists of four isoform proteins (p45, p42, p40, and p37) which are generated by alternative splicing. The present study was therefore undertaken to clarify any isoform-specific differences in terms of their functions and nucleocytoplasmic localization. All isoforms primarily localized in the nucleus. However, heterokaryon analysis and a study using RNA polymerase II inhibitor revealed that p40/p37 exhibited a continuous shuttling between the nucleus and cytoplasm. Constant nuclear retention activity was mapped to the p45/p42-specific sequence at the C-terminal region, which is retained by alternative splicing. Using this domain as a probe, we performed a yeast two-hybrid screening and we found that scaffold attachment factor B (SAF-B), a nuclear matrix-associated protein, exhibits protein-protein interaction to this region. Colocalization of p45/p42 and SAF-B was observed as a speckle in the nucleus. Interestingly, p45/p42 isoforms appeared to act as a negative regulator in gene expression by forming a complex with SAF-B. Thus, the present study revealed that the isoform-specific functions of AUF1/hnRNP D are defined by intracellular shuttling capacity.

3T3 Cells↗