Colocalization of a high molecular mass phosphoprotein of the nuclear matrix (p255) with spliceosomes

1995 ◽  
Vol 108 (5) ◽  
pp. 1873-1882 ◽  
Author(s):  
S. Bisotto ◽  
P. Lauriault ◽  
M. Duval ◽  
M. Vincent
Keyword(s):  
Monoclonal Antibody ◽  
Nuclear Matrix ◽  
Nuclear Protein ◽  
High Ionic Strength ◽  
High Molecular Mass ◽  
Splicing Factor ◽  
Nuclear Speckles ◽  
Ionic Detergents ◽  
Interphase Cells ◽  
Nuclear Structures

It was previously demonstrated that monoclonal antibody CC-3 binds to a phosphorylation dependent epitope present on a 255 kDa nuclear protein (p255). We show here that in interphase cells, p255 distributes to typical nuclear speckles that correspond to the localization of spliceosome components as revealed by antibodies to the m3G cap of snRNAs or to the non-snRNP splicing factor SC-35. Immunofluorescence and immunoblot studies indicated that p255 is resistant to extraction with non-ionic detergents, nucleases and high ionic strength buffers and may thus be defined biochemically as a nuclear matrix phosphoprotein. To determine the nature of the association of p255 with the nuclear structure, its distribution was studied at different stages of the cell cycle and after the cells were treated with nucleases or heat shocked. We found that the antigen diffused into the cytoplasm during metaphase but was reorganized into cytoplasmic speckles during anaphase-telophase transition, where it colocalized with SC-35. Nuclear matrix preparations that were digested with DNases and RNases showed that interphasic p255 still localized to nuclear speckles even though snRNA and snRNP antigens were removed. Heat-shocked cells labelled with monoclonal antibody CC-3 exhibited more rounded and less interconnected speckles, identical to those decorated by anti-SC-35 antibody under such conditions. These results indicate that p255 and SC-35 are present in the same nuclear structures, to which they are more tightly bound than the snRNP antigens. They further suggest that both proteins are implicated in spliceosome assembly or attachment.

The analysis of 40 kDa nuclear protein, p40, in interphase cells and mitotic cells

1993 ◽  
Vol 106 (3) ◽  
pp. 741-748 ◽  
Author(s):  
Y. Kaneda ◽  
K. Kinoshita ◽  
M. Sato ◽  
K. Tanaka ◽  
Y. Kaneda
Keyword(s):  
Monoclonal Antibody ◽  
Nuclear Envelope ◽  
Nuclear Protein ◽  
Dnase I ◽  
High Salt ◽  
The Other ◽  
Interphase Nuclei ◽  
Interphase Cells ◽  
Detergent Treatment ◽  
Sequential Treatments

We previously reported that the monoclonal antibody M108 recognized a 40 kDa protein both in the nucleus and the cytoplasm. This nuclear 40 kDa antigen was located in the nuclear envelope in interphase cells and in the perichromosomal region during mitosis. Now, we have analyzed this nuclear 40 kDa protein (p40) further, through morphological and biochemical approaches. At the beginning of mitosis, the perinuclear p40 detached from the nuclear envelope and moved to surround the condensing chromatin, while in the late stage of mitosis, the perichromosomal p40 moved back to the reassembled nuclear envelope. Most of the perichromosomal p40 on the metaphase chromosome was solubilized only by DNase I treatment, not by either high salt or detergent treatment. On the other hand, the perinuclear p40 was not solubilized by DNase1 alone, or high salt detergent alone. Sequential treatments with DNase I and high salt detergent were required to extract p40 in interphase nuclei. These results suggest that p40 was associated both with the nuclear envelope and chromatin DNA in interphase nuclei, while it bound only to chromatin DNA in mitosis.

A monoclonal antibody recognizing nuclear matrix-associated nuclear bodies

1992 ◽  
Vol 101 (4) ◽  
pp. 773-784 ◽  
Author(s):  
N. Stuurman ◽  
A. de Graaf ◽  
A. Floore ◽  
A. Josso ◽  
B. Humbel ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Nuclear Matrix ◽  
Nuclear Bodies ◽  
Biliary Cirrhosis ◽  
Nuclear Speckles ◽  
Metaphase Chromosomes ◽  
Double Labeling ◽  
T24 Cells ◽  
Bladder Carcinoma Cell Line ◽  
Spherical Structures

We have isolated a monoclonal antibody, 5E10, that labels discrete spots in the interphase nucleus. By immunoblotting mAb 5E10 recognized predominantly a 126 kDa polypeptide with an isoelectric point of 5.5. Indirect immunofluorescence showed that mAb 5E10 labeled spots in many cell lines and tissues from rat or human origin, but not in cells from mouse, chicken, African green monkey, or the lower eukaryotes Saccharomyces and Dictyostelium. In the human bladder carcinoma cell line T24 the number of nuclear spots were found to be 21 +/− 10 (n = 132). In many cells spots were found also in the cytoplasm. In a small fraction of T24 cells the mAb revealed thread-like structures in addition to spots. Throughout mitosis the antigen was found to be clustered in the cytoplasm, not associated with metaphase chromosomes. The spherical structures that contain the antigen were tightly bound to the nuclear matrix. Immunogold labeling with mAb 5E10 showed that the antigen is localized in 0.3 microns diameter spherical, electron-dense structures, reminiscent of nuclear bodies. Double-labeling experiments showed that these spots do not colocalize with U1 snRNPs and centromeres. The spots did colocalize with nuclear speckles recognized by a primary biliary cirrhosis autoimmune serum, which is thought to recognize nuclear bodies. On the basis of these observations we conclude that mAb 5E10 recognizes discrete nuclear substructures, most likely nuclear bodies.

Nuclear matrix bound fibroblast growth factor receptor is associated with splicing factor rich and transcriptionally active nuclear speckles

2003 ◽  
Vol 90 (4) ◽  
pp. 856-869 ◽  
Author(s):  
Suryanarayan Somanathan ◽  
Ewa K. Stachowiak ◽  
Alan J. Siegel ◽  
Michal K. Stachowiak ◽  
Ronald Berezney
Keyword(s):  
Growth Factor ◽  
Fibroblast Growth Factor ◽  
Nuclear Matrix ◽  
Fibroblast Growth Factor Receptor ◽  
Growth Factor Receptor ◽  
Splicing Factor ◽  
Fibroblast Growth ◽  
Nuclear Speckles ◽  
Matrix Bound

Functional association of nuclear protein 4.1 with pre-mRNA splicing factors

1998 ◽  
Vol 111 (14) ◽  
pp. 1963-1971 ◽  
Author(s):  
M.J. Lallena ◽  
C. Martinez ◽  
J. Valcarcel ◽  
I. Correas
Keyword(s):  
Nuclear Protein ◽  
Transmembrane Proteins ◽  
Mrna Splicing ◽  
Splicing Factor ◽  
Splicing Factors ◽  
Protein 4.1 ◽  
Nuclear Extracts ◽  
Nuclear Structures ◽  
The Relationship

Protein 4.1 is a multifunctional polypeptide that links transmembrane proteins with the underlying spectrin/actin cytoskeleton. Recent studies have shown that protein 4.1 is also present in the nucleus, localized in domains enriched in splicing factors. Here we further analyze the relationship between protein 4. 1 and components of the splicing machinery. Using HeLa nuclear extracts capable of supporting the splicing of pre-mRNAs in vitro, we show that anti-4.1 antibodies specifically immunoprecipitate pre-mRNA and splicing intermediates. Immunodepletion of protein 4.1 from HeLa nuclear extracts results in inhibition of their splicing activity, as assayed with two different pre-mRNA substrates. Coprecipitation of protein 4.1 from HeLa nuclear extracts with proteins involved in the processing of pre-mRNA further suggests an association between nuclear protein 4.1 and components of the splicing apparatus. The molecular cloning of a 4.1 cDNA encoding the isoform designated 4.1E has allowed us to show that this protein is targeted to the nucleus, that it associates with the splicing factor U2AF35, and that its overexpression induces the redistribution of the splicing factor SC35. Based on our combined biochemical and localization results, we propose that 4.1 proteins are part of nuclear structures to which splicing factors functionally associate, most likely for storage purposes.

A monoclonal antibody recognizes a human nuclear protein resembling Xenopus oocyte nucleoplasmin

1987 ◽  
Vol 87 (5) ◽  
pp. 713-722
Author(s):  
J. Lord ◽  
G. Brown ◽  
C. Bunce ◽  
P. Nathan
Keyword(s):  
Monoclonal Antibody ◽  
Xenopus Oocytes ◽  
Nuclear Protein ◽  
Chinese Hamster ◽  
Nucleosome Assembly ◽  
3T3 Cells ◽  
Nucleosome Assembly Protein ◽  
Subunit Molecular Weight ◽  
Interphase Cells ◽  
A Subunit

The monoclonal antibody 10BG2 (IgG3) was derived from a mouse immunized with human pre B cells. In immunofluorescence studies the antibody revealed a human nuclear-associated determinant, which in interphase cells was entirely restricted to the nucleus. In metaphase cells 10BG2 antigen was detected throughout the cytoplasm with intensified staining at the periphery of chromosomes. 10BG2 antibody stained all human normal and transformed cells tested. In contrast, the antibody did not stain mouse 3T3 cells or Chinese hamster ovary (CHO) cells. Electrophoresis under denaturing and non-denaturing conditions revealed the 10BG2 antigen to be a 130 X 10(3) to 140 X 10(3) Mr protein with a subunit molecular weight of 29.5 X 10(3). This suggests the protein is at least a tetramer. On two-dimensional gels the 10BG2 antigen had a streaked appearance and separated into two isoelectric variants (pI5.2, 5.4). The protein was also shown to be phosphorylated and thermostable, and remained in solution at pH 3.6. 10BG2 antigen was highly soluble in aqueous buffers and co-migrated on non-denaturing gels with the nucleosome-assembly protein, nucleoplasmin, purified from Xenopus oocytes. The similarity of 10BG2 antigen to nucleoplasmin is discussed.

scholarly journals Splicing factor USP39 promotes ovarian cancer malignancy through maintaining efficient splicing of oncogenic HMGA2

2021 ◽  
Vol 12 (4) ◽  
Author(s):  
Shourong Wang ◽  
Zixiang Wang ◽  
Jieyin Li ◽  
Junchao Qin ◽  
Jianping Song ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Cancer Cells ◽  
Malignant Tumors ◽  
Splicing Factor ◽  
Ovarian Cancer Cells ◽  
Nuclear Speckles ◽  
Aberrant Expression ◽  
Intergenic Regions

AbstractAberrant expression of splicing factors was found to promote tumorigenesis and the development of human malignant tumors. Nevertheless, the underlying mechanisms and functional relevance remain elusive. We here show that USP39, a component of the spliceosome, is frequently overexpressed in high-grade serous ovarian carcinoma (HGSOC) and that an elevated level of USP39 is associated with a poor prognosis. USP39 promotes proliferation/invasion in vitro and tumor growth in vivo. Importantly, USP39 was transcriptionally activated by the oncogene protein c-MYC in ovarian cancer cells. We further demonstrated that USP39 colocalizes with spliceosome components in nuclear speckles. Transcriptomic analysis revealed that USP39 deletion led to globally impaired splicing that is characterized by skipped exons and overrepresentation of introns and intergenic regions. Furthermore, RNA immunoprecipitation sequencing showed that USP39 preferentially binds to exon-intron regions near 5′ and 3′ splicing sites. In particular, USP39 facilitates efficient splicing of HMGA2 and thereby increases the malignancy of ovarian cancer cells. Taken together, our results indicate that USP39 functions as an oncogenic splicing factor in ovarian cancer and represents a potential target for ovarian cancer therapy.

Characterization of nuclear structures containing superhelical DNA

1976 ◽  
Vol 22 (2) ◽  
pp. 303-324 ◽  
Author(s):  
P.R. Cook ◽  
I.A. Brazell ◽  
E. Jost
Keyword(s):  
Protein Content ◽  
Ionic Detergents ◽  
High Concentrations ◽  
Nuclear Structures ◽  
Intercalating Agents ◽  
Made In

Structures resembling nuclei but depleted of protein may be released by gently lysing cells in solutions containing non-ionic detergents and high concentrations of salt. These nucleoids sediment in gradients containing intercalating agents in a manner characteristic of DNA that is intact, supercoiled and circular. The concentration of salt present during isolation of human nucleoids affects their protein content. When made in I-95 M NaCl they lack histones and most of the proteins characteristic of chromatin; in 1-0 M NaCl they contain variable amounts of histones. The effects of various treatments on nucleoid integrity were investigated.

scholarly journals Isolation of monoclonal antibodies specific for products of avian oncogene myb.

1984 ◽  
Vol 4 (12) ◽  
pp. 2843-2850 ◽  
Author(s):  
G I Evan ◽  
G K Lewis ◽  
J M Bishop
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Nuclear Protein ◽  
Gene Products ◽  
Viral Oncogenes ◽  
Myb Gene

We isolated a series of monoclonal antibodies which were raised against a bacterially expressed protein, bp37v-myb, and coded for by part of the avian v-myb gene. These monoclonal antibodies recognized a range of antigenic specificities on bp37v-myb, and this was reflected in their differing specificities for the gene products of the v-myb, c-myb, and E26 viral oncogenes. One monoclonal antibody recognized, in addition to the v-myb and c-myb gene products, a conserved nuclear protein found in all tested cells. We describe the characterization of these monoclonal antibodies.

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