B23 Protein Research Articles

The RNA binding activity of a ribosome biogenesis factor, nucleophosmin/B23, is modulated by phosphorylation with a cell cycle-dependent kinase and by association with its subtype.

Nucleophosmin/B23 is a nucleolar phosphoprotein. It has been shown that B23 binds to nucleic acids, digests RNA, and is localized in nucleolar granular components from which preribosomal particles are transported to cytoplasm. The intracellular localization of B23 is significantly changed during the cell cycle. Here, we have examined the cellular localization of B23 proteins and the effect of mitotic phosphorylation of B23.1 on its RNA binding activity. Two splicing variants of B23 proteins, termed B23.1 and B23.2, were complexed both in vivo and in vitro. The RNA binding activity of B23.1 was impaired by hetero-oligomer formation with B23.2. Both subtypes of B23 proteins were phosphorylated during mitosis by cyclin B/cdc2. The RNA binding activity of B23.1 was repressed through cyclin B/cdc2-mediated phosphorylation at specific sites in B23. Thus, the RNA binding activity of B23.1 is stringently modulated by its phosphorylation and subtype association. Interphase B23.1 was mainly localized in nucleoli, whereas B23.2 and mitotic B23.1, those of which were incapable of binding to RNA, were dispersed throughout the nucleoplasm and cytoplasm, respectively. These results suggest that nucleolar localization of B23.1 is mediated by its ability to associate with RNA.

Identification of nucleophosmin/B23, an acidic nucleolar protein, as a stimulatory factor for in vitro replication of adenovirus DNA complexed with viral basic core proteins

The processes governing chromatin remodeling and assembly, which occur prior to and/or after transcription and replication, are not completely understood. To understand the mechanisms of transcription and replication from chromatin templates, we have established in vitro replication and transcription systems using adenovirus (Ad) DNA complexed with viral basic core proteins, called Ad core, as a template. Using this system, we have previously identified, from HeLa cells, template activating factor-I as a stimulatory factor for the Ad core DNA replication. Here, using this system as a tool, we identified and purified a novel template activating factor activity that consists of two acidic polypeptides whose apparent molecular masses are 38 kDa and 37 kDa. These two polypeptides correspond to two splicing variants of nucleolar phosphoprotein, nucleophosmin/B23. Recombinant B23 proteins stimulate the Ad core DNA replication, and the acidic regions of B23 proteins are important for its activity. In addition, B23 proteins directly bind to core histones and transfer them to naked DNA. Furthermore, chromatin components such as histones and topoisomerase II are co-immunoprecipitated with B23 from cell extracts. These observations lead to a hypothesis that nucleophosmin/B23 is involved in structural changes of chromatin, thereby regulating transcription and replication within the ribosomal DNA region or maintaining the nucleolar structure.

Immunocytochemical localization of nucleophosmin and RH-II/Gu protein in nucleoli of HeLa cells after treatment with actinomycin D

HeLa cells were studied with the use of light microscopical immunocytochemistry to obtain more information on the nucleolar localization of nucleophosmin (B23 protein) and RH-II/Gu protein. In control cells these proteins are colocalized. After structural rearrangement of nucleoli induced by actinomycin D, nucleophosmin was present in the nucleolar periphery and nucleolar peripheral caps. In contrast, RH-II/Gu protein was observed mainly in central regions of nucleoli as a compact body. However, in some nucleoli the proteins colocalized in a small portion of the peripheral nucleolar cap. The proteins were also colocalized in micronucleoli but occasionally RH-II/Gu protein was absent or both proteins were not present.

Multiple Subcellular Localizations of PCTAIRE-1 in Brain

We developed a selective antibody to a synthetic peptide corresponding to an N-terminal sequence of the PCTAIRE-1 protein. In rodent brain extracts it recognized only the protein doublet characteristic of PCTAIRE-1, and this signal is completely abolished by preincubation of the antibody with the immunopeptide. Immunolabeling experiments done with this PCTAIRE-1-specific antibody reveal that the protein is widely distributed in the rodent brain as are the mRNAs visualized using an antisense riboprobe corresponding to the entire PCTAIRE-1 open reading frame. Two types of PCTAIRE-1 protein localizations were observed: first a diffuse labeling of almost all brain regions, particularly intense in the molecular layer of the cerebellum and the mossy fiber region of the hippocampus, and second a spot-like localization in the nuclei of large neurons such as cerebellar Purkinje cells and pyramidal cells of the hippocampus. Colocalization with the B23 protein allows one to identify these compartments as nucleoli. Our results suggest a nucleolar function of PCTAIRE-1 in large neurons and a role in regions containing important granule cell projections.

AgNOR proteins from morphologically intact isolated nucleoli

AgNOR staining has been proposed as a useful tool for the diagnosis and prognosis of cancer. The AgNOR proteins, however, have not yet been clearly identified and characterized, possibly due to the partial character of the results obtained when studying the proteins extracted from altered nucleoli isolated by “standard” methods. In the present study, we analysed, on western blots, the AgNOR staining profiles obtained with protein extracts from Ehrlich tumor cell nucleoli isolated by a recent procedure that preserves the nucleolar ultrastructure. In addition to the well-known C23 and B23 protein bands, we readily detected an extra band at approximately 125 Kda. By immunoblotting, we showed that this polypeptide may be related to the nucleolar phosphoprotein pp135 evidenced in rat-cell nucleoli. By immunoelectron microscopy, we detected this protein in the dense fibrillar component and fibrillar center of the nucleoli as well as the coiled bodies. The distribution coincides with the cytochemical AgNOR staining pattern obtained at the ultrastructural level.

Effects of Sex Steroids on Silver Stained Proteins of Nucleolar Organizer Regions (Ag-NOR) in the Rabbit Uterus

Silver staining of argyrophilic proteins of nucleolar organizer regions (Ag-NOR) has been used to evaluate a hyperactive state of cells. We studied the effects of sex steroids on the Ag-NOR proteins in the rabbit uterus. Estradiol-17β (E2) caused uterine hypertrophy and increased the number of Ag-NOR dots in epithelial and stromal cells of the endometrium and smooth muscles cells in a dose dependent manner without proliferative change in the endometrium. Progestins including progesterone, medroxyprogesterone acetate (MPA), and norethindrone following E2 caused progestational proliferation of the endometrium with an increase in the Ag-NOR number to various extents, whereas methyltestosterone, a synthetic androgen, caused only a minimal proliferative change in the endometrium without an increase in the Ag-NOR number. Western blot analysis revealed that progesterone and MPA increased the amounts of a 100 kDa protein (nucleolin) and a 37 kDa protein (B23 protein) in the endometium and a marked proliferative change, whereas E2 did not. These results suggest that although the Ag-NOR number was enhanced by both progestin and estrogen, only progestins substantially increase the Ag-NOR protein in the rabbit uterus. This implies that an increase in the amount of nucleolin and B23 protein is associated with the proliferative potential of the cells.

Experimental induction of prenucleolar bodies (PNBs) in interphase cells: interphase PNBs show similar characteristics as those typically observed at telophase of mitosis in untreated cells.

Recently, it was shown that a short exposure of living mammalian cells to low ionic strength buffers (hypotonic shock) caused partial or almost complete unraveling of interphase nucleoli. However, when the cells were released from the hypotonic shock and transferred to normal isotonic medium, functionally active and structurally integral nucleoli were reassembled at their initial positions within interphase nuclei. Here, we show further that this process is accompanied by the appearance of numerous discrete extranucleolar bodies, which have striking similarities to the prenucleolar bodies (PNBs) observed in untreated cells at telophase of mitosis. (1) Like PNBs at mitosis, hypotonically induced interphase PNBs are composed of RNA-positive granules and fibrils, contain the major nucleolar protein B23 and silver-binding proteins, but lack DNA and RNA polymerase I transcription factor UBF. (2) As for mitotic PNBs, disappearance of the interphase PNB counterparts coincides with the increase in size of reconstructed nucleoli. (3) Addition of actinomycin D does not prevent assembly of interphase PNBs, but does arrest their coalescence with the chromosomal nucleolus-organizing regions and blocks the complete reformation of nucleoli. It is concluded that the assembly of PNBs generally observed at telophase of mitosis can be induced experimentally in nuclei of interphase mammalian cells in vivo. At interphase, this process is probably initiated by changes in the intracellular ionic environment.

Major internal nuclear matrix proteins are common to different human cell types.

The nuclear matrix may be involved in the structural and functional organization of the cell nucleus. However, we still do not understand the molecular basis of the intranuclear fibrogranular network that is part of the nuclear matrix. We recently described a method to identify internal nuclear matrix proteins [Mattern et al. (1996): J Cell Biochem 62:275-289], which was done by comparing two nuclear matrix preparations: one with and one without the internal structure by using quantitative two-dimensional gel electrophoresis. In the present study, we use the same approach to compare the nuclear matrix proteins of four different human cell types to investigate whether they have a similar internal nuclear matrix protein composition. Major nuclear matrix proteins present in all these cell types likely represent the base of the internal nuclear matrix. We demonstrate that the 25 most abundant internal nuclear matrix proteins are common to all four cell types. Together, these common proteins represent more than 75% of the total internal nuclear matrix protein mass in each cell type. This set of proteins includes B23 and most hnRNP proteins. The quantity of most of these proteins is very similar in the four cell types. The fact that the internal nuclear matrix consists mainly of hnRNP proteins, which may be involved in transcription, transport, and processing of hnRNA, supports the idea that the internal nuclear matrix is the result of these processes.

Spatial association of HIV-1 tat protein and the nucleolar transport protein B23 in stably transfected Jurkat T-cells.

The human immunodeficiency virus (HIV-1) encodes a transactivator protein, the product of the tat gene (tat), which is essential for virus replication. In this study, immunogold electron microscopy was used in a stably transfected Jurkat T-cell line that constitutively expresses HIV-1 tat protein to determine the subcellular and intranuclear distribution of tat protein. Two nucleocytoplasmic shuttle proteins C23/nucleolin and B23 and a third nucleolar antigen that was detected by monoclonal antibody MAb 1277 were also examined. In addition, spatial association of C23 and B23 with tat protein at several subcellular locations was examined in dual-labeling experiments. The results showed that tat protein was found in both the cytoplasm and nucleus but was especially prominent within the dense fibrillar and granular components of the nucleolus. There was little labeling of tat protein in the fibrillar centers where MAb 1277 antigen was localized at a comparatively high level. The subcellular and intranucleolar distribution of tat protein was virtually identical to the pattern seen with C23 and B23. Although the intranuclear distributions of C23, B23 and tat protein were very similar, C23 and tat protein were seldom spatially associated. In contrast, B23 and tat protein were frequently spatially associated in the nucleolus and in several other subcellular locations including the cytoplasm, nucleoplasm, at the nuclear envelope and plasma membrane. While a physical association was not directly demonstrated in this study, the spatial association between B23 and tat protein strongly suggest that such an association may exist.

The chromosome periphery during mitosis

A complex structure, visible by electron microscopy, surrounds each chromosome during mitosis. The organization of this structure is distinct from that of the chromosomes and the cytoplasm. It forms a perichromosomal layer that can be isolated together with the chromosomes. This layer covers the chromosomes except in centromeric regions. The perichromosomal layer includes nuclear and nucleolar proteins as well as ribonucleoproteins (RNPs). The list of proteins and RNAs identified includes nuclear matrix proteins (perichromin, peripherin), nucleolar proteins (perichro-monucleolin, Ki-67 antigen, B23 protein, fibrillarin, p103, p52), ribosomal proteins (S1) and snRNAs (U3 RNAs). Only limited information is available about how and when the perichromosomal layer is formed. During early prophase, the proteins extend from the nucleoli towards the periphery of the nucleus. Thin cordon-like structures reach the nuclear envelope delimiting areas in which chromosomes condense. At telophase, the proteins are associated with the part of the chromosomes remaining condensed and accumulate in newly formed nucleoli in regions where chromatin is already decondensed. The perichromosomal layer contains several different classes of proteins and RNPs and it has been attributed various roles: (1) in chromosome organization, (2) as a barrier around the chromosomes, (3) involvement in compartmentation of the cells in prophase and telophase and (4) a binding site for chromosomal passenger proteins necessary to the early process of nuclear assembly.

Relief of YY1-induced transcriptional repression by protein-protein interaction with the nucleolar phosphoprotein B23.

Previous studies have shown that the transcription factor YY1 can both activate and repress transcription of many mammalian genes (reviewed by Hahn (Hahn, S. (1992) Curr. Biol. 2, 152-154)). Given the diverse effects of the YY1 protein, it seems likely that its function depends on interaction with other cellular factors. We have used the yeast two-hybrid system to isolate mouse cDNAs encoding proteins capable of directly binding to YY1. Sequence analysis of one clone revealed it had an open reading frame with the potential to code for a protein nearly identical to the previously published mouse nucleolar phosphoprotein B23. The YY1.B23 complex is specific, and occurs in vivo and in vitro. Overexpression of the B23 protein can reverse the transcriptional repression exerted by YY1. These results suggest a role for a nucleolar protein as a component in transcription and provide a possible mechanism for transcriptional regulation by YY1.

Specific Complex of Human Immunodeficiency Virus Type 1 Rev and Nucleolar B23 Proteins: Dissociation by the Rev Response Element

The human immunodeficiency virus type 1 (HIV) Rev protein is thought to be involved in the export of unspliced or singly spliced viral mRNAs from the nucleus to the cytoplasm. This function is mediated by a sequence-specific interaction with a cis-acting RNA element, the Rev response element (RRE), present in these intron-containing RNAs. To identify possible host proteins involved in Rev function, we fractionated nuclear cell extracts with a Rev affinity column. A single, tightly associated Rev-binding protein was identified; this protein is the mammalian nucleolar protein B23. The interaction between HIV Rev and B23 is very specific, as it was observed in complex cell extracts. The complex is also very stable toward dissociation by high salt concentrations. Despite the stability of the Rev-B23 protein complex, the addition of RRE, but not control RNA, led to the displacement of B23 and the formation of a specific Rev-RRE complex. The mammalian nucleolar protein B23 or its amphibian counterpart No38 is believed to function as a shuttle receptor for the nuclear import of ribosomal proteins. B23 may also serve as a shuttle for the import of HIV Rev from the cytoplasm into the nucleus or nucleolus to allow further rounds of export of RRE-containing viral RNAs.

Specific complex of human immunodeficiency virus type 1 rev and nucleolar B23 proteins: dissociation by the Rev response element.

The human immunodeficiency virus type 1 (HIV) Rev protein is thought to be involved in the export of unspliced or singly spliced viral mRNAs from the nucleus to the cytoplasm. This function is mediated by a sequence-specific interaction with a cis-acting RNA element, the Rev response element (RRE), present in these intron-containing RNAs. To identify possible host proteins involved in Rev function, we fractionated nuclear cell extracts with a Rev affinity column. A single, tightly associated Rev-binding protein was identified; this protein is the mammalian nucleolar protein B23. The interaction between HIV Rev and B23 is very specific, as it was observed in complex cell extracts. The complex is also very stable toward dissociation by high salt concentrations. Despite the stability of the Rev-B23 protein complex, the addition of RRE, but not control RNA, led to the displacement of B23 and the formation of a specific Rev-RRE complex. The mammalian nucleolar protein B23 or its amphibian counterpart No38 is believed to function as a shuttle receptor for the nuclear import of ribosomal proteins. B23 may also serve as a shuttle for the import of HIV Rev from the cytoplasm into the nucleus or nucleolus to allow further rounds of export of RRE-containing viral RNAs.

Isolation and characterization of a molecular cDNA clone of a human mRNA from interferon-treated cells encoding nucleolar protein B23, numatrin

A cDNA clone encoding human nucleolar phosphoprotein B23, numatrin, was isolated from a library prepared with mRNA from human U cells. The complete nucleotide sequence was determined; it revealed a single open reading frame of 294 amino acids that included four in-frame AUG codons as potential sites of translation initiation. Comparison of the human B23 nucleotide sequence with the rat and mouse B23 sequences revealed 91% homology (hum:rat, and hum:mus) in the coding region; the predicted B23 proteins displayed 94% amino acid identity. Northern gel blot analysis revealed a single B23 mRNA species of ∼1.5 kb. The level of B23 mRNA in U cells was not detectably altered by treatment with either alpha or gamma interferon. Southern gel blot analysis revealed polymorphism within the human B23 gene structure, and suggested the presence of multiple B23 genes and/or extensive splicing of B23 RNA transcripts.