RESUMEN
Transient transfection of HeLa cells with a plasmid encoding the full-length human fibrillarin fused to a green fluorescent protein (GFP) resulted in two major patterns of intensity of the nucleolar labeling for the chimeric protein: weak and strong. Both patterns were maintained in fibrillarin-GFP expressing cells after fixation with formaldehyde. When the fixed fibrillarin-GFP expressing cells were used for immunolabeling with antibodies to fibrillarin, only the nucleoli with a weak GFP-signal became strongly labeled, whereas those with the heavy signals were only lightly stained, if at all. A similar pattern was observed if the cells were immunolabeled with antibodies to GFP. These observations suggest that an increase in antigen accumulation within the nucleolus, which could take place under various physiological or experimental conditions, could prevent the antigen from being recognized by specific antibodies. These results have implications regarding contradictory data on localization of various nucleolar antigens obtained by conventional immunocytochemistry.
Asunto(s)
Anticuerpos/inmunología , Antígenos Nucleares/inmunología , Antígenos Nucleares/metabolismo , Nucléolo Celular/inmunología , Nucléolo Celular/metabolismo , Complejo Antígeno-Anticuerpo/inmunología , Artefactos , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Genes Reporteros/genética , Células HeLa , Humanos , Inmunohistoquímica , TransfecciónRESUMEN
The cell nucleolus is the subnuclear body in which ribosomal subunits are assembled, and it is also the location of several processes not related to ribosome biogenesis. Recent studies have revealed that nucleolar components move about in a variety of ways. One class of movement is associated with ribosome assembly, which is a vectorial process originating at the sites of transcription in the border region between the fibrillar center and the dense fibrillar component. The nascent preribosomal particles move outwardly to become the granular components where further maturation takes place. These particles continue their travel through the nucleoplasm for eventual export to the cytoplasm to become functional ribosomes. In a second kind of motion, many nucleolar components rapidly exchange with the nucleoplasm. Thirdly, nucleolar components engage in very complex movements when the nucleolus disassembles at the beginning of mitosis and then reassembles at the end of mitosis. Finally, many other cellular and viral macromolecules, which are not related to ribosome assembly, also pass through or are retained by the nucleolus. These are involved in nontraditional roles of the nucleolus, including regulation of tumor suppressor and oncogene activities, signal recognition particle assembly, modification of small RNAs, control of aging, and modulating telomerase function.
Asunto(s)
Nucléolo Celular/fisiología , Sustancias Macromoleculares/metabolismo , Ribosomas/metabolismo , Animales , Nucléolo Celular/metabolismo , Nucléolo Celular/ultraestructura , VIH-1/metabolismo , Células HeLa , Humanos , Microscopía Fluorescente , Mitosis/fisiología , Proteínas Virales/metabolismoRESUMEN
The protein B23 is a major nucleolar phosphoprotein comprising two isoforms, B23.1 and B23.2, which differ only in their carboxyl-terminal short sequences, the N-terminal 255 residues being identical in both forms. Both B23.1 and B23.2 stimulated immunoaffinity-purified calf thymus DNA polymerase alpha in a dose-dependent manner. The stimulatory effect of protein B23.1, the longer isoform, was found to be 2-fold greater than that of B23.2. Purified DNA polymerase alpha bound tightly to a protein B23.1-immobilized column, while it bound weakly to a protein B23.2-immobilized column. Surface plasmon resonance studies by BIAcore further showed that protein B23.1 bound to the DNA polymerase alpha-(dA).(dT) complex more tightly than did protein B23.2. The protein B23 isoforms appear to interact directly with the DNA polymerase alpha protein and not through the bound nucleic acid. These observations indicated that protein B23 physically bound to the DNA polymerase alpha and stimulated the enzyme activity. Product analyses showed that protein B23 greatly enhanced the reaction both in amount and length of product DNA, whereas it did not significantly alter the processivity of polymerization. In contrast, protein B23 effectively protected DNA polymerase alpha from heat inactivation. These results suggest that protein B23 stabilizes DNA polymerase alpha that is detached from product DNA, allowing the enzyme to be recruited for further elongation. Moreover, experiments using various C-terminal deletion mutants of protein B23 indicated that 12 amino acids at the C-terminal end of B23.1, which are absent in B23.2, may be essential for the full stimulation of the DNA polymerase alpha.
Asunto(s)
ADN Polimerasa I/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Secuencia de Aminoácidos , Animales , Estabilidad de Enzimas , Datos de Secuencia Molecular , Mutación , Proteínas Nucleares/genética , Nucleofosmina , Unión Proteica , Isoformas de Proteínas , Estructura Terciaria de Proteína , Ratas , Proteínas Recombinantes/metabolismo , Resonancia por Plasmón de SuperficieRESUMEN
Mammalian cell nucleoli disassemble at the onset of M-phase and reassemble during telophase. Recent studies showed that partially processed preribosomal RNA (pre-rRNA) is preserved in association with processing components in the perichromosomal regions (PRs) and in particles called nucleolus-derived foci (NDF) during mitosis. Here, the dynamics of nucleolar reassembly were examined for the first time in living cells expressing fusions of the processing-related proteins fibrillarin, nucleolin, or B23 with green fluorescent protein (GFP). During telophase the NDF disappeared with a concomitant appearance of material in the reforming nuclei. Prenucleolar bodies (PNBs) appeared in nuclei in early telophase and gradually disappeared as nucleoli formed, strongly suggesting the transfer of PNB components to newly forming nucleoli. Fluorescence recovery after photobleaching (FRAP) showed that fibrillarin-GFP reassociates with the NDF and PNBs at rapid and similar rates. The reentry of processing complexes into telophase nuclei is suggested by the presence of pre-rRNA sequences in PNBs. Entry of specific proteins into the nucleolus approximately correlated with the timing of processing events. The mitotically preserved processing complexes may be essential for regulating the distribution of components to reassembling daughter cell nucleoli.
Asunto(s)
Nucléolo Celular/fisiología , Mitosis/fisiología , Proteínas Nucleares/aislamiento & purificación , Precursores del ARN/metabolismo , Procesamiento Postranscripcional del ARN , ARN Ribosómico/metabolismo , Animales , Nucléolo Celular/ultraestructura , Núcleo Celular/ultraestructura , Células Cultivadas , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/aislamiento & purificación , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/genética , Proteínas Nucleares/genética , Nucleofosmina , Proteínas Recombinantes de Fusión , Telofase/fisiologíaRESUMEN
Protein B23 is a multifunctional nucleolar protein whose cellular location and characteristics strongly suggest that it is a ribosome assembly factor. The protein has nucleic acid binding, ribonuclease, and molecular chaperone activities. To determine the contributions of unique polypeptide segments enriched in certain classes of amino acid residues to the respective activities, several constructs that produced N- and C-terminal deletion mutant proteins were prepared. The C-terminal quarter of the protein was shown to be necessary and sufficient for nucleic acid binding. Basic and aromatic segments at the N- and C-terminal ends, respectively, of the nucleic acid binding region were required for activity. The molecular chaperone activity was contained in the N-terminal half of the molecule, with important contributions from both nonpolar and acidic regions. The chaperone activity also correlated with the ability of the protein to form oligomers. The central portion of the molecule was required for ribonuclease activity and possibly contains the catalytic site; this region overlapped with the chaperone-containing segment of the molecule. The C-terminal, nucleic acid-binding region enhanced the ribonuclease activity but was not essential for it. These data suggest that the three activities reside in mainly separate but partially overlapping segments of the polypeptide chain.
Asunto(s)
Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Animales , Cromatografía en Gel , Clonación Molecular , Cinética , Mutagénesis , Proteínas Nucleares/aislamiento & purificación , Nucleofosmina , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Desnaturalización Proteica , Ratas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Eliminación de Secuencia , TermodinámicaRESUMEN
The function of the nucleolus as a factory for assembling ribosomal subunits is well established, but many unrelated activities have been discovered over the past decade. Our understanding of the dynamics of nucleolar structure and its reassembly at the end of mitosis has recently advanced and the small nucleolar RNAs have been shown to be major players in the processing and modification of preribosomal RNA. Unexpectedly, the nucleolus also seems to play a role in nuclear export, sequestering regulatory molecules, modifying small RNAs, assembling ribonucleoprotein (RNP) and controlling aging.
Asunto(s)
Nucléolo Celular/fisiología , Nucléolo Celular/ultraestructura , Animales , Nucléolo Celular/genética , Humanos , Mitosis , Proteínas Nucleares , ARN Polimerasa I , Procesamiento Postranscripcional del ARN , ARN Ribosómico/metabolismo , Ribosomas/genética , Ribosomas/fisiología , Ribosomas/ultraestructuraRESUMEN
Protein B23 is an abundant, multifunctional nucleolar phosphoprotein whose activities are proposed to play a role in ribosome assembly. Szebeni et al. (1997) showed stimulation of nuclear import in vitro by protein B23 and suggested that this effect was due to a molecular chaperone-like activity. Protein B23 was tested for chaperone activities using several protein substrates. The temperature-dependent and -independent aggregation of the HIV-1 Rev protein was measured using a zero angle light scattering (turbidity) assay. Protein B23 inhibited the aggregation of the Rev protein, with the amount of inhibition proportional to the concentration of B23 added. This activity was saturable with nearly complete inhibition when the molar ratio of B23:Rev was slightly above one. Protein B23 also protected liver alcohol dehydrogenase (LADH), carboxypeptidase A, citrate synthase, and rhodanese from aggregation during thermal denaturation and preserved the enzyme activity of LADH under these conditions. In addition, protein B23 was able to promote the restoration of activity of LADH previously denatured with guanidine-HCl. Protein B23 preferentially bound denatured substrates and exposed hydrophobic regions when complexed with denatured proteins. Thus, by several criteria, protein B23 behaves like a molecular chaperone; these activities may be related to its role in ribosome biogenesis.
Asunto(s)
Chaperonas Moleculares/fisiología , Proteínas Nucleares/química , Proteínas Nucleares/fisiología , Alcohol Deshidrogenasa/metabolismo , Relación Dosis-Respuesta a Droga , Productos del Gen rev/química , VIH-1/química , Hígado/metabolismo , Nucleofosmina , Desnaturalización Proteica/efectos de los fármacos , Pliegue de Proteína , Temperatura , Tiosulfato Azufretransferasa/metabolismo , Factores de Tiempo , Ultracentrifugación , Productos del Gen rev del Virus de la Inmunodeficiencia HumanaRESUMEN
B23 is a major phosphoprotein in the interphasic nucleolus where it is involved in the assembly of pre-ribosomes. Using several cultured animal cells, we report that, in addition to the known redistribution of the protein during mitosis, B23 also becomes associated with mitotic spindle poles starting from early prometaphase onwards. Colocalization of B23 with the protein NuMA (Nuclear Mitotic Apparatus protein) was studied in mitotic cells and taxol-arrested cells. During the onset of mitosis, we observed that a fraction of B23 associates with, and dissociates from, the poles later than NuMA. At metaphase, both proteins are colocalized at the poles. The polar redistribution of both B23 and NuMA is mediated by microtubules. In taxol-treated cells, B23 is associated with the microtubule minus ends in the center of mitotic asters together with NuMA. Association of B23 with microtubule minus ends of mitotic asters was further confirmed with an in vitro assay, where B23 was found by western blotting to co-sediment with taxol-induced microtubule asters formed in a mitotic cell extract. Immunolabeling demonstrated that B23 and NuMA were both present at the center of the asters. Furthermore, an additional hyperphosphorylated form of B23 appeared when microtubule asters formed and associated with the asters. Immunodepletion of B23 from the mitotic extract revealed that taxol-induced microtubule asters were still observed in B23-immunodepleted mitotic extract, indicating that the presence of B23 at the poles is unlikely to be essential for spindle formation or stabilisation.
Asunto(s)
Mitosis/fisiología , Proteínas Nucleares/metabolismo , Huso Acromático/metabolismo , Animales , Antígenos Nucleares , Autoantígenos/metabolismo , Proteínas de Ciclo Celular , División Celular/efectos de los fármacos , División Celular/fisiología , Línea Celular , Nucléolo Celular/efectos de los fármacos , Nucléolo Celular/metabolismo , Sistema Libre de Células/metabolismo , Células HeLa , Humanos , Macropodidae , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Mitosis/efectos de los fármacos , Nocodazol/farmacología , Proteínas Asociadas a Matriz Nuclear , Región Organizadora del Nucléolo/metabolismo , Nucleofosmina , Paclitaxel/farmacología , Fosfoproteínas/metabolismo , Fosforilación , Isoformas de Proteínas/metabolismo , RatasRESUMEN
Protein B23 is an abundant nucleolar protein and a putative ribosome assembly factor which possesses an intrinsic ribonuclease activity. In the current work, the effects of RNA sequence and secondary structure on the cleavage preference by protein B23 were studied. Protein B23 ribonuclease preferentially cleaved the single-stranded homopolymers poly(A), poly(U) and poly(C). However, double-stranded co-polymers and poly(G) were resistant to cleavage. No base specificity was observed with an oligoribonucleotide substrate. The action of protein B23 ribonuclease on different regions of pre-rRNA was studied using transcripts synthesized in vitro from cloned rDNA segments. Although no specific cleavages were detected in transcripts containing sequences from the 5' external transcribed spacer or the first internal transcribed spacer, the enzyme preferentially cleaved the second internal transcribed spacer (ITS2) approximately 250 nt downstream from the 3'-end of 5.8S rRNA. Preferential cleavage was retained when the transcript was extended by 100 nt at the 3'-end, but abolished in a transcript lacking this cleavage site. Furthermore, this site was not susceptible to cleavage by RNase A or RNase T1. These results, in conjunction with the sub-nucleolar localization of the protein with elements of the processing machinery, suggest that the protein B23 endoribonuclease could play a role in pre-rRNA processing in ITS2.
Asunto(s)
Proteínas Nucleares/genética , Precursores del ARN/genética , ARN Ribosómico/genética , Animales , Proteínas Nucleares/metabolismo , Conformación de Ácido Nucleico , Nucleofosmina , Plásmidos/genética , ARN Ribosómico/metabolismo , Ratas , Ribonucleasas/genética , Ribonucleasas/metabolismo , Análisis de SecuenciaRESUMEN
Previous studies showed that components implicated in pre-rRNA processing, including U3 small nucleolar (sno)RNA, fibrillarin, nucleolin, and proteins B23 and p52, accumulate in perichromosomal regions and in numerous mitotic cytoplasmic particles, termed nucleolus-derived foci (NDF) between early anaphase and late telophase. The latter structures were analyzed for the presence of pre-rRNA by fluorescence in situ hybridization using probes for segments of pre-rRNA with known half-lives. The NDF did not contain the short-lived 5'-external transcribed spacer (ETS) leader segment upstream from the primary processing site in 47S pre-rRNA. However, the NDF contained sequences from the 5'-ETS core, 18S, internal transcribed spacer 1 (ITS1), and 28S segments and also had detectable, but significantly reduced, levels of the 3'-ETS sequence. Northern analyses showed that in mitotic cells, the latter sequences were present predominantly in 45S-46S pre-rRNAs, indicating that high-molecular weight processing intermediates are preserved during mitosis. Two additional essential processing components were also found in the NDF: U8 snoRNA and hPop1 (a protein component of RNase MRP and RNase P). Thus, the NDF appear to be large complexes containing partially processed pre-rRNA associated with processing components in which processing has been significantly suppressed. The NDF may facilitate coordinated assembly of postmitotic nucleoli.
Asunto(s)
Proteínas Portadoras , Mitosis/fisiología , Precursores del ARN , Procesamiento Postranscripcional del ARN , Regiones no Traducidas 5' , Animales , Proteínas Reguladoras de la Apoptosis , Línea Celular , Nucléolo Celular/metabolismo , Endorribonucleasas/metabolismo , Haplorrinos , Humanos , ARN Catalítico/metabolismo , ARN Ribosómico 18S , ARN Ribosómico 28S , ARN Nuclear Pequeño , Ribonucleasa P , Ribonucleoproteínas/análisisRESUMEN
The myeloid cell nuclear differentiation antigen (MNDA) is a nuclear protein expressed specifically in developing cells of the human myelomonocytic lineage, including the end-stage monocytes/macrophages and granulocytes. Nuclear localization, lineage- and stage-specific expression, association with chromatin, and regulation by interferon alpha indicate that this protein is involved in regulating gene expression uniquely associated with the differentiation process and/or function of the monocyte/macrophage. MNDA does not bind specific DNA sequences, but rather a set of nuclear proteins that includes nucleolin (C23). Both in vitro binding assays and co-immunoprecipitation were used to demonstrate that MNDA also binds protein B23 (nucleophosmin/NPM). Three reciprocal chromosome translocations found in certain cases of leukemia/lymphoma involve fusions with the NPM/B23 gene, t(5;17) NPM-RARalpha, t(2;5) NPM-ALK, and the t(3;5) NPM-MLF1. In the current study, MNDA was not able to bind the NPM-ALK chimera originating from the t(2;5) and containing residues 1-117 of NPM. However, MNDA did bind the NPM-MLF1 product of the t(3;5) that contains the N-terminal 175 residues of NPM. The additional 58 amino acids (amino acids 117-175) of the NPM sequence that are contained in the product of the NPM-MLF1 fusion gene relative to the product of the NPM-ALK fusion appear responsible for MNDA binding. This additional NPM sequence contains a nuclear localization signal and clusters of acidic residues believed to bind nuclear localization signals of other proteins. Whereas NPM and nucleolin are primarily localized within the nucleolus, MNDA is distributed throughout the nucleus including the nucleolus, suggesting that additional interactions define overall MNDA localization.
Asunto(s)
Antígenos de Diferenciación Mielomonocítica/metabolismo , Leucemia Mieloide/genética , Síndromes Mielodisplásicos/genética , Proteínas Nucleares/genética , Proteínas/genética , Proteínas Recombinantes de Fusión/metabolismo , Factores de Transcripción/metabolismo , Translocación Genética , Enfermedad Aguda , Proteínas de Ciclo Celular , Mapeo Cromosómico , Cromosomas Humanos Par 2 , Cromosomas Humanos Par 3 , Cromosomas Humanos Par 5 , Proteínas de Unión al ADN , Humanos , Proteínas Nucleares/biosíntesis , Nucleofosmina , Biosíntesis de Proteínas , Proteínas Tirosina Quinasas/biosíntesis , Proteínas Tirosina Quinasas/genética , Proteínas Recombinantes de Fusión/genética , Células Tumorales CultivadasRESUMEN
. The subcellular location of several nonribosomal nucleolar proteins was examined at various stages of mitosis in synchronized mammalian cell lines including HeLa, 3T3, COS-7 and HIV-1 Rev-expressing CMT3 cells. Nucleolar proteins B23, fibrillarin, nucleolin and p52 as well as U3 snoRNA were located partially in the peripheral regions of chromosomes from prometaphase to early telophase. However, these proteins were also found in large cytoplasmic particles, 1-2 microm in diameter, termed nucleolus-derived foci (NDF). The NDF reached maximum numbers (as many as 100 per cell) during mid- to late anaphase, after which their number declined to a few or none during late telophase. The decline in the number of NDF approximately coincided with the appearance of prenucleolar bodies and reforming nucleoli. The HIV-1 Rev protein and a mutant Rev protein defective in its nuclear export signal were also found in the NDF. The mutant Rev protein precisely followed the pattern of localization of the above nucleolar proteins, whereas the wild-type Rev did not enter nuclei until G1 phase. The nucleolar shuttling phosphoprotein Nopp140 did not follow the above pattern of localization during mitosis: it dispersed in the cytoplasm from prometaphase through early telophase and was not found in the NDF. Although the NDF and mitotic coiled bodies disappeared from the cytoplasm at approximately the same time during mitosis, protein B23 was not found in mitotic coiled bodies, nor was p80 coilin present in the NDF. These results suggest that a class of proteins involved in preribosomal RNA processing associate with chromosome periphery and with NDF as part of a system to conserve and deliver preexisting components to reforming nucleoli during mitosis.
Asunto(s)
Anafase/fisiología , Núcleo Celular/ultraestructura , Proteínas Nucleares/metabolismo , Telofase/fisiología , Animales , Línea Celular , Núcleo Celular/química , Núcleo Celular/genética , Cromosomas/química , Citoplasma/genética , Citoplasma/ultraestructura , Citoplasma/virología , Productos del Gen rev/metabolismo , VIH-1/genética , Humanos , Mamíferos , Ratones , Mitosis , Proteínas Nucleares/análisis , Fosfoproteínas/análisis , Fosfoproteínas/metabolismo , ARN Nuclear Pequeño/genética , ARN Nuclear Pequeño/metabolismo , Productos del Gen rev del Virus de la Inmunodeficiencia HumanaRESUMEN
Nucleolar phosphoprotein B23 is a putative ribosome assembly factor with a relatively high affinity for peptides containing sequences of nuclear localization signals (NLSs) of the SV40 T-antigen type [Szebeni, A., Herrera, J. E., & Olson, O. J. (1995) Biochemistry 34, 8037-8042]. The effects of protein B23 on nuclear import were determined by an in vitro assay [Dean, D. A., & Kasamatsu, H. (1994) J. Biol. Chem. 269, 4910-4916] using NLS peptide-conjugated bovine serum albumin (NLS-BSA) or the HIV-1 Rev protein as substrates for import into isolated rat liver nuclei. The import was ATP-dependent and inhibited by wheat germ agglutinin or by an antibody against p97, a component of the nuclear import system. The rate of import of either substrate was increased if protein B23 was added to the incubation medium. Similar enhancements of import were seen with both isoforms (B23.1 and B23.2). The stimulatory effect on Rev protein import was saturable with maximum stimulation (2-3-fold) at a molar ratio of protein B23:Rev of approximately 1:1. Phosphorylation of protein B23.1 by casein kinase II produced an additional doubling of the import rate. This effect was not seen if protein B23.1 was phosphorylated with a cdc2 type protein kinase. Mutant forms of protein B23.1 in which the nuclear localization signal was either deleted or altered did not stimulate import of the substrates. These results suggest that protein B23 plays a role as an accessory factor in the nuclear import of the NLS-containing proteins and that phosphorylation at sites in the highly acidic segments of the protein enhances the stimulatory effect.
Asunto(s)
Núcleo Celular/metabolismo , Productos del Gen rev/metabolismo , VIH-1 , Proteínas Nucleares/metabolismo , Albúmina Sérica/metabolismo , Adenosina Trifosfato/farmacología , Animales , Anticuerpos Monoclonales/inmunología , Antígenos Transformadores de Poliomavirus/metabolismo , Quinasa de la Caseína II , Reactivos de Enlaces Cruzados/metabolismo , Fluoresceínas , Colorantes Fluorescentes/metabolismo , Hígado/metabolismo , Microscopía Fluorescente , Mutación , Señales de Localización Nuclear , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/inmunología , Proteínas Nucleares/farmacología , Nucleofosmina , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Ratas , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Aglutininas del Germen de Trigo/farmacología , Productos del Gen rev del Virus de la Inmunodeficiencia HumanaRESUMEN
The HIV-1 Rev protein localizes predominantly to the nucleolus of HIV-1-infected or Rev-expressing cells. The subcellular location of Rev during mitotic nucleolar disintegration was examined at various stages of mitosis in synchronized Rev-expressing CMT3 cells. During early prophase Rev was predominantly located in disintegrating nucleoli and began to accumulate at the peripheral regions of chromosomes in late prophase, eventually distributing uniformly on all chromosomes in prometaphase. In anaphase Rev remained associated with the perichromosomal regions, but significant amounts of Rev were also seen in numerous nucleolus-derived foci. The movement of Rev from disintegrating nucleoli to perichromosomal regions and foci was similar to that of nonribosomal nucleolar proteins, including fibrillarin, nucleolin, protein B23 and p52 of the granular component. During telophase Rev remained associated with perichromosomal regions and mitotic foci until the nuclear envelope started to reform. When nuclear envelope formation was complete in late telophase, nonribosomal nucleolar proteins were present in prenucleolar bodies (PNBs) which were eventually incorporated into nucleoli; at the same time, Rev was excluded from nuclei. In contrast, a trans-dominant negative Rev protein containing an inactive nuclear export signal reentered nuclei by the nonribosomal nucleolar protein pathway in late telophase, associating with PNBs and reformed nucleoli. Rev protein reentry into postmitotic nuclei was delayed until early G1 phase, but before the arrival of ribosomal protein S6. Thus, Rev behaves like a nonribosomal nucleolar protein through mitosis until early telophase; however, its nuclear reentry seems to require reestablishment of both a nuclear import system and active nucleoli.
Asunto(s)
Productos del Gen rev/metabolismo , VIH-1/metabolismo , Mitosis/fisiología , Animales , Transporte Biológico Activo , Línea Celular , Nucléolo Celular/metabolismo , Núcleo Celular/metabolismo , Productos del Gen rev/genética , VIH-1/genética , Haplorrinos , Microscopía Fluorescente , Proteínas Nucleares/metabolismo , Eliminación de Secuencia , Transducción de Señal , Productos del Gen rev del Virus de la Inmunodeficiencia HumanaRESUMEN
Protein B23 is a major nonribosomal nucleolar protein and putative ribosome assembly factor that has been demonstrated to form oligomers. Sedimentation velocity and equilibrium analyses were used to examine the oligomerization properties of recombinant proteins B23.1 and B23.2. Under low ionic strength conditions protein B23.1 was predominantly a 2.1S monomer with small amounts of a 7.1S oligomer. At NaCl concentrations of 0.1 M and above the protein was almost exclusively the 7.1S oligomer. The oligomer remained the predominant species in NaCl concentrations as high as 1 M, suggesting that oligomers are not stabilized by electrostatic interactions. Low concentrations of divalent metal ions (0.1 - 1mM Ca2+ or Mg2+) also promoted oligomerization. Reducing agents had no effect on oligomerization, indicating that disulfide bridges are not important in oligomer formation. Protein B23.2, the carboxyl-terminal truncated isoform, had sedimentation characteristics similar to that of protein B23.1, suggesting that the carboxyl-terminal end of protein B23.1 is not essential for oligomerization. Protein B23.1 was previously shown to bind nucleic acids [Wang, D., Baumann, A., Szebeni, A., & Olson, M. O. J.(1995) J. Biol. Chem. 269, 30994-30998]. The effect of protein B23.1 oligomerization on its interaction with a 230 base pair DNA fragment was examined by sedimentation analyses. Under conditions where significant amounts of monomer were present, protein B23.1 was capable of binding DNA, whereas conditions that strongly favored oligomerization caused a nearly complete abolition of DNA binding activity. These studies suggest that protein B23 exists in an equilibrium between monomer and oligomer and that the quaternary structure of the protein may regulate its DNA binding properties.
Asunto(s)
Proteínas Nucleares/aislamiento & purificación , Animales , Cationes Bivalentes , Cationes Monovalentes , ADN/metabolismo , Técnicas In Vitro , Metales , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Nucleofosmina , Unión Proteica , Conformación Proteica , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Sales (Química) , UltracentrifugaciónRESUMEN
DNA topoisomerase I (topo I) is involved in the regulation of DNA supercoiling, gene transcription, and rDNA recombination. However, little is known about interactions between topo I and other nuclear proteins. We used affinity chromatography with a topo I fusion protein to screen U-937 leukemic cell extracts and have identified nucleolin as a topo I-binding protein. Coimmunoprecipitation and other studies demonstrate that the interaction between topo I and nucleolin is direct. Furthermore, deletion analyses have identified the 166-210-amino acid region of topo I as sufficient for the interaction with nucleolin. Since nucleolin has been implicated in nuclear transport and in a variety of transcriptional processes, the interaction with topo I may relate to the cellular localization of topo I or to the known role of this topoisomerase in transcription.
Asunto(s)
ADN-Topoisomerasas de Tipo I/química , Proteínas Nucleares/química , Fosfoproteínas/química , Proteínas de Unión al ARN , Secuencia de Aminoácidos , Sitios de Unión , Compartimento Celular , ADN-Topoisomerasas de Tipo I/metabolismo , ADN Superhelicoidal/metabolismo , Humanos , Datos de Secuencia Molecular , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Células Tumorales Cultivadas , NucleolinaRESUMEN
The human myeloid cell nuclear differentiation antigen (MNDA) is a nuclear protein expressed specifically in cells of the myelomonocytic lineage and regulated by interferon alpha in a cell-specific fashion. MNDA is also a member of a family of interferon-regulated genes of unknown function. In an effort to elucidate the function of MNDA, three techniques (affinity purification, coimmunoprecipitation, and protein blot assay) were used to characterize its specific protein binding activities. Microsequence analysis showed that MNDA bound the 100 kDa nucleolin protein. The identification of nucleolin was confirmed by immunoreaction with specific antibodies. MNDA contains motifs which could account for specific binding to nucleolin. Nucleolin binds other macromolecules and exhibits features consistent with roles in signal transduction, production of ribosomes, nuclear matrix structure, and regulation of transcription. The present results indicate that the function of MNDA is most likely related to interactions with other proteins. Through these associations, MNDA could contribute cell/lineage- and differentiation-specific limits to the function of ubiquitous proteins such as nucleolin. Further analysis of MNDA protein binding could be critical to elucidating the function of MNDA and could contribute to understanding the function of the products of other members of this interferon-inducible family of genes.
Asunto(s)
Antígenos de Diferenciación Mielomonocítica/fisiología , Proteínas Nucleares/metabolismo , Región Organizadora del Nucléolo , Fosfoproteínas/metabolismo , Proteínas de Unión al ARN , Factores de Transcripción/fisiología , Secuencia de Aminoácidos , Antígenos de Diferenciación Mielomonocítica/genética , Antígenos de Diferenciación Mielomonocítica/metabolismo , Secuencia de Bases , Western Blotting , Proteínas Portadoras/análisis , Epítopos , Humanos , Datos de Secuencia Molecular , Peso Molecular , Pruebas de Precipitina , Unión Proteica , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Transducción de Señal/fisiología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Células Tumorales Cultivadas , NucleolinaRESUMEN
Protein B23 is an abundant nucleolar protein and putative ribosome assembly factor. The protein was analyzed for ribonuclease activity using RNA-embedded gels and perchloric acid precipitation assays. Three purified bacterially expressed forms of the protein, B23.1, B23.2 and an N-terminal polyhistidine tagged B23.1 as well as the natural protein were found to have ribonuclease activity. However, the specific activity of recombinant B23.1 was approximately 5-fold greater than that of recombinant B23.2. The activity was insensitive to human placental ribonuclease inhibitor, but was inhibited by calf thymus DNA in a dose dependent manner. The enzyme exhibited activity over a broad range of pH with an apparent optimum at pH 7.5. The activity was stimulated by but not dependent on the presence of low concentrations of Ca2+, Mg2+ or NaCl. The Ca2+ effect was saturable and only stimulatory in nature. In contrast, Mg2+ and NaCl exhibited optimal concentrations for stimulation and both inhibited the ribonuclease at concentrations above these optima. These data suggest that protein B23 has intrinsic ribonuclease activity. The location of protein B23 in subcompartments of the nucleolus that contain preribosomal RNA suggests that its ribonuclease activity plays a role in the processing of preribosomal RNA.
Asunto(s)
Proteínas Nucleares/metabolismo , Ribonucleasas/metabolismo , Animales , Calcio/farmacología , Cationes Bivalentes , ADN/farmacología , Inhibidores Enzimáticos/farmacología , Humanos , Concentración de Iones de Hidrógeno , Neoplasias Hepáticas Experimentales , Magnesio/farmacología , Nucleofosmina , Nucleótidos/metabolismo , Concentración Osmolar , Hormonas Placentarias/farmacología , Ratas , Proteínas Recombinantes/metabolismo , Ribonucleasas/antagonistas & inhibidores , Cloruro de Sodio/farmacología , Células Tumorales CultivadasRESUMEN
Protein B23 is a nucleolar and nuclear matrix-associated phosphoprotein that is involved in ribosome synthesis. Its expression and phosphorylation in rat ventral prostate, an androgen target organ, are profoundly influenced by androgens. Induction of programmed cell death (apoptosis) in the prostatic epithelium by androgen deprivation in the animal induces an early decline in protein B23 in the absence of a corresponding loss of protein B23 mRNA. We have now demonstrated that prostatic nuclei retain the ability to transcribe the B23 mRNA and that a significant amount of this mRNA persists even after 7 days of androgen deprivation when > 80% of the prostatic epithelial cells have undergone apoptosis. The B23 mRNA from these nuclei is also translatable in vitro. However, the majority of the B23 mRNA is associated with free and short-stretch polysomes, which may account for the castration-induced decline in synthesis of protein B23 in vivo. In addition, the mechanism of down-regulation of protein B23 in apoptotic prostatic cells appears to relate to two coordinate signals, which include loss of phosphorylation of the protein as well as the expression of a protease active toward dephosphorylated protein B23, under these conditions.
Asunto(s)
Apoptosis , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Próstata/metabolismo , Procesamiento Proteico-Postraduccional , Andrógenos/deficiencia , Animales , Hidrólisis , Masculino , Proteínas Nucleares/genética , Nucleofosmina , Fosfoproteínas/genética , Fosforilación , Próstata/patología , Biosíntesis de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Transcripción GenéticaRESUMEN
The human immunodeficiency virus 1 (HIV-1) Rev transactivator protein plays a critical role in the regulation of expression of structural proteins by controlling the pathway of mRNA transport. The Rev protein is located predominantly in the nucleoli of HIV-1 infected or Rev-expressing cells. Previous studies demonstrated that the Rev protein forms a specific complex in vitro with protein B23 which is suggested to be a nucleolar receptor and/or carrier for the Rev protein. To study the role of the nucleolus and nucleolar proteins in Rev function, transfected COS-7 or transformed CMT3 cells expressing the Rev protein were examined for subcellular locations of Rev and other proteins using indirect immunofluorescence and immunoelectron microscopy. One day after transfection the Rev protein was found in most cells only in the nucleolar dense fibrillar and granular components where it colocalized with protein B23. These were designated class 1 cells. In a second class of cells Rev and B23 accumulated in the nucleoplasm as well as in nucleoli. Treatment of class 1 cells with actinomycin D (AMD) under conditions that blocked only RNA polymerase I transcription caused Rev to completely redistribute from nucleoli to the cytoplasm. Simultaneously, protein B23 was partially released from nucleoli, mostly into the nucleoplasm, with detectable amounts in the cytoplasm. In cells recovering from AMD treatment in the presence of cycloheximide Rev and B23 showed coincident relocation to nucleoli. Class 2 cells were resistant to AMD-induced Rev redistribution. Selective inhibition of RNA polymerase II transcription by alpha-amanitin or by DRB did not cause Rev to be released into the cytoplasm suggesting that active preribosomal RNA transcription is required for the nucleolar location of Rev. However, treatment with either of the latter two drugs at higher doses and for longer times caused partial disruption of nucleoli accompanied by translocation of the Rev protein to the cytoplasm. These results suggest that the nucleolar location of Rev depends on continuous preribosomal RNA transcription and a substantially intact nucleolar structure.