RESUMO
Activation of ribosomal RNA (rRNA) synthesis is pivotal during cell growth and proliferation, but its aberrant upregulation may promote tumorigenesis. Here, we demonstrate that the candidate oncoprotein, LYAR, enhances ribosomal DNA (rDNA) transcription. Our data reveal that LYAR binds the histone-associated protein BRD2 without involvement of acetyl-lysine-binding bromodomains and recruits BRD2 to the rDNA promoter and transcribed regions via association with upstream binding factor. We show that BRD2 is required for the recruitment of the MYST-type acetyltransferase KAT7 to rDNA loci, resulting in enhanced local acetylation of histone H4. In addition, LYAR binds a complex of BRD4 and KAT7, which is then recruited to rDNA independently of the BRD2-KAT7 complex to accelerate the local acetylation of both H4 and H3. BRD2 also helps recruit BRD4 to rDNA. By contrast, LYAR has no effect on rDNA methylation or the binding of RNA polymerase I subunits to rDNA. These data suggest that LYAR promotes the association of the BRD2-KAT7 and BRD4-KAT7 complexes with transcription-competent rDNA loci but not to transcriptionally silent rDNA loci, thereby increasing rRNA synthesis by altering the local acetylation status of histone H3 and H4.
Assuntos
Proteínas de Ciclo Celular/genética , Proteínas de Ligação a DNA/genética , Histona Acetiltransferases/genética , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Acetilação , Carcinogênese/genética , Cromatina/genética , Metilação de DNA/genética , DNA Ribossômico/genética , Histonas/genética , Humanos , RNA Polimerase I/genética , RNA Ribossômico/biossíntese , RNA Ribossômico/genética , Transcrição GênicaRESUMO
Ribosome biogenesis is an essential process for cell growth and proliferation and is enhanced in cancer and embryonic stem cells. Mouse Ly-1 antibody reactive clone product (Lyar) is expressed at very high levels in many tumor, leukemia or embryonic stem cells; is a novel nucleolar protein with zinc-finger DNA-binding motifs and is involved in cell growth regulation. However, cellular function of Lyar remains unexplored. Here, we show that human homologue of Lyar (LYAR) accelerates ribosome biogenesis at the level of processing of preribosomal RNA (pre-rRNA). We show that LYAR is excluded from the nucleolus after actinomycin D treatment and is present in preribosomal fraction of the nuclear extract as well as in the fractions with 40S, 60S and 90S sedimentation coefficients. LYAR is required for processing of 47S/45S, 32S, 30S and 21S pre-rRNAs. In addition, we show that over-expression of LYAR increases cell proliferation without affecting the expression of c-Myc or p53. Combined, these results suggest that some rapidly growing cells enhance ribosome biogenesis by increasing the expression of LYAR.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Precursores de RNA/metabolismo , RNA Ribossômico/metabolismo , Animais , Proliferação de Células , Proteínas de Ligação a DNA/genética , Dactinomicina/farmacologia , Células HEK293 , Células HeLa , Humanos , Camundongos , Proteínas Nucleares/genética , Inibidores da Síntese de Ácido Nucleico/farmacologia , Processamento Pós-Transcricional do RNA , Homologia Estrutural de ProteínaRESUMO
Mature osteoclasts are multinuclear, macrophage-like cells derived from hematopoietic stem cells in the bone marrow. Several transcription factors regulating osteoclast differentiation have been identified. However, the molecular basis of transcriptional regulation in osteoclasts at epigenetic levels is largely unknown. In fact, no osteoclast-specific transcriptional co-regulators have been characterized. Recently, selective ablation of estrogen receptor alpha (ERalpha) in mature osteoclasts derived from female mice (ERalpha(Deltaoc/Deltaoc)) exhibited trabecular bone loss due to induced apoptosis via upregulated expression of Fas ligand mRNA. In general, the component composition of the ERalpha-associated co-activator complex and its expression levels are distinct among tissues. However, ERalpha transcriptional co-regulators in mature osteoclasts remain unclear. In the present study, we achieved large-scale cultivation of mature, multinucleated osteoclasts and established a purification system for ERalpha-associated proteins. In addition to co-regulators previously found in other ERalpha target cells, several unexpected factors were found such as CAP-H. The mRNA expression level of CAP-H was high during osteoclast differentiation. These results demonstrate the existence of osteoclast-specific transcriptional co-regulators supporting ERalpha function.
Assuntos
Proteínas Correpressoras/isolamento & purificação , Receptor alfa de Estrogênio/metabolismo , Osteoclastos/metabolismo , Transativadores/isolamento & purificação , Animais , Clonagem Molecular , Proteínas Correpressoras/genética , Proteínas Correpressoras/metabolismo , Receptor alfa de Estrogênio/isolamento & purificação , Feminino , Humanos , Camundongos , Modelos Biológicos , Osteoclastos/química , Transativadores/genética , Transativadores/metabolismo , Ativação Transcricional/genéticaRESUMO
Chromatin reorganization is essential for transcriptional control by sequence-specific transcription factors. However, the molecular link between transcriptional control and chromatin reconfiguration remains unclear. By colocalization of the nuclear ecdysone receptor (EcR) on the ecdysone-induced puff in the salivary gland, Drosophila DEK (dDEK) was genetically identified as a coactivator of EcR in both insect cells and intact flies. Biochemical purification and characterization of the complexes containing fly and human DEKs revealed that DEKs serve as histone chaperones via phosphorylation by forming complexes with casein kinase 2. Consistent with the preferential association of the DEK complex with histones enriched in active epigenetic marks, dDEK facilitated H3.3 assembly during puff formation. In some human myeloid leukemia patients, DEK was fused to CAN by chromosomal translocation. This mutation significantly reduced formation of the DEK complex, which is required for histone chaperone activity. Thus, the present study suggests that at least one histone chaperone can be categorized as a type of transcriptional coactivator for nuclear receptors.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas Oncogênicas/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores da Família Eph/metabolismo , Ativação Transcricional/genética , Animais , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/genética , Sequência Conservada , Proteínas de Drosophila/genética , Ecdisona/metabolismo , Evolução Molecular , Chaperonas de Histonas/metabolismo , Humanos , Leucemia Mieloide Aguda/fisiopatologia , Nucleossomos/metabolismo , Proteínas Oncogênicas/genética , Proteínas de Ligação a Poli-ADP-Ribose , Receptores da Família Eph/genéticaRESUMO
Vitamin D plays an important role in regulating bone and calcium metabolism. The actions of vitamin D are mediated through the nuclear vitamin D receptor (VDR), and gene disruption of the VDR in mice causes skeletal disorders. However, the precise role of the VDR in each stage of osteoblastogenesis is not well understood. To address this issue, we used a biochemical approach to identify an osteoblast-specific coregulator of the VDR. Using a GST-fused VDR ligand-binding domain as bait, proteins associated with liganded VDR were purified from nuclear extracts of HOS osteoblastic cells and compared with those of HeLa cells. Among the interactants identified by mass fingerprinting, CCAAT displacement protein (CDP) was found as a novel ligand-dependent VDR interactant in HOS cells, together with other previously reported DRIP/TRAP complex components. Further biochemical analysis showed that complex formation between the VDR and CDP was distinct from the previously known DRIP/TRAP complex and the p160 family coactivator complexes. Transient expression of CDP potentiated VDR-mediated transcriptional activation in HOS cells. Furthermore, modulation of CDP expression levels in osteoblastic SaM-1 cells affected vitamin D-dependent osteoblast differentiation before the maturation (mineralization) stage. These findings suggest that CDP is a novel differentiation stage-specific coactivator of the VDR in osteoblasts.
Assuntos
Proteínas de Homeodomínio/fisiologia , Proteínas Nucleares/fisiologia , Receptores de Calcitriol/fisiologia , Proteínas Repressoras/fisiologia , Diferenciação Celular/efeitos dos fármacos , Humanos , Osteoblastos/metabolismo , Osteossarcoma/metabolismo , Receptores de Calcitriol/genética , Fatores de Transcrição , Células Tumorais CultivadasRESUMO
Histone acetyl transferases (HATs) play a crucial role in eukaryotes by regulating chromatin architecture and locus-specific transcription. The GCN5 HAT was identified as a subunit of the SAGA (Spt-Ada-Gcn5-Acetyltransferase) multiprotein complex. Vertebrate cells express a second HAT, PCAF, that is 73% identical to GCN5. Here, we report the characterization of the mammalian ATAC (Ada-Two-A-Containing) complexes containing either GCN5 or PCAF in a mutually exclusive manner. In vitro ATAC complexes acetylate lysine 14 of histone H3. Moreover, ATAC- or SAGA-specific knock-down experiments suggest that both ATAC and SAGA are involved in the acetylation of histone H3K9 and K14 residues. Despite their catalytic similarities, SAGA and ATAC execute their coactivator functions on distinct sets of inducible target genes. Interestingly, ATAC strongly influences the global phosphorylation level of histone H3S10, suggesting that in mammalian cells a cross-talk exists linking ATAC function to H3S10 phosphorylation.
Assuntos
Regulação da Expressão Gênica , Histona Acetiltransferases/metabolismo , Complexos Multienzimáticos/metabolismo , Fatores de Transcrição de p300-CBP/metabolismo , Acetilação , Animais , Catálise , Drosophila , Técnicas de Silenciamento de Genes , Células HeLa , Histona Acetiltransferases/genética , Histonas/metabolismo , Humanos , Complexos Multienzimáticos/genética , Fosforilação , Fatores de Transcrição de p300-CBP/genéticaRESUMO
Epigenetic modifications at the histone level affect gene regulation in response to extracellular signals. However, regulated epigenetic modifications at the DNA level, especially active DNA demethylation, in gene activation are not well understood. Here we report that DNA methylation/demethylation is hormonally switched to control transcription of the cytochrome p450 27B1 (CYP27B1) gene. Reflecting vitamin-D-mediated transrepression of the CYP27B1 gene by the negative vitamin D response element (nVDRE), methylation of CpG sites ((5m)CpG) is induced by vitamin D in this gene promoter. Conversely, treatment with parathyroid hormone, a hormone known to activate the CYP27B1 gene, induces active demethylation of the (5m)CpG sites in this promoter. Biochemical purification of a complex associated with the nVDRE-binding protein (VDIR, also known as TCF3) identified two DNA methyltransferases, DNMT1 and DNMT3B, for methylation of CpG sites, as well as a DNA glycosylase, MBD4 (ref. 10). Protein-kinase-C-phosphorylated MBD4 by parathyroid hormone stimulation promotes incision of methylated DNA through glycosylase activity, and a base-excision repair process seems to complete DNA demethylation in the MBD4-bound promoter. Such parathyroid-hormone-induced DNA demethylation and subsequent transcriptional derepression are impaired in Mbd4(-/-) mice. Thus, the present findings suggest that methylation switching at the DNA level contributes to the hormonal control of transcription.
Assuntos
Metilação de DNA/efeitos dos fármacos , Hormônio Paratireóideo/farmacologia , Transcrição Gênica/efeitos dos fármacos , 25-Hidroxivitamina D3 1-alfa-Hidroxilase/genética , Animais , Linhagem Celular , Ilhas de CpG/genética , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/metabolismo , DNA Glicosilases/metabolismo , Regulação para Baixo/efeitos dos fármacos , Endodesoxirribonucleases/deficiência , Endodesoxirribonucleases/genética , Camundongos , Fosforilação , Proteína Quinase C/metabolismo , Elementos de Resposta/genética , Vitamina D/farmacologia , DNA Metiltransferase 3BRESUMO
Steroid hormones and their cognate nuclear receptors exert a wide spectrum of biological actions through regulation of transcriptional and posttranscriptional processes. However, the underlying molecular mechanism by which steroid hormones control posttranscriptional processes is largely unknown. We now report that estrogen receptor alpha (ERalpha) inhibits the maturation of a particular microRNA (miRNA) and thereby stabilizes the mRNA of an ERalpha target gene through the 3'UTR. Estrogen-bound ERalpha downregulated expression of a set of miRNAs in both animals and cultured cells. Activated ERalpha attenuated the processing of primary miRNAs into pre-miRNAs through estrogen-dependent association with the Drosha complex, resulting in stabilization of the transcript of an ERalpha target gene through its 3'UTR. Thus, a steroid hormone achieves posttranscriptional control by regulating the maturation of miRNA.
Assuntos
Receptor alfa de Estrogênio/metabolismo , Estrogênios/farmacologia , MicroRNAs/genética , Regiões 3' não Traduzidas , Animais , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Humanos , Camundongos , Ribonuclease III/metabolismo , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Spinal and bulbar muscular atrophy (SBMA) is a neurodegenerative disorder caused by a polyglutamine repeat (polyQ) expansion within the human androgen receptor (AR). Unlike other neurodegenerative diseases caused by abnormal polyQ expansion, the onset of SBMA depends on androgen binding to mutant human polyQ-AR proteins. This is also observed in Drosophila eyes ectopically expressing the polyQ-AR mutants. We have genetically screened mediators of androgen-induced neurodegeneration caused by polyQ-AR mutants in Drosophila eyes. We identified Rbf (Retinoblastoma-family protein), the Drosophila homologue of human Rb (Retinoblastoma protein), as a neuroprotective factor. Androgen-dependent association of Rbf or Rb with AR was remarkably potentiated by aberrant polyQ expansion. Such potentiated Rb association appeared to attenuate recruitment of histone deacetyltransferase 1 (HDAC1), a corepressor of E2F function. Either overexpression of Rbf or E2F deficiency in fly eyes reduced the neurotoxicity of the polyQ-AR mutants. Induction of E2F function by polyQ-AR-bound androgen was suppressed by Rb in human neuroblastoma cells. We conclude that abnormal expansion of polyQ may potentiate innate androgen-dependent association of AR with Rb. This appears to lead to androgen-dependent onset of SBMA through aberrant E2F transactivation caused by suppressed histone deacetylation.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Fatores de Transcrição E2F/metabolismo , Atrofia Muscular Espinal/metabolismo , Atrofia Muscular Espinal/patologia , Degeneração Neural/patologia , Peptídeos/metabolismo , Receptores Androgênicos/metabolismo , Androgênios/farmacologia , Animais , Proteínas de Drosophila/genética , Fatores de Transcrição E2F/genética , Humanos , Ligantes , Proteínas Mutantes/metabolismo , Degeneração Neural/metabolismo , Ligação Proteica , Proteína do Retinoblastoma/metabolismo , Fatores de Transcrição/metabolismo , Ativação TranscricionalRESUMO
Ligand-bound nuclear receptors (NR) activate transcription of the target genes. This activation is coupled with histone modifications and chromatin remodeling through the function of various coregulators. However, the nature of the dependence of a NR coregulator action on the presence of the chromatin environment at the target genes is unclear. To address this issue, we have developed a modified position effect variegation experimental model system that includes an androgen-dependent reporter transgene inserted into either a pericentric heterochromatin region or a euchromatic region of Drosophila chromosome. Human androgen receptor (AR) and its constitutively active truncation mutant (AR AF-1) were transcriptionally functional in both chromosomal regions. Predictably, the level of AR-induced transactivation was lower in the pericentric heterochromatin. In genetic screening for AR AF-1 coregulators, Drosophila CREB binding protein (dCBP) was found to corepress AR transactivation at the pericentric region whereas it led to coactivation in the euchromatic area. Mutations of Sir2 acetylation sites or deletion of the CBP acetyltransferase domain abrogated dCBP corepressive action for AR at heterochromatic areas in vivo. Such a CBP corepressor function for AR was observed in the transcriptionally silent promoter of an AR target gene in cultured mammalian cells. Thus, our findings suggest that the action of NR coregulators may depend on the state of chromatin at the target loci.
Assuntos
Proteína de Ligação a CREB/metabolismo , Efeitos da Posição Cromossômica/genética , Drosophila melanogaster/genética , Heterocromatina/metabolismo , Modelos Genéticos , Receptores Androgênicos/genética , Ativação Transcricional/genética , Acetilação , Sequência de Aminoácidos , Animais , Proteína de Ligação a CREB/química , Domínio Catalítico , Linhagem Celular Tumoral , Sequência Conservada , Proteínas de Drosophila/química , Eucromatina/metabolismo , Histona Desacetilases/química , Histonas/metabolismo , Humanos , Lisina/metabolismo , Metilação , Dados de Sequência Molecular , Regiões Promotoras Genéticas/genética , Receptores Androgênicos/química , Receptores Androgênicos/metabolismo , Proteínas Repressoras/metabolismo , Sirtuínas/químicaRESUMO
H2A.Z is an evolutionarily highly conserved non-allelic variant of histone H2A. H2A.Z and its homologues have been shown to involve in both chromatin silencing and activation. Although much of our knowledge of H2A.Z biological activity has come from studies on its yeast homologue Htz1, H2A.Z appears to have more complex and diverse functions in higher eukaryotes. To investigate the involvement of H2AvD, a Drosophila homologue of mammalian H2A.Z, in mechanisms of conditional activation of facultatively silenced genes, we generated transgenic Drosophila lines expressing H2AvD fused at the C- or N-terminus with the green fluorescent protein (GFP). Using heat shock-induced gene activation and polytene chromosome puff formation as an in vivo model system, we analyzed effects of H2AvD termini modifications on transcription. We found that N-terminally fused GFP inhibited H2AvD acetylation and impaired heat shock-induced puff formation and hsp70 gene activation. Our data suggest that the N-terminal region of H2AvD plays a pivotal role in transcriptional activation and that induction of transiently silenced Drosophila loci associates with increased acetylation of H2AvD.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Inativação Gênica , Variação Genética , Histonas/metabolismo , Acetilação , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Cromossomos/química , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/química , Drosophila melanogaster/genética , Proteínas de Choque Térmico HSP70/genética , Histonas/química , Histonas/genética , Dados de Sequência MolecularRESUMO
Abnormal polyglutamine (polyQ) expansion in the N-terminal domain of the human androgen receptor (hAR) is known to cause spinobulbar muscular atrophy (SBMA), a hereditary human neurodegenerative disorder. To explore the molecular mechanisms of neurodegeneration in SBMA, we genetically screened modulators of neurodegeneration in a Drosophila SBMA experimental model system. We identified hoip as an accelerator of polyQ-induced neurodegeneration. We found that hoip forms a complex with 18s rRNA together nop56 and nop5 proteins, whose human homologs are known to form a snoRNP complex involved in ribosomal RNA processing. Significantly, the levels of mutant polyQ-hAR were up-regulated in a mutant line overexpressing hoip. Consistently, severe neurodegeneration phenotype (rough eye) was also observed in both nop56 and nop5 overexpression mutant lines. These findings suggest that the process of neurodegeneration induced by abnormal polyQ expansion in the hAR may be regulated by the activity of snoRNP complex.
Assuntos
Proteínas de Drosophila/genética , Drosophila/genética , Genes de Insetos , Degeneração Neural/metabolismo , Peptídeos/metabolismo , Proteínas de Ligação a RNA/genética , Animais , Animais Geneticamente Modificados , Células Cultivadas , Modelos Animais de Doenças , Drosophila/citologia , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Olho/metabolismo , Olho/ultraestrutura , Transtornos Musculares Atróficos/genética , Transtornos Musculares Atróficos/metabolismo , Mutação , Degeneração Neural/genética , Proteínas de Ligação a RNA/metabolismo , Receptores Androgênicos , TransfecçãoRESUMO
Although several murine mAbs that have been humanized became useful therapeutic agents against a few malignancies, therapeutic Abs are not yet available for the majority of the human cancers because of our lack of knowledge of which antigens (Ags) can become useful targets. In the present study we established a procedure for comprehensive identification of such Ags through the extensive isolation of human mAbs that may become therapeutic. Using the phage-display Ab library we isolated a large number of human mAbs that bind to the surface of tumor cells. They were individually screened by immunostaining, and clones that preferentially and strongly stained the malignant cells were chosen. The Ags recognized by those clones were isolated by immunoprecipitation and identified by MS. We isolated 2,114 mAbs with unique sequences and identified 21 distinct Ags highly expressed on several carcinomas. Of those 2,114 mAbs 356 bound specifically to one of the 21 Ags. After preparing complete IgG(1) Abs the in vitro assay for Ab-dependent cell-mediated cytotoxicity (ADCC) and the in vivo assay in cancer-bearing athymic mice were performed to examine antitumor activity. The mAbs converted to IgG(1) revealed effective ADCC as well as antitumor activity in vivo. Because half of the 21 Ags showed distinct tumor-specific expression pattern and the mAbs isolated showed various characteristics with strong affinity to the Ag, it is likely that some of the Ags detected will become useful targets for the corresponding carcinoma therapy and that several mAbs will become therapeutic agents.
Assuntos
Anticorpos Monoclonais/química , Carcinoma/imunologia , Neoplasias/imunologia , Animais , Antígenos de Neoplasias/química , Antineoplásicos/farmacologia , Carcinoma/diagnóstico , Linhagem Celular Tumoral , Receptores ErbB/metabolismo , Humanos , Imunoglobulina G/metabolismo , Imunoterapia/instrumentação , Imunoterapia/métodos , Camundongos , Camundongos Nus , Modelos Biológicos , Neoplasias/diagnóstico , Biblioteca de PeptídeosRESUMO
MicroRNAs (miRNAs) control cell proliferation, differentiation and fate through modulation of gene expression by partially base-pairing with target mRNA sequences. Drosha is an RNase III enzyme that is the catalytic subunit of a large complex that cleaves pri-miRNAs with distinct structures into pre-miRNAs. Here, we show that both the p68 and p72 DEAD-box RNA helicase subunits in the mouse Drosha complex are indispensable for survival in mice, and both are required for primary miRNA and rRNA processing. Gene disruption of either p68 or p72 in mice resulted in early lethality, and in both p68(-/-) and p72(-/-) embryos, expression levels of a set of, but not all, miRNAs and 5.8S rRNA were significantly lowered. In p72(-/-) MEF cells, expression of p72, but not a mutant lacking ATPase activity, restored the impaired expression of miRNAs and 5.8S rRNA. Furthermore, we purified the large complex of mouse Drosha and showed it could generate pre-miRNA and 5.8S rRNA in vitro. Thus, we suggest that DEAD-box RNA helicase subunits are required for recognition of a subset of primary miRNAs in mDrosha-mediated processing.
Assuntos
RNA Helicases DEAD-box/metabolismo , Embrião de Mamíferos/metabolismo , MicroRNAs/metabolismo , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , RNA Ribossômico/metabolismo , Ribonuclease III/metabolismo , Animais , Diferenciação Celular , Linhagem da Célula , Proliferação de Células , Células Cultivadas , Imunoprecipitação da Cromatina , RNA Helicases DEAD-box/deficiência , RNA Helicases DEAD-box/genética , Embrião de Mamíferos/citologia , Embrião de Mamíferos/enzimologia , Fibroblastos/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Isoenzimas/metabolismo , Camundongos , Camundongos Knockout , Análise de Sequência com Séries de Oligonucleotídeos , Interferência de RNA , RNA Ribossômico 5,8S/metabolismoRESUMO
Proteomic technologies powered by advancements in mass spectrometry and bioinformatics and coupled with accumulated genome sequence data allow a comprehensive study of cell function through large-scale and systematic protein identifications of protein constituents of the cell and tissues, as well as of multi-protein complexes that carry out many cellular function in a higher-order network in the cell. One of the most extensively analyzed cellular functions by proteomics is the production of ribosome, the protein-synthesis machinery, in the nucle(ol)us--the main site of ribosome biogenesis. The use of tagged proteins as affinity bait, coupled with mass spectrometric identification, enabled us to isolate synthetic intermediates of ribosomes that might represent snapshots of nascent ribosomes at particular stages of ribosome biogenesis and to identify their constituents--some of which showed dynamic changes for association with the intermediates at various stages of ribosome biogenesis. In this review, in conjunction with the results from yeast cells, our proteomic approach to analyze ribosome biogenesis in mammalian cells is described.
Assuntos
Proteoma/metabolismo , Proteômica , Ribonucleoproteínas/química , Ribonucleoproteínas/metabolismo , Ribossomos/química , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Animais , Substâncias MacromolecularesRESUMO
Human parvulin (hParvulin; Par14/EPVH) belongs to the third family of peptidylprolyl cis-trans isomerases that exhibit an enzymatic activity of interconverting the cis-trans conformation of the prolyl peptide bond, and shows sequence similarity to the regulator enzyme for cell cycle transitions, human Pin1. However, the cellular function of hParvulin is entirely unknown. Here, we demonstrate that hParvulin associates with the preribosomal ribonucleoprotein (pre-rRNP) complexes, which contain preribosomal RNAs, at least 26 ribosomal proteins, and 26 trans-acting factors involved in rRNA processing and assembly at an early stage of ribosome biogenesis. Since an amino-terminal domain of hParvulin, which is proposed to be a putative DNA-binding domain, was alone sufficient to associate in principle with the pre-rRNP complexes, the association is probably through protein-RNA interaction. In addition, hParvulin co-precipitated at least 10 proteins not previously known to be involved in ribosome biogenesis. Coincidentally, most of these proteins are implicated in regulation of microtubule assembly or nucleolar reformation during the mitotic phase of the cell. Thus, these results, coupled with the preferential nuclear localization of hParvulin, suggest that hParvulin may be involved in ribosome biogenesis and/or nucleolar re-assembly of mammalian cells.