RESUMO
SAF-A/hnRNP U is an abundant nuclear protein that interacts specifically with nuclear matrix attachment region DNA (MAR) and RNA as a component of hnRNPs. SAF-A/hnRNP U was also shown to specifically bind mouse major satellite DNA (satMa). Antibodies against SAF-A and GFP-fusion constructs were used in the current work in order to trace SAF-A localization. In accordance with its diverse nucleic acid binding specificity, SAF-A was found to be localized in three different domains: outside the chromosomes, on the surface of the chromosome arms (probably MARs), and in the centromere region where it apparently binds specifically to the satMa. GFP-fusion constructs with different SAF-A/hnRNP U domains confirms the functional significance of the protein's functional domains in interphase cells. In telophase cells, the anti-SAF-A antibody signal appeared as a kind of network covering unfolded chromosomes.
Assuntos
Núcleo Celular/metabolismo , Cromossomos de Mamíferos/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo U/metabolismo , Animais , Apoptose , Células COS , Núcleo Celular/ultraestrutura , Chlorocebus aethiops , Ribonucleoproteínas Nucleares Heterogêneas Grupo U/ultraestrutura , Interfase/fisiologia , Metáfase/fisiologia , Camundongos , Telófase/fisiologiaRESUMO
Several oncogenic proteins and tumour suppressors target the RNA polymerase I and interfere with rRNA synthesis. Here, we show that the glycogen synthase kinase (GSK) 3beta, which phosphorylates the tumour suppressor PTEN (phosphatase and tensin homologue deleted on chromosome 10), is selectively enriched in nucleoli of RAS-transformed cells. Immunoprecipitation and chromatin immunoprecipitation assays performed on epithelial and endothelial cells transformed with oncogenic RAS show that GSK3beta and PTEN are part of the same complex and associate with promoter and coding region of the rDNA. An active GSK3beta mutant abolished nucleolar BrUTP incorporation and associated with the member of the selectivity factor 1 complex TAF(I)110. Finally, GSK3beta inhibition upregulated 45S, 18S and 28S rRNA synthesis in RAS-transformed epithelial cells as revealed by semiquantitative real-time PCR and promoted cellular proliferation. Our results underscore a repressive function for GSK3beta in rRNA biogenesis supporting its role as a tumour supressor.
Assuntos
Quinase 3 da Glicogênio Sintase/metabolismo , RNA Polimerase I/genética , Transcrição Gênica , Nucléolo Celular/metabolismo , Imunofluorescência , Quinase 3 da Glicogênio Sintase/genética , Glicogênio Sintase Quinase 3 beta , Mutação , PTEN Fosfo-Hidrolase/metabolismoRESUMO
Recent developments in the field of gene transcription regulation have unfolded a key role for actin as an important co-factor for all three eukaryotic RNA polymerases. In this review article we discuss the latest findings on actin in transcription of protein-coding and ribosomal genes, in complex with specific hnRNP proteins and a form of myosin 1beta which is entirely localized to the cell nucleus. Based on these recent studies, we propose a general model where actin may function in basal gene transcription as an allosteric regulator, to recruit transcriptional co-activators on active genes. A future challenge will be the identification of the polymerization state of actin in gene transcription and how it is mechanistically regulated.
Assuntos
Actinas/fisiologia , Transcrição Gênica , Regulação Alostérica , Núcleo Celular/química , Modelos Biológicos , Miosinas/fisiologia , Proteínas Nucleares/fisiologia , RNA Polimerase II/metabolismo , Ribonucleoproteínas/fisiologiaRESUMO
NAP57 has been found as a component of nuclear matrix protein complex with ability to specifically bind alphoid DNA. Polyclonal antibodies against NAP57 were raised in order to investigate intranuclear localization and interactions of the protein. Two types of localization were observed: a) nucleoplasmic and b) nucleolar. A bulk of nucleoplasmic fraction is present in splicing factors compartments (SFC). The type of localization pattern does not depend on the cell cycle phase, but we revealed changes in NAP57 localization pattern during S phase. According to immunoprecipitation and immunofluorescence assays, NAP57 specifically interacts with DEAD RNA helicase p68 in vitro and co-localizes with helicase p68 in the nucleus of cultured cells. We suppose participation of both proteins in processing of small nuclear RNA on the SFC periphery, and positioning of the nucleolus according to centromere regions of chromosomes.
Assuntos
Núcleo Celular/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Quinases/metabolismo , RNA Helicases/metabolismo , Animais , Linhagem Celular , Nucléolo Celular/metabolismo , RNA Helicases DEAD-box , Imunofluorescência , Células HeLa , Humanos , Imunoprecipitação , Interfase , CamundongosRESUMO
A complex of three proteins (of 80, 70, 58 kDa-p80, p70, and p58, respectively) with the ability to bind alphoid DNA (alpha-satDNA) was revealed by gel mobility shift assay (GMSA) in human nuclear matrix. The probes of the alpha-satDNA bound in the GMSA with the greatest specificity, but the complex was capable of binding human satellite 3 fragment. According to ion exchange and affinity chromatography, the complex includes two DNA-binding proteins, p70 and p80, and a non-DNA-binding one, p58, which enhances the specificity of binding to the alpha-satDNA. GMSA, SDS-PAGE and immunoblotting showed that the lamins, as well as constitutive centromeric proteins (CENP-A, CENP-B, CENP-C, CENP-G), were not incorporated into the complex. It was demonstrated by immunoprecipitation assay that p70 and, probably p58, share a common antigen determinant with the rod domain of intermediate filaments (IF) proteins. The results obtained indicate that the nuclear matrix contains at least one IF-related protein that is able to bind specifically to alpha-satDNA in vitro and that this protein is distinct from the lamins.