RESUMO
RNA interference (RNAi) is a widespread gene-silencing mechanism and is required for heterochromatin assembly in a variety of organisms. The RNA-induced transcriptional silencing complex (RITS), composed of Ago1, Tas3 and Chp1, is a key component of RNAi machinery in fission yeast that connects short interference RNA (siRNA) and heterochromatin formation. However, the process by which RITS is assembled is not well understood. Here, we identified Sgf73, a subunit of the SAGA co-transcriptional complex, is required for pericentromeric heterochromatin silencing and the generation of siRNA. This novel role of Sgf73 is independent of enzymatic activities or structural integrity of SAGA. Instead, Sgf73 is physically associated with Ago1 and Chp1. The interactions among the subunits of the RITS, including those between Tas3 and Chp1, between Chp1 and Ago1, between Ago1 and Tas3, were all impaired by the deletion of sgf73(+). Consistently, the recruitment of Ago1 and Chp1 to the pericentromeric region was abolished in sgf73Δ cells. Our study unveils a moonlighting function of a SAGA subunit. It suggests Sgf73 is a novel factor that promotes assembly of RITS and RNAi-mediated heterochromatin formation.
Assuntos
Acetiltransferases/metabolismo , Proteínas Argonautas/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Complexo de Inativação Induzido por RNA/química , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Acetiltransferases/genética , Proteínas Argonautas/genética , Proteínas de Transporte/genética , Proteínas de Ciclo Celular/genética , Núcleo Celular/genética , Centrômero/genética , Montagem e Desmontagem da Cromatina/genética , Imunoprecipitação da Cromatina , Heterocromatina/genética , Imunoprecipitação , RNA Fúngico/genética , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Schizosaccharomyces/genética , Schizosaccharomyces/crescimento & desenvolvimento , Proteínas de Schizosaccharomyces pombe/genéticaRESUMO
The deregulation of microRNAs has been demonstrated in various tumor processes. Here, we report that microRNA-544 (miR-544) is decreased in cervical cancer tissues compared with normal cervical tissues. To identify the mechanisms involved in miR-544 deregulation, we studied the regulation of miR-544 expression at the transcriptional level. We first identified the transcriptional start site of miR-544 by 5' rapid amplification of cDNA ends and subsequently determined the miR-544 promoter. We discovered that the transcription factor Krueppel-like factor 4 (KLF4) is involved in the transcriptional regulation of miR-544 through interaction with the miR-544 promoter. In addition, we found that miR-544 directly targets the YWHAZ oncogene and functions as a tumor suppressor in cervical cancer cells. miR-544 is involved in cell cycle regulation and suppresses cervical cancer cell proliferation, colony formation, migration and invasion in a manner associated with YWHAZ downregulation. In summary, our findings demonstrate that KLF4 upregulates miR-544 transcription by activating the miR-544 promoter and that miR-544 functions as a tumor suppressor by targeting YWHAZ. Therefore, miR-544 may be a potential novel therapeutic target and prognostic marker for cervical cancer.
RESUMO
MicroRNAs (miRNAs) play important roles in various biological processes and are closely associated with the development of cancer. In fact, aberrant expression of miRNAs has been implicated in numerous cancers. In cervical cancer, miR-203 levels are decreased, although the cause of this aberrant expression remains unclear. In this study, we investigate the molecular mechanisms regulating miR-203 gene transcription. We identify the miR-203 transcription start site by 5' rapid amplification of cDNA ends and subsequently identify the miR-203 promoter region. Promoter analysis revealed that IRF1, a transcription factor, regulates miR-203 transcription by binding to the miR-203 promoter. We also demonstrate that miR-203 targets the 3' untranslated region of BANF1, thus downregulating its expression, whereas miR-203 expression is driven by IRF1. MiR-203 is involved in cell cycle regulation and overexpression of miR-203 suppresses cervical cancer cell proliferation, colony formation, migration and invasion. The inhibitory effect of miR-203 on the cancer cells is partially mediated by downregulating its target, BANF1, since knockdown of BANF1 also suppresses colony formation, migration and invasion.
Assuntos
Proteínas de Ligação a DNA/genética , Regulação para Baixo , Fator Regulador 1 de Interferon/metabolismo , MicroRNAs/metabolismo , Proteínas Nucleares/genética , Neoplasias do Colo do Útero/genética , Regiões 3' não Traduzidas , Animais , Sequência de Bases , Sítios de Ligação , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/metabolismo , Feminino , Células HeLa , Humanos , Fator Regulador 1 de Interferon/química , MicroRNAs/genética , Dados de Sequência Molecular , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , RNA Interferente Pequeno/metabolismo , Alinhamento de Sequência , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo , Neoplasias do Colo do Útero/patologiaRESUMO
Human serum albumin (HSA) is the major protein component of human plasma. To date, HSA for clinical uses is mostly produced by fractionation of human whole blood, which is accompanied by a lot of limitations. To obtain long-term bioactive albumin, we used hsa as a foreign gene and constructed a recombinant plasmid pJS700-HSA which carries a recombinant gene cotC-hsa under the control of cotC promoter. Plasmid pJS700-HSA was transformed into Bacillus subtilis by double cross-over and an amylase inactivated mutant was produced. After induction of spore formation, western blot and fluorescence immunoassay were used to monitor HSA surface expression on spores. We estimated that HSA displayed on the spore accounted for 0.135 % of the total spore proteins and about 0.023 fg HSA were exposed on the surface of each spore. Oral administration to mice with spores displaying HSA implied that the recombinant spores may have potential ability to increase the serum albumin level in vivo due to the resistant characters of spores.
Assuntos
Bacillus subtilis/metabolismo , Expressão Gênica , Albumina Sérica/metabolismo , Esporos Bacterianos/metabolismo , Administração Oral , Animais , Bacillus subtilis/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos ICR , Plasmídeos , Regiões Promotoras Genéticas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Albumina Sérica/genética , Albumina Sérica Humana , Esporos Bacterianos/genéticaRESUMO
A spectrophotometric method for the detection of tyrosinase activity is developed by utilizing the product-triggered aggregation of boronic acid-functionalized gold nanoparticles. Based on the changes of absorbance in UV-visible spectra, the assay shows extremely high sensitivity and lowered limit of detection of 1 × 10(-10) u mL(-1).
Assuntos
Nanopartículas Metálicas/química , Monofenol Mono-Oxigenase/análise , Espectrofotometria Ultravioleta/métodos , Ácidos Borônicos , Ouro/química , Monofenol Mono-Oxigenase/químicaRESUMO
Natural wild-type strains of Bacillus subtilis spore is regarded as a non-pathogenic for both human and animal, and has been classified as a novel food which is currently being used as probiotics added in the consumption. To identify B. subtilis spore proteins, we have accomplished a preliminary proteomic analysis of B. subtilis spore, with a combination of two-dimensional electrophoretic separations and matrix-assisted laser desorption ionization tandem time of flight mass spectrometry (MALDI-TOF-MS). In this article, we presented a reference map of 158 B. subtilis spore proteins with an isoelectric point (pI) between 4 and 7. Followed by mass spectrometry (MS) analysis, we identified 71 B. subtilis spore proteins with high level of confidence. Database searches, combined with hydropathy analysis and GO analysis revealed that most of the B. subtilis spore proteins were hydrophilic proteins related to catalytic function. These results should accelerate efforts to understand the resistance of spore to harsh conditions.
