RESUMO
Luciferase reporter assays are widely used to study promoter activity, transcription factors, intracellular signaling, protein interactions (Jia et al., PloS One 6:e26414), miRNA processing (Allegra and Mertens, Biochem Biophys Res Commun 406:501-505), and target recognition (Jin et al., Methods Mol Biol 936:117-127). Here we describe the use of a dual-luciferase reporter system to evaluate the enzymatic activity of a key enzyme involved in RNA maturation-DROSHA. This dual system is a simple and fast method for the quantification of the DROSHA processing activity in live cells.
Assuntos
Ensaios Enzimáticos/métodos , Genes Reporter/genética , Luciferases/genética , Ribonuclease III/metabolismo , Regiões 3' não Traduzidas/genética , Adesão Celular , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Sequências Repetidas Invertidas , MicroRNAs/genética , MicroRNAs/metabolismo , Plasmídeos/genética , Clivagem do RNA , Precursores de RNA/genética , Precursores de RNA/metabolismo , RNA Interferente Pequeno/genética , Ribonuclease III/deficiência , Ribonuclease III/genética , TransfecçãoRESUMO
Non-coding RNAs are much more common than previously thought. However, for the vast majority of non-coding RNAs, the cellular function remains enigmatic. The two long non-coding RNA (lncRNA) genes DLEU1 and DLEU2 map to a critical region at chromosomal band 13q14.3 that is recurrently deleted in solid tumors and hematopoietic malignancies like chronic lymphocytic leukemia (CLL). While no point mutations have been found in the protein coding candidate genes at 13q14.3, they are deregulated in malignant cells, suggesting an epigenetic tumor suppressor mechanism. We therefore characterized the epigenetic makeup of 13q14.3 in CLL cells and found histone modifications by chromatin-immunoprecipitation (ChIP) that are associated with activated transcription and significant DNA-demethylation at the transcriptional start sites of DLEU1 and DLEU2 using 5 different semi-quantitative and quantitative methods (aPRIMES, BioCOBRA, MCIp, MassARRAY, and bisulfite sequencing). These epigenetic aberrations were correlated with transcriptional deregulation of the neighboring candidate tumor suppressor genes, suggesting a coregulation in cis of this gene cluster. We found that the 13q14.3 genes in addition to their previously known functions regulate NF-kB activity, which we could show after overexpression, siRNA-mediated knockdown, and dominant-negative mutant genes by using Western blots with previously undescribed antibodies, by a customized ELISA as well as by reporter assays. In addition, we performed an unbiased screen of 810 human miRNAs and identified the miR-15/16 family of genes at 13q14.3 as the strongest inducers of NF-kB activity. In summary, the tumor suppressor mechanism at 13q14.3 is a cluster of genes controlled by two lncRNA genes that are regulated by DNA-methylation and histone modifications and whose members all regulate NF-kB. Therefore, the tumor suppressor mechanism in 13q14.3 underlines the role both of epigenetic aberrations and of lncRNA genes in human tumorigenesis and is an example of colocalization of a functionally related gene cluster.
Assuntos
Metilação de DNA/genética , Leucemia , RNA Longo não Codificante , Proteínas Supressoras de Tumor , Adulto , Idoso , Idoso de 80 Anos ou mais , Transformação Celular Neoplásica , Cromatina/genética , Cromossomos Humanos Par 13/genética , Regulação para Baixo , Epigênese Genética/genética , Feminino , Células HEK293 , Humanos , Leucemia/sangue , Leucemia/genética , Leucemia/fisiopatologia , Masculino , Pessoa de Meia-Idade , Mutação , NF-kappa B/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Sítio de Iniciação de Transcrição , Transferases , Proteínas Supressoras de Tumor/sangue , Proteínas Supressoras de Tumor/genética , Regulação para CimaRESUMO
The RNAse III Drosha is responsible for the first step of microRNA maturation, the cleavage of primary miRNA to produce the precursor miRNA. Processing by Drosha is finely regulated and influences the amount of mature microRNA in a cell. We describe in the present work a method to quantify Drosha processing activity in-vivo, which is applicable to any microRNA. With respect to other methods for measuring Drosha activity, our system is faster and scalable, can be used with any cellular system and does not require cell sorting or use of radioactive isotopes. This system is useful to study regulation of Drosha activity in physiological and pathological conditions.
Assuntos
MicroRNAs/análise , MicroRNAs/metabolismo , Processamento Pós-Transcricional do RNA , Ribonuclease III/metabolismo , Genes Reporter , Células HEK293 , Humanos , Luciferases/análise , Luciferases/genética , Métodos , MicroRNAs/química , Ribonuclease III/genéticaRESUMO
Loss of a critical region in 13q14.3 [del(13q)] is the most common genomic aberration in chronic lymphocytic leukemia (CLL), occurring in more than 50% of patients (Stilgenbauer et al., Oncogene 1998;16:1891 - 1897, Dohner et al., N Engl J Med 2000;343:1910 - 1916). Despite extensive investigations, no point mutations have been found in the remaining allele that would inactivate one of the candidate tumor suppressor genes and explain the pathomechanism postulated for this region. However, the genes in the region are significantly down-regulated in CLL cells, more than would be expected by gene dosage, and recently a complex epigenetic regulatory mechanism was identified for 13q14.3 in non-malignant cells that involves asynchronous replication timing and monoallelic expression of candidate tumor suppressor genes. Here, we propose a model of a multigenic pathomechanism in 13q14.3, where several tumor suppressor genes, including the miRNA genes miR-16-1 and miR-15a, are co-regulated by the two long non-coding RNA genes DLEU1 and DLEU2 that span the critical region. Furthermore, we propose these co-regulated genes to be involved in the same molecular pathways, thereby also forming a functional gene cluster. Elucidating the molecular and cellular function of the 13q14.3 candidate genes will shed light on the underlying pathomechanism of CLL.
Assuntos
Cromossomos Humanos Par 13 , Leucemia Linfocítica Crônica de Células B/genética , MicroRNAs/genética , Proteínas Supressoras de Tumor/genética , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Humanos , Leucemia Linfocítica Crônica de Células B/etiologia , Modelos Biológicos , RNA Longo não Codificante , TransferasesRESUMO
The identification of protein-protein interaction networks has often given important information about the functions of specific proteins and on the cross-talk among metabolic and regulatory pathways. The availability of entire genome sequences has rendered feasible the systematic screening of collections of proteins, often of unknown function, aimed to find the cognate ligands. Once identified by genetic and/or biochemical approaches, the interaction between two proteins should be validated in the physiologic environment. Herein we describe an experimental strategy to screen collections of protein-protein interaction domains to find and validate candidate interactors. The approach is based on the assumption that the overexpression in cultured cells of protein-protein interaction domains, isolated from the context of the whole protein, could titrate the endogenous ligand and, in turn, exert a dominant negative effect. The identification of the ligand could provide us with a tool to check the relevance of the interaction because the contemporary overexpression of the isolated domain and of its ligand could rescue the dominant negative phenotype. We explored this approach by analyzing the possible dominant negative effects on the cell cycle progression of a collection of phosphotyrosine binding (PTB) domains of human proteins. Of 47 PTB domains, we found that the overexpression of 10 of them significantly interfered with the cell cycle progression of NIH3T3 cells. Four of them were used as baits to identify the cognate interactors. Among these proteins, CARM1, interacting with the PTB domain of RabGAP1, and EF1alpha, interacting with RGS12, were able to rescue the block of the cell cycle induced by the isolated PTB domain of the partner protein, thus confirming in vivo the relevance of the interaction. These results suggest that the described approach can be used for the systematic screening of the ligands of various protein-protein interaction domains also by using different biological assays.
Assuntos
Ligantes , Mapeamento de Interação de Proteínas/métodos , Estrutura Terciária de Proteína , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Ciclo Celular , Linhagem Celular , Proteínas do Citoesqueleto/metabolismo , Fibroblastos/metabolismo , Humanos , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Camundongos , Complexos Multiproteicos/metabolismo , Células NIH 3T3 , Fosfotirosina/metabolismo , Proteínas RGS/metabolismo , Receptores de LDL/metabolismo , Proteínas Supressoras de Tumor/metabolismoRESUMO
The molecular adaptor Fe65 is one of the cytosolic ligands of the Alzheimer's beta-amyloid precursor protein (APP), and this complex is believed to play important roles in mammalian cells. Upon cleavage of APP by specific processing activities, the complex between Fe65 and the APP intracellular domain (AICD) translocates to the nucleus. Experimental evidence suggests that the Fe65-AICD complex regulates gene transcription. In Caenorhabditis elegans the orthologue of the Fe65 gene, feh-1, regulates pharyngeal activity. In fact, the rate of pharyngeal contraction is increased following transient or stable suppression of the feh-1 gene expression. Here we show that the increased contraction rate of the pharynx in feh-1 mutant worms is associated to decreased acetylcholinesterase activity. The decreased activity is accompanied by reduced expression of ace-1 and ace-2 transcripts, coding for the two major acetylcholinesterase activities in the nematode. These results indicate a target of the regulatory mechanisms based on the Fe65-APP complex that could be relevant for the pathogenesis of Alzheimer's disease.