RESUMO
Gene silencing by RNA interference (RNAi) has proven to be a powerful tool for investigating gene function in mammalian cells. Combination of several short interfering RNA (siRNA) targeting the same gene is commonly used to improve RNA interference. However, in contrary to the well-described mechanism of RNAi, efficiency of single siRNA compared to pool remains poorly documented. We addressed this issue using several active and inactive siRNA targeting Eg5, a kinesin-related motor involved in mitotic spindle assembly. These siRNA, used alone or in combination, were tested for their silencing efficiency in several cancer cell lines. Here we show that presence of inactive Eg5 siRNA in a pool dramatically decreases knockdown efficacy in a cell line- and dose-dependent manner. Lack of inhibition by unrelated siRNA suggests that a competition may occur during siRNA incorporation into RNA-induced silencing complexes (RISCs) along with the target mRNA. Altogether, our results, which need to be confirmed with additional inactive siRNA, indicate that combination of siRNA may not increase but instead decrease silencing efficiency.
Assuntos
Cinesinas/antagonistas & inibidores , Cinesinas/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Sequência de Bases , Linhagem Celular Tumoral , DNA Complementar/genética , Humanos , Mitose/efeitos dos fármacos , Mitose/genética , Interferência de RNA , RNA Interferente Pequeno/metabolismo , TransfecçãoRESUMO
Histone acetylation and deacetylation are chromatin-modifying processes that have fundamental importance for transcriptional regulation. Transcriptionally active chromatin regions show a high degree of histone acetylation, whereas deacetylation events are generally linked to transcriptional silencing. Many of the acetylating and deacetylating enzymes were originally identified as transcriptional coactivators or repressors. Their histone-modifying enzymatic activity was discovered more recently, opening up a whole new area of research. Histone acetyltransferases such as CREB-binding protein (CBP) and PCAF are involved in processes as diverse as promoting cell cycle progression and regulating differentiation. A controlled balance between histone acetylation and deacetylation seems to be essential for normal cell growth. Both histone acetyltransferases and deacetylases are involved in the development of diseases, including neurodegenerative disorders and cancer. Treatments that target these enzymes are already under clinical investigation.
Assuntos
Acetiltransferases/fisiologia , Diferenciação Celular/fisiologia , Divisão Celular/fisiologia , Histona Desacetilases/fisiologia , Histonas/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Acetilação , Acetiltransferases/genética , Animais , Ciclo Celular/fisiologia , Transformação Celular Neoplásica/genética , Cromatina/metabolismo , Cromatina/ultraestrutura , Inativação Gênica/fisiologia , Hematopoese , Histona Acetiltransferases , Histona Desacetilases/genética , Humanos , Doença de Huntington/metabolismo , Camundongos , Família Multigênica , Proteínas Musculares/fisiologia , Proteínas de Neoplasias/fisiologia , Neoplasias/enzimologia , Processamento de Proteína Pós-Traducional , Proteínas de Saccharomyces cerevisiae/genética , Transcrição Gênica/fisiologiaRESUMO
We describe a system designed to express biotinylated proteins in mammalian cells in vivo and its application to the study of protein-DNA interactions in vivo by chromatin immunoprecipitation (ChIP). The system is based on coexpression of the target protein fused to a short biotin acceptor domain together with the biotinylating enzyme BirA from Escherichia coli. The superior strength of the biotin-avidin interaction allows one to employ more stringent washing conditions in the ChIP protocol, resulting in a better signal/noise ratio.
Assuntos
Biotinilação , Carbono-Nitrogênio Ligases/química , Cromatina/química , Proteínas de Escherichia coli/química , Testes de Precipitina , Proteínas Repressoras/química , Fatores de Transcrição/química , Animais , Carbono-Nitrogênio Ligases/genética , Proteínas de Ciclo Celular/genética , Cromatina/genética , Cromatina/imunologia , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona/genética , Proteínas de Ligação a DNA/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Fluorescência Verde , Células HeLa , Humanos , Imunoadsorventes , Proteínas Luminescentes/genética , Camundongos , Células NIH 3T3 , Proteínas Nucleares/química , Proteínas Nucleares/genética , Regiões Promotoras Genéticas/genética , Ligação Proteica/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Repressoras/genética , Fator de Transcrição DP1 , Fatores de Transcrição/genética , Transdução GenéticaRESUMO
Single base pair mutations that alter the function of tumor suppressor genes and oncogenes occur frequently during oncogenesis. The guardian of the genome, p53, is inactivated by point mutation in more than 50% of human cancers. Synthetic small inhibiting RNAs (siRNAs) can suppress gene expression in mammalian cells, although their degree of selectivity might be compromised by an amplification mechanism. Here, we demonstrate that a single base difference in siRNAs discriminates between mutant and WT p53 in cells expressing both forms, resulting in the restoration of WT protein function. Therefore, siRNAs may be used to suppress expression of point-mutated genes and provide the basis for selective and personalized antitumor therapy.