RESUMO
Across kingdoms, RNA interference (RNAi) has been shown to control gene expression at the transcriptional- or the post-transcriptional level. Here, we describe a mechanism which involves both aspects: truncated transgenes, which fail to produce intact mRNA, induce siRNA accumulation and silencing of homologous loci in trans in the ciliate Paramecium We show that silencing is achieved by co-transcriptional silencing, associated with repressive histone marks at the endogenous gene. This is accompanied by secondary siRNA accumulation, strictly limited to the open reading frame of the remote locus. Our data shows that in this mechanism, heterochromatic marks depend on a variety of RNAi components. These include RDR3 and PTIWI14 as well as a second set of components, which are also involved in post-transcriptional silencing: RDR2, PTIWI13, DCR1 and CID2. Our data indicates differential processing of nascent un-spliced and long, spliced transcripts thus suggesting a hitherto-unrecognized functional interaction between post-transcriptional and co-transcriptional RNAi. Both sets of RNAi components are required for efficient trans-acting RNAi at the chromatin level and our data indicates similar mechanisms contributing to genome wide regulation of gene expression by epigenetic mechanisms.
Assuntos
Heterocromatina/metabolismo , Paramecium/genética , Proteínas de Protozoários/genética , Interferência de RNA , RNA de Cadeia Dupla/genética , Transgenes , Montagem e Desmontagem da Cromatina , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Ontologia Genética , Heterocromatina/química , Anotação de Sequência Molecular , Paramecium/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , Polinucleotídeo Adenililtransferase/genética , Polinucleotídeo Adenililtransferase/metabolismo , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/metabolismo , RNA de Cadeia Dupla/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Breast cancer is the most common cancer among women around the world. Human Epidermal growth factor Receptor-2 (HER2) is a membrane tyrosine kinase overexpressed in 30% of human breast cancers; thus, it serves as an important drug target. Currently available HER2 inhibitor lapatinib targets the ATP binding site of the cytoplasmic kinase domain, blocking autophosphorylation and activation of HER-2. However, it causes side effects like diarrhea, nausea, rash and possible liver toxicity. As phytochemicals have fewer side effects and are relatively affordable, they offer an effective alternative. Hence, we aimed to identify potential phytochemicals that could act as HER2 inhibitors employing computational methods such as molecular docking, molecular dynamic simulation, and ADMET prediction. Out of 1500 phytochemicals docked to the ATP binding site of the HER2 kinase domain, luxenchalcone, rhinacanthin Q, subtrifloralacton D, and 7,7â³-dimethyllanaraflavone exhibited higher binding affinity than the reference inhibitor and satisfied the Lipinski's rule of five. Analysis of molecular dynamics simulation trajectory showed that Rhinacanthin Q, subtrifloralacton D, and 7,7â³-dimethyllanaraflavone formed a stable and compact complex without vast conformational fluctuations. MM/PBSA binding free energy analysis revealed that Rhinacanthin Q, subtrifloralacton D, and 7,7â³-dimethyllanaraflavone have high binding affinity to HER2. Therefore, Rhinacanthin Q, subtrifloralacton D, and 7,7â³-dimethyllanaraflavone could be potential bioactive molecules to act as inhibitor of HER2 protein. Eventually, experimental studies are needed to evaluate the potentials of these phytochemicals further. The development of drug for HER2 positive breast cancer could be accelerated with the findings of our research. Communicated by Ramaswamy H. Sarma.