RESUMO
BACKGROUND: Heterochromatin is the tightly packaged dynamic region of the eukaryotic chromosome that plays a vital role in cellular processes such as mitosis and meiotic recombination. Recent experiments in Schizosaccharomyces pombe have revealed the structure of centromeric heterochromatin is affected in RNAi pathway mutants. It has also been shown in fission yeast that the heterochromatin barrier is traversed by RNA Pol II and that the passage of RNA Pol II through heterochromatin is important for heterochromatin structure. Thus, an intricate interaction between the RNAi machinery and RNA Pol II affects heterochromatin structure. However, the role of the RNAi machinery and RNA Pol II on the metazoan heterochromatin landscape is not known. This study analyses the interaction of the small RNA machinery and RNA Pol II on Drosophila heterochromatin structure. RESULTS: The results in this paper show genetic and biochemical interaction between RNA Pol II (largest and second largest subunit) and small RNA silencing machinery components (dcr-2, ago1, ago2, piwi, Lip [D], aub and hls). Immunofluorescence analysis of polytene chromosomes from trans-heterozygotes of RNA Pol II and different mutations of the small RNA pathways show decreased H3K9me2 and mislocalization of Heterochromatin protein-1. A genetic analysis performed on these mutants showed a strong suppression of white-mottled4h position effect variegation. This was further corroborated by a western blot analysis and chromatin immunoprecipitation, which showed decreased H3K9me2 in trans-heterozygote mutants compared to wild type or single heterozygotes. Co-immunoprecipitation performed using Drosophila embryo extracts showed the RNA Pol II largest subunit interacting with Dcr-2 and dAGO1. Co-localization performed on polytene chromosomes showed RNA Pol II and dAGO1 overlapping at some sites. CONCLUSION: Our experiments show a genetic and biochemical interaction between RNA Pol II (largest and second largest subunits) and the small RNA silencing machinery in Drosophila. The interaction has functional aspects in terms of determining H3K9me2 and HP-1 deposition at the chromocentric heterochromatin. Thus, RNA Pol II has an important role in establishing heterochromatin structure in Drosophila.
RESUMO
BACKGROUND: CG11033 (dKDM2) is the Drosophila homolog of the gene KDM2B. dKDM2 has been known to possess histone lysine demethylase activity towards H3K36me2 in cell lines and it regulates H2A ubiquitination. The human homolog of the gene has dual activity towards H3K36me2 as well as H3K4me3, and plays an important role in cellular senescence. FINDINGS: We have used transgenic flies bearing an RNAi construct for the dKDM2 gene. The knockdown of dKDM2 gene was performed by crossing UAS-RNAi-dKDM2 flies with actin-Gal4 flies. Western blots of acid extracted histones and immunofluoresence analysis of polytene chromosome showed the activity of the enzyme dKDM2 to be specific for H3K4me3 in adult flies. Immunofluoresence analysis of polytene chromosome also revealed the presence of multiple nucleoli in RNAi knockdown mutants of dKDM2 and decreased H3-acetylation marks associated with active transcription. CONCLUSION: Our findings indicate that dKDM2 is a histone lysine demethylase with specificity for H3K4me3 and regulates nucleolar organization.
Assuntos
Drosophila melanogaster/genética , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Animais , Proteínas Argonautas , Elementos de DNA Transponíveis , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo , Fatores de Iniciação em Eucariotos , Células Germinativas/metabolismo , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Mutação , RNA Helicases/genética , RNA Helicases/metabolismo , Interferência de RNA , Complexo de Inativação Induzido por RNA/genética , Complexo de Inativação Induzido por RNA/metabolismo , Ribonuclease III/metabolismoRESUMO
RNA interference (RNAi) is the technique employing double-stranded RNA to target the destruction of homologous messenger RNAs. It has gained wide usage in genetics. While having the potential for many practical applications, it is a reflection of a much broader spectrum of small RNA-mediated processes in the cell. The RNAi machinery was originally perceived as a defense mechanism against viruses and transposons. While this is certainly true, small RNAs have now been implicated in many other aspects of cell biology. Here we review the current knowledge of the biochemistry of RNAi in Drosophila and the involvement of small RNAs in RNAi, transposon silencing, virus defense, transgene silencing, pairing-sensitive silencing, telomere function, chromatin insulator activity, nucleolar stability, and heterochromatin formation. The discovery of the role of RNA molecules in the degradation of mRNA transcripts leading to decreased gene expression resulted in a paradigm shift in the field of molecular biology. Transgene silencing was first discovered in plant cells (Matzke et al. 1989; van der Krol et al. 1990; Napoli et al. 1990) and can occur on both the transcriptional and posttranscriptional levels, but both involve short RNA moieties in their mechanism. RNA interference (RNAi) is a type of gene silencing mechanism in which a double-stranded RNA (dsRNA) molecule directs the specific degradation of the corresponding mRNA (target RNA). The technique of RNAi was first discovered in Caenorhabditis elegans in 1994 (Guo and Kemphues 1994). Later the active component was found to be a dsRNA (Fire et al. 1998). In subsequent years, it has been found to occur in diverse eukaryotes
Assuntos
Drosophila/genética , Interferência de RNA , Animais , Nucléolo Celular/química , Nucléolo Celular/metabolismo , Reparo do DNA , Elementos de DNA Transponíveis/genética , Drosophila/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Heterocromatina/genética , RNA Interferente Pequeno/química , RNA Interferente Pequeno/metabolismo , Complexo de Inativação Induzido por RNA/química , Complexo de Inativação Induzido por RNA/metabolismo , Telômero/genéticaRESUMO
In Drosophila, the RNA interference (RNAi) genes participate in Polycomb (Pc)-mediated transgene silencing. Recently, the involvement of the RNAi genes in Pc silencing, pairing-sensitive silencing and long-range contacts among Pc-associated sequences has been explored. These Pc-associated sequences are involved with the control of the proper expression of developmental HOX genes.
Assuntos
Pareamento Cromossômico , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Proteínas/metabolismo , Interferência de RNA , Animais , Proteínas Argonautas , Núcleo Celular/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/metabolismo , Modelos Genéticos , Complexo Repressor Polycomb 1 , Complexo de Inativação Induzido por RNARESUMO
The sex chromosomes of many species differ in dosage but the total gene expression output is similar, a phenomenon referred to as dosage compensation. Previously, diverse mechanisms were postulated to account for compensation in distantly related taxa. However, two recent papers present evidence that dosage compensation in Drosophila, mammals and nematodes share the property that there is an approximately two-fold upregulation of the single active X chromosome in each case.(1,2) The results suggest that a common mechanism might operate in these different cases.
Assuntos
Cromossomos Sexuais/genética , Animais , Cromatina/genética , Genoma/genética , HumanosRESUMO
The RNAi machinery is not only involved with post-transcriptional degradation of messenger RNAs, but also used for targeting of chromatin changes associated with transcriptional silencing. Two recent papers determine the global patterns of gene expression and chromatin modifications produced by the RNAi machinery in fission yeast.(9, 10) The major sites include the outer centromere repeats, the mating-type locus and subtelomeric regions. By comparison, studies of Arabidopsis heterochromatin also implicate transposons as a major target for silencing. Analyses of siRNA libraries from Drosophila, nematodes and Arabidopsis indicate that major repeats at centromeres, telomeres and transposable elements are likely targets of RNAi. Also, intergenic regions are implicated as targets in Arabidopsis.
Assuntos
Inativação Gênica , RNA Interferente Pequeno/metabolismo , Transcrição Gênica/genética , Animais , Elementos de DNA Transponíveis/genética , Perfilação da Expressão Gênica , Heterocromatina/genética , RNA Interferente Pequeno/genéticaRESUMO
Knowledge of the role of RNA in affecting gene expression has expanded in the past several years. Small RNAs serve as homology guides to target messenger RNAs for destruction at the post-transcriptional level in the experimental technique known as RNA interference and in the silencing of some transgenes. These small RNAs are also involved in sequence-specific targeting of chromatin modifications for transcriptional silencing of transgenes, transposable elements, heterochromatin and some cases of Polycomb-mediated gene silencing. RNA silencing processes in Drosophila are described.
Assuntos
Drosophila melanogaster/fisiologia , Regulação da Expressão Gênica , Interferência de RNA , RNA Interferente Pequeno , Animais , Cromatina/metabolismo , DNA/genética , Reparo do DNA , Elementos de DNA Transponíveis , Drosophila melanogaster/genética , Inativação Gênica , Heterocromatina/química , Modelos Biológicos , Modelos Genéticos , Processamento de Proteína Pós-Traducional , RNA/química , RNA/genética , Edição de RNA , RNA Mensageiro/metabolismo , Transcrição Gênica , TransgenesRESUMO
Mutation of the multi-KH domain protein DPP1, which has single-stranded nucleic acid binding activity, suppresses heterochromatin-mediated silencing in Drosophila; it also disrupts the modification of histone H3 at lysine 9, and association of heterochromatin protein 1 on the heterochromatic regions, suggesting a role for DDP1 in heterochromatin formation.