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Interaction of RNA polymerase II and the small RNA machinery affects heterochromatic silencing in Drosophila

机译：RNA聚合酶II和小型RNA机制的相互作用影响果蝇中的异色沉默

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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.
机译：背景异染色质是真核染色体紧密包装的动态区域，在诸如有丝分裂和减数分裂重组等细胞过程中起着至关重要的作用。最近在粟酒裂殖酵母中的实验表明，着丝粒异染色质的结构在RNAi途径突变体中受到影响。在裂变酵母中还显示出异染色质屏障被RNA Pol II穿过，RNA Pol II通过异染色质对于异染色质结构很重要。因此，RNAi机制与RNA Pol II之间的复杂相互作用会影响异染色质结构。但是，尚不清楚RNAi机制和RNA Pol II在后生异染色质景观上的作用。这项研究分析了小RNA机制和RNA Pol II对果蝇异染色质结构的相互作用。结果本文的结果显示了RNA Pol II（最大和第二大亚基）与小的RNA沉默机制组件（dcr-2，ago1，ago2，piwi，Lip [D]，aub和hls）之间的遗传和生化相互作用。从RNA Pol II的反杂合体和小RNA途径的不同突变中提取的多聚烯染色体的免疫荧光分析显示，H3K9me2的减少和异染色质蛋白-1的定位错误。对这些突变体进行的遗传分析显示，强烈抑制了白斑驳的4h位置效应变异。 Western印迹分析和染色质免疫沉淀进一步证实了这一点，与野生型或单杂合子相比，反杂合子突变体中的H3K9me2减少。使用果蝇胚胎提取物进行的共免疫沉淀表明，RNA Pol II最大的亚基与Dcr-2和dAGO1相互作用。在聚丙烯染色体上进行的共定位显示RNA Pol II和dAGO1在某些位点重叠。结论我们的实验表明果蝇中的RNA Pol II（最大和第二大亚基）与小型RNA沉默机制之间存在遗传和生化相互作用。在确定H3K9me2和HP-1在发色中心异染色质上的沉积方面，该相互作用具有功能方面。因此，RNA Pol II在果蝇中建立异染色质结构中具有重要作用。

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