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DNA-RNA hybrid formation mediates RNAi-directed heterochromatin formation

机译：DNA-RNA杂合体形成介导RNAi定向的异染色质形成

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摘要

Certain noncoding RNAs (ncRNAs) implicated in the regulation of chromatin structure associate with chromatin. During the formation of RNAi-directed heterochromatin in fission yeast, ncRNAs transcribed from heterochromatin are thought to recruit the RNAi machinery to chromatin for the formation of heterochromatin; however, the molecular details of this association are not clear. Here, using RNA immunoprecipitation assay, we showed that the heterochromatic ncRNA was associated with chromatin via the formation of a DNARNA hybrid and bound to the RNA-induced transcriptional silencing (RITS) complex. The presence of DNARNA hybrid in the cell was also confirmed by immunofluorescence analysis using anti-DNARNA hybrid antibody. Over-expression and depletion of RNase H in vivo decreased and increased the amount of DNARNA hybrid formed, respectively, and both disturbed heterochromatin. Moreover, DNARNA hybrid was formed on, and over-expression of RNase H inhibited the formation of, artificial heterochromatin induced by tethering of RITS to mRNA. These results indicate that heterochromatic ncRNAs are retained on chromatin via the formation of DNARNA hybrids and provide a platform for the RNAi-directed heterochromatin assembly and suggest that DNARNA hybrid formation plays a role in chromatic ncRNA function.
机译：涉及染色质结构调节的某些非编码RNA（ncRNA）与染色质相关。在裂变酵母中形成RNAi指导的异染色质的过程中，从异染色质转录的ncRNA被认为募集了RNAi机器到染色质中以形成异染色质。但是，这种关联的分子细节尚不清楚。在这里，使用RNA免疫沉淀测定法，我们表明异色ncRNA通过形成DNARNA杂种与染色质相关联，并与RNA诱导的转录沉默（RITS）复合物结合。通过使用抗DNARNA杂合抗体的免疫荧光分析也证实了细胞中DNARNA杂合体的存在。体内RNase H的过度表达和耗竭分别减少和增加了形成的DNARNA杂种的数量，并且都干扰了异染色质。此外，DNARNA杂化物形成于RNAase H上，而RNase H的过度表达则抑制了将RITS束缚于mRNA诱导的人工异染色质的形成。这些结果表明，异色的ncRNA通过形成DNARNA杂种而保留在染色质上，并为RNAi定向的异染色质装配提供了平台，并暗示DNARNA杂种的形成在有色ncRNA功能中起作用。

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