DNA-RNA hybrid formation mediates RNAi-directed heterochromatin formation
DNA-RNA hybrid formation mediates RNAi-directed heterochromatin formation
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DOI：
10.1111/j.1365-2443.2012.01583.x
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发表时间：
2012-03-01
期刊：
影响因子：
2.1
通讯作者：
Murakami, Yota
中科院分区：
文献类型：
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作者：
Nakama, Mina;Kawakami, Kei;Murakami, Yota
某些参与染色质结构调节的非编码RNA（ncRNA）与染色质相关。在分裂酵母中RNAi指导的异染色质形成期间，从异染色质转录的ncRNA被认为将RNAi机制募集到染色质以形成异染色质;然而，这种关联的分子细节尚不清楚。在这里，使用RNA免疫沉淀试验，我们表明，异染色质ncRNA通过形成DNARNA杂交体与染色质结合，并结合到RNA诱导的转录沉默（RITS）复合物。细胞中DNARNA杂合体的存在也通过使用抗DNARNA杂合体抗体的免疫荧光分析来证实。体内RNase H的过度表达和缺失分别减少和增加DNARNA杂交体形成的量，并且都扰乱异染色质。此外，DNARNA杂合的形成，并过度表达RNase H抑制，人工异染色质的形成诱导的RITS的mRNA的束缚。这些结果表明异染色质ncRNA通过DNARNA杂交体的形成保留在染色质上，并为RNAi指导的异染色质组装提供了平台，并表明DNARNA杂交体的形成在染色质ncRNA功能中起作用。
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.