Laboratory of Bioorganic Chemistry, Department of Chemistry, Hokkaido University, Sapporo, Japan.
Genes Dev. 2012 Aug 15;26(16):1811-24. doi: 10.1101/gad.190272.112.
In fission yeast, siRNA is generated from pericentromeric noncoding RNA by the RNAi machinery. siRNA synthesis and heterochromatin formation are interdependent, forming a self-reinforcing loop on chromatin. In this system, siRNA is amplified by the RNA-dependent RNA polymerase complex (RDRC) and the endoribonuclease Dcr1, which synthesizes dsRNA and processes the dsRNA, respectively. The amplification is essential for stable heterochromatin formation. Here, a novel gene, dsh1(+) (defect of the gene silencing at centromeric heterochromatin), is identified as an essential component of RNAi-directed heterochromatin assembly. Loss of dsh1(+) abolishes normal RNAi function and heterochromatic gene silencing at pericentromeres. Dsh1 interacts with Dcr1 and RDRC and couples the reactions of both proteins to the effective production of siRNA in vivo. Dsh1 binds to heterochromatin in the absence of RDRC, while RDRC requires Dsh1 for its chromatin-binding activity, suggesting that Dsh1 recruits RDRC to chromatin. Immunofluorescence analysis shows that Dsh1 forms foci at the nuclear periphery, and some Dsh1 foci colocalize with Dcr1 and RDRC. Dsh1 is required for the colocalization of Dcr1 and RDRC. Moreover, loss of the nuclear periphery localization of Dsh1 abolishes Dsh1 function. Taken together, these results suggest that Dsh1 assembles the RNAi machinery on heterochromatin and forms a perinuclear compartment for amplification of heterochromatic siRNA.
在裂殖酵母中，siRNA 是由 RNAi 机制从着丝粒周围的非编码 RNA 产生的。siRNA 的合成和异染色质的形成是相互依赖的，在染色质上形成一个自我强化的循环。在这个系统中，siRNA 由 RNA 依赖性 RNA 聚合酶复合物（RDRC）和内切核酸酶 Dcr1 扩增，它们分别合成 dsRNA 和加工 dsRNA。扩增对于稳定的异染色质形成是必不可少的。在这里，鉴定了一个新基因 dsh1(+)（着丝粒异染色质基因沉默缺陷），它是 RNAi 指导的异染色质组装的必需组成部分。dsh1(+)的缺失会导致正常的 RNAi 功能丧失和着丝粒周围异染色质基因沉默。Dsh1 与 Dcr1 和 RDRC 相互作用，并将这两种蛋白质的反应与体内有效产生 siRNA 偶联。在没有 RDRC 的情况下，Dsh1 与异染色质结合，而 RDRC 需要 Dsh1 才能发挥其染色质结合活性，表明 Dsh1 将 RDRC 招募到染色质上。免疫荧光分析表明，Dsh1 在核周形成焦点，并且一些 Dsh1 焦点与 Dcr1 和 RDRC 共定位。Dsh1 对于 Dcr1 和 RDRC 的共定位是必需的。此外，Dsh1 的核周定位缺失会使 Dsh1 功能丧失。综上所述，这些结果表明 Dsh1 将 RNAi 机制组装在异染色质上，并形成一个核周隔室，用于扩增异染色质 siRNA。
