A stepwise pathway for biogenesis of 24-nt secondary siRNAs and spreading of DNA methylation
A stepwise pathway for biogenesis of 24-nt secondary siRNAs and spreading of DNA methylation
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DOI：
10.1038/emboj.2008.260
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发表时间：
2009-01-07
期刊：
影响因子：
11.4
通讯作者：
Matzke, Marjori
中科院分区：
文献类型：
--
作者：
Daxinger, Lucia;Kanno, Tatsuo;Matzke, Marjori
我们利用转基因系统研究了拟南芥转录基因沉默过程中RNA指导的DNA甲基化（RdDM）的传播。使用该系统的正向和反向遗传学方法描绘了二级siRNA的生物发生和甲基化从上游增强子元件到下游序列的单向扩散的逐步途径。反式作用的发夹衍生的初级siRNA在靶增强子区域诱导初级RdDM，独立于增强子相关的“新生”RNA。初级RdDM是该途径中的关键步骤，因为它吸引次级siRNA生成机制，包括RNA聚合酶IV、RNA依赖性RNA聚合酶2和Dicer样3（DCL 3）。这些因子在涉及新生RNA的周转途径中起作用，新生RNA通常在非沉默植物中稳定积累，以产生顺式作用的次级siRNA，其诱导下游区域的甲基化。在沉默缺陷突变体的正向遗传筛选中鉴定DCL 3证明了对24-nt siRNA的严格要求以指导甲基化。类似的DNA甲基化扩散的逐步过程可能发生在哺乳动物基因组中，其在上游调控区广泛转录。
We used a transgene system to study spreading of RNA-directed DNA methylation (RdDM) during transcriptional gene silencing in Arabidopsis thaliana. Forward and reverse genetics approaches using this system delineated a stepwise pathway for the biogenesis of secondary siRNAs and unidirectional spreading of methylation from an upstream enhancer element into downstream sequences. Trans-acting, hairpin-derived primary siRNAs induce primary RdDM, independently of an enhancer-associated 'nascent' RNA, at the target enhancer region. Primary RdDM is a key step in the pathway because it attracts the secondary siRNA-generating machinery, including RNA polymerase IV, RNA-dependent RNA polymerase2 and Dicer-like3 (DCL3). These factors act in a turnover pathway involving a nascent RNA, which normally accumulates stably in non-silenced plants, to produce cis-acting secondary siRNAs that induce methylation in the downstream region. The identification of DCL3 in a forward genetic screen for silencing-defective mutants demonstrated a strict requirement for 24-nt siRNAs to direct methylation. A similar stepwise process for spreading of DNA methylation may occur in mammalian genomes, which are extensively transcribed in upstream regulatory regions.