A stepwise pathway for biogenesis of 24-nt secondary siRNAs and spreading of DNA methylation

作者：Daxinger Lucia; Kanno Tatsuo; Bucher Etienne; van der Winden Johannes; Naumann Ulf; Matzke Antonius J M; Matzke Marjori

来源：The EMBO Journal, 2009, 28(1): 48-57.

DOI：10.1038/emboj.2008.260

• Dicer-like3
• methylation spreading
• Pol IV
• RNA-directed DNA methylation
• secondary siRNAs

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.

• 出版日期2009-1-7