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EMBO J. 2009 Jan 7;28(1):48-57. doi: 10.1038/emboj.2008.260. Epub 2008 Dec 11.

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

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  • 1Gregor Mendel Institute of Molecular Plant Biology, Austrian Academy of Sciences, Vienna, Austria.

Abstract

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.

PMID:
19078964
[PubMed - indexed for MEDLINE]
PMCID:
PMC2633084
Free PMC Article

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