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  • The EMBO Journal (2009) 28, 48 - 57
  • doi:10.1038/emboj.2008.260

Published online: 11 December 2008

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

Lucia Daxinger1,a, Tatsuo Kanno1,a, Etienne Bucher1,b, Johannes van der Winden1, Ulf Naumann1, Antonius J M Matzke1 and Marjori Matzke1

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

Correspondence to:

Marjori Matzke, Gregor Mendel Institute of Molecular Plant Biology, Austrian Academy of Sciences, Dr Bohr-Gasse 3, Vienna A-1030, Austria. Tel.: +43 1 79044 9810; Fax: +43 1 79044 9800; E-mail: marjori.matzke@gmi.oeaw.ac.at

aThese authors contributed equally to this work

bPresent address: Laboratory of Plant Genetics, University of Geneva, CH-1211 Geneva 4, Switzerland

Received 8 October 2008; Accepted 17 November 2008

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.

  • Keywords:

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