title: A stepwise pathway for biogenesis of 24-nt secondary siRNAs and spreading of DNA methylation.
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.
authors.affiliation: Gregor Mendel Institute of Molecular Plant Biology, Austrian Academy of Sciences, Vienna, Austria.
authors.forename: Lucia
authors.initials: L
authors.lastname: Daxinger
authors.forename: Tatsuo
authors.initials: T
authors.lastname: Kanno
authors.forename: Etienne
authors.initials: E
authors.lastname: Bucher
authors.forename: Johannes
authors.initials: J
authors.lastname: van der Winden
authors.forename: Ulf
authors.initials: U
authors.lastname: Naumann
authors.forename: Antonius J M
authors.initials: AJ
authors.lastname: Matzke
authors.forename: Marjori
authors.initials: M
authors.lastname: Matzke
mesh_terms: D017360:Arabidopsis / Q000378:metabolism* / Q000502:physiology*; D029681:Arabidopsis Proteins / Q000378:metabolism; D019175:DNA Methylation; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D020868:Gene Silencing; D011485:Protein Binding; D034741:RNA, Small Interfering / Q000378:metabolism*; D012324:RNA-Dependent RNA Polymerase / Q000378:metabolism; D043244:Ribonuclease III / Q000378:metabolism
chemical_list: D029681:Arabidopsis Proteins; D034741:RNA, Small Interfering; C499181:RNA polymerase IV, Arabidopsis; D012324:RNA-Dependent RNA Polymerase; D012321:DNA-Directed RNA Polymerases; C519654:DCL3 protein, Arabidopsis; D043244:Ribonuclease III