Heterochromatic siRNAs and DDM1 Independently Silence Aberrant 5S rDNA Transcripts in Arabidopsis Todd Blevins1¤, Olga Pontes2, Craig S. Pikaard2, Frederick Meins, Jr.1* 1 Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland, 2 Department of Biology, Washington University in St. Louis, St. Louis, Missouri, United States of America Abstract 5S ribosomal RNA gene repeats are arranged in heterochromatic arrays (5S rDNA) situated near the centromeres of Arabidopsis chromosomes. The chromatin remodeling factor DDM1 is known to maintain 5S rDNA methylation patterns while silencing transcription through 5S rDNA intergenic spacers (IGS). We mapped small-interfering RNAs (siRNA) to a composite 5S rDNA repeat, revealing a high density of siRNAs matching silenced IGS transcripts. IGS transcript repression requires proteins of the heterochromatic siRNA pathway, including RNA polymerase IV (Pol IV), RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) and DICER-LIKE 3 (DCL3). Using molecular and cytogenetic approaches, we show that the DDM1 and siRNA-dependent silencing effects are genetically independent. DDM1 suppresses production of the siRNAs, however, thereby limiting RNA-directed DNA methylation at 5S rDNA repeats. We conclude that DDM1 and siRNA-dependent silencing are overlapping processes that both repress aberrant 5S rDNA transcription and contribute to the heterochromatic state of 5S rDNA arrays. Citation: Blevins T, Pontes O, Pikaard CS, Meins F Jr (2009) Heterochromatic siRNAs and DDM1 Independently Silence Aberrant 5S rDNA Transcripts in Arabidopsis. PLoS ONE 4(6): e5932. doi:10.1371/journal.pone.0005932 Editor: Beth A. Sullivan, Duke University, United States of America Received March 3, 2009; Accepted May 11, 2009; Published June 16, 2009 Copyright: 2009 Blevins et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Todd Blevins was supported by a Friedrich Miescher Institute student fellowship, and postdoctoral fellowships from the Swiss National Foundation, Novartis Foundation and Keck Foundation. Olga Pontes was supported by an Edward Mallinckrodt Foundation Award. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: Frederick.Meins@fmi.ch ¤ Current address: Department of Biology, Washington University in St. Louis, St. Louis, Missouri, United States of America Introduction RNA silencing collectively refers to processes by which specialized RNA-protein complexes repress gene expression, guide repressive covalent modifications to histones or mediate DNA methylation within specific DNA regions [1–4]. Short-interfering RNAs (siRNAs) are the ,20–25 nt guide RNAs that afford sequence-specificity to RNA silencing. Most Arabidopsis siRNAs depend on RNA polymerase IV (Pol IV), a plant-specific enzyme composed of subunits homologous to RNA polymerases I, II and III [5–10]. DNA methylation of repetitive elements correlates with transcriptional silencing and formation of a heterochromatic state, conditions which are thought to preserve genome integrity in eukaryotes [11–15]. In current models, Pol IV generates precursors of ‘‘heterochro- matic’’ siRNAs from repeat regions, such as the retroelement, AtSN1 [16,17]. Pol IV is speculated to produce single-stranded RNA transcripts that are converted into double-stranded RNA (dsRNA) by RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) and processed into siRNA duplexes by DICER-LIKE 3 (DCL3) [7,18,19]. The effector protein AGO4 preferentially binds heterochromatic siRNAs [20,21], which appear to guide RNA- directed DNA methylation (RdDM) [4]. The cytosine methyl- transferase, DRM2, is thought to methylate cytosines targeted for RdDM [22]. A second plant-specific RNA polymerase, Pol V (previously known as Pol IVb), mediates production of non-coding transcripts critical for steps downstream of siRNA biogenesis [8,9,19,23]. Additional proteins have also been implicated in these downstream steps [24,25]. In addition to their role in silencing transposable elements, Pol IV-dependent siRNAs also arise from essential genes, including 5S rRNA gene repeats [18,26]. 5S rRNA is a structurally conserved, integral component of the ribosome large subunit in all organisms and is essential for ribosome function [27–29]. 5S rRNA genes are arranged in tandem repeat arrays (5S rDNA) situated in pericentromeric regions of Arabidopsis chromosomes, totaling approximately 1000 repeat copies [30,31]. Genic portions of active repeats, located on Chromosomes 4 and 5 in the A. thaliana ecotype Col-0, are transcribed by Pol III to produce 120 nucleotide (nt) 5S RNA transcripts [31–33]. It is not yet clear what proportion of these 5S genes is active at any one time. Circumstantial evidence indicates that Pol IV may play a role in silencing at 5S rDNA loci. For example, siRNAs matching 5S rDNA sequences associate with AGO4 in vivo [5,21] and 5S rDNA methylation is reduced in mutants deficient for the heterochro- matic siRNA pathway, such as nrpd1 (Pol IV largest subunit), rdr2, dcl3, ago4 and drm2 [6,7,18,19]. Moreover, Pol IV physically colocalizes with 5S gene loci, as well as 45S rRNA gene loci [19]. The consequences of DNA methylation changes remain unclear, however, because steady-state levels of major 5S rRNA species are not affected by reduced 5S rDNA methylation [34]. Studies with deficiency mutants have identified multiple systems that maintain DNA methylation in Arabidopsis [35–38]. Methyl- cytosine in a ‘CG’ sequence context (CG methylation) is PLoS ONE | www.plosone.org 1 June 2009 | Volume 4 | Issue 6 | e5932