5S rDNA Silencing by siRNAs Figure 2. DDM1 limits IGS siRNA accumulation and asymmetric methylation. A) Detection of IGS siRNAs (siR1003) in dicer-like (dcl) mutant combinations. Blot analysis of small RNA isolated from leaves of wild type (WT); single mutants dcl2, dcl3, dcl4; double mutants dcl2 dcl3 (dcl2/3), dcl2 dcl4 (dcl2/4), dcl3 dcl4 (dcl3/4); and the triple mutant dcl2 dcl3 dcl4 (dcl2/3/4). B) Localization of IGS-related RNA in interphase nuclei by fluorescent in situ hybridization with an RNA probe for siR1003 (red). DNA was stained with DAPI (white). The size bar corresponds to 5 mm. C) Overaccumulation of IGS siRNAs in the ddm1 background. Blot analysis of small RNA from dcl3 and double mutant dcl3 ddm1 using probes for siR1003 and the larger 39flanking region (Figure 1A, diagram). WT and ddm1 signals from the same membrane are provided for comparison. D) IGS siRNA overaccumulation persists in out-crossed ddm1. Blot analysis of small RNA isolated from WT, nrpd1 (2/2), ddm1 (2/2) and F1 heterozygotes (+/2) of each mutant crossed to the WT. E) Asymmetric cytosines in the IGS are hypermethylated in met1 and ddm1. Southern blot analysis was performed on Alu I-digested genomic DNA isolated from WT, nrpd1, rdr2, two alleles of dcl3, met1 and ddm1. The probe corresponds to 5S LT1, shown aligned to a representative 5S rDNA repeat unit from Chromosome 5. Alu I and Hae III sites in the IGS region are indicated. doi:10.1371/journal.pone.0005932.g002 complement of siR1003 (data not shown). Together, our data suggest that dsRNA derived from aberrant 5S rDNA transcripts can be processed by multiple DCLs when DCL3 is mutated, generating IGS siRNAs that contribute to silencing of 5S rDNA. DDM1 attenuates IGS siRNA production and RdDM We confirmed earlier reports that IGS transcript (5S LT1) and siRNA levels are elevated in ddm1 [7,52]. Noting this phenome- non, we sought to localize IGS-derived RNA in interphase nuclei using RNA fluorescent in situ hybridization (RNA-FISH). In 83% of wild type nuclei (n = 136) the siR1003 probe detected RNA restricted to a distinct, sub-compartment of the nucleolus, a prominent interphase structure devoid of DAPI staining (Figure 2B). In contrast, the 5S RNA-FISH signal in 94% of ddm1 nuclei (n = 158) was typically dispersed throughout the nucleolus and nucleoplasm (see also Table S1). These results suggest that IGS-derived siRNAs or their precursors overaccu- mulate and/or mislocalize in ddm1 nuclei. Based on the RNA-FISH result and siRNA overaccumulation observed in both ddm1 and met1 mutants [7,54], we hypothesized that DDM1 and MET1 repress formation of dsRNA precursors that are processed by DCLs to generate IGS siRNAs. If this hypothesis is correct, a double mutant ddm1 dcl3 should overexpress alternate siRNA size-classes (i.e., those noted in Figure 2A). We generated ddm1 dcl3 by genetic cross and, indeed, detected elevated siRNA levels in ddm1 dcl3 relative to dcl3 (Figure 2C). DDM1 and MET1 are required to maintain a silent chromatin state associated with CG methylation [40,41,43], which is not immediately re-established upon replacement of functional DDM1 or MET1 [40,65,66]. To test whether this silent chromatin state PLoS ONE | www.plosone.org 4 June 2009 | Volume 4 | Issue 6 | e5932