maintained by the methyltransferase MET1 acting in concert with a SWI2/SNF2-like chromatin remodeling factor, DDM1 [39–45]. Maintenance of methylcytosine at ‘CHG’ sites (CHG methylation, where H is A, T or C) depends primarily on the methyltransferase CMT3 [46,47]. Remaining methylcytosine at ‘CHH’ sites (asymmetric methylation) involves DRM2-dependent de novo methylation [37,48]. DDM1 also contributes to the maintenance of CHG and CHH methylation in a subset of genomic sequences [39,49]. Mutations in MET1, DDM1 and CMT3 can release silencing of transposable elements [13,15,47,50], whereas their effects on 5S rDNA are more subtle: they derepress ‘‘minor’’ 5S rRNA species whose sequences diverge from ‘‘major’’ species at only one or two positions [51–53]. In addition, atypical long transcripts greater than 120 nt in length are generated at 5S rDNA loci in met1 and ddm1 mutants [52,54]. Thus, CG and CHG methylation are implicated in silencing specific subsets of 5S rRNA genes and for preventing production of aberrant transcripts at 5S rDNA loci [31]. The large-scale organization of heterochromatin in the nucleus depends on DDM1 and MET1; nuclei of ddm1 and met1 mutants generally show reductions in heterochromatin content relative to wild type nuclei [51,54,55]. In ddm1 mutants, portions of 5S rDNA arrays become localized outside of their normal positions associated with compact ‘‘chromocenters’’ [51]. Heterochromatic marks such as 5-methylcytosine and Histone 3 Lysine 9 methylation also become dispersed relative to the chromocenters in inbred met1 lines; this is coincident with a progressive, ectopic increase in asymmetric methylation, which is thought to be an siRNA-directed process [54]. The overaccumulation of 5S rDNA-derived siRNAs in DDM1 and MET1-deficient mutants and 5S rDNA decondensation in Pol IV- deficient mutants suggests a role for siRNAs in 5S rDNA chromatin organization [7,19,54,56]. Although DDM1 and heterochromatic siRNAs both appear to function in 5S rDNA condensation, how their functions are coordinated is not known. Here we demonstrate that Pol IV and other proteins of the heterochromatic siRNA pathway are required for silencing of aberrantly long 5S rDNA transcripts that extend into intergenic spacers downstream of 5S rRNA genes. Production of siRNAs matching these transcripts depends on proteins of this pathway and is limited by the chromatin-remodeling factor DDM1. Our genetic analyses of these phenomena lead us to propose that DDM1-dependent maintenance of silent chromatin, and Pol IV- dependent RNA silencing are overlapping processes that repress aberrant 5S rDNA transcription and contribute to 5S rDNA heterochromatic organization. Results Distribution of siRNAs representing the 5S rDNA unit repeat Heterochromatic siRNAs in Arabidopsis are typically 23 to 24 nt long and match both strands of corresponding genetic loci [5,16,18,57]. A known 5S rDNA-related species, siR1003, was first characterized by Xie et al. (2004) and matches specific repeats on Chromosomes 3 and 5 [18]. In the present study, we identified 5S rDNA siRNAs in existing high-throughput sequencing data and mapped these to the ,500 bp unit repeat. Analysis of the origin of siRNAs is complicated by natural variation among the ,1000 5S rDNA repeats. To resolve this problem, we generated a gapped alignment of 283 individual 5S rDNA repeats derived from an A. thaliana YAC library [58]. Next, 59-end positions of individually matched small RNAs were aligned with a composite 530-bp repeat. Figure 1A summarizes our analysis of small RNA datasets from leaves obtained by 5S rDNA Silencing by siRNAs Rajagopalan et al. (2006) and Kasschau et al. (2007) [57,59]. Interestingly, 60% of the 3662 matched sequencing reads represented the intergenic spacer (IGS). A cluster of small RNAs (Figure 1A, **) in the IGS matched both strands (44% and 56%, respectively) and included siR1003 (Figure 1B). This IGS cluster was also apparent in the maps we generated for other Arabidopsis tissues (Figure S1, seedlings and inflorescences). The remaining sequences that matched 5S rDNA, 40%, correspond to the 120-bp 5S rRNA genes: these reads were disproportionately 20 to 22-nt long and corresponded predominantly to the 5S rRNA-encoding strand (92% of 1481 genic reads). Analysis of inflorescence data yielded a spike of reads with 59-ends corresponding to the 5S rRNA 59-terminus (Figure S1). Together with the abundant reads with 39-ends aligned to the 5S rRNA 39-terminus (Figure 1A and Figure S1), this strongly suggests that most genic small RNAs are products of 5S rRNA degradation. Pol IV and heterochromatic siRNA production silence 5S rDNA transcripts Numerous IGS siRNAs occur at positions consistent with the possibility that they derive from aberrant run-on transcripts of 5S rRNA genes that extend into the IGS. To test this hypothesis, we used RT-PCR to measure levels of two long transcripts known to be derived from 5S rDNA, namely: 5S LT1, which is 210 nt in length and extends 90 nt into the 39 IGS, and 5S LT2, which has the same 59 and 39 ends as 5S LT1, but is 70 nt shorter due to an internal deletion ([52,53], Figure 1A). Mutants homozygous for alleles of the two largest Pol IV subunits, nrpd1 and nrpd2, showed higher levels of 5S LT1 than wild type plants; these elevated levels were comparable to 5S LT1 derepression in ddm1 (Figure 1C). Other mutants disrupting heterochromatic siRNA biogenesis, namely rdr2 and dcl3, also showed higher 5S LT1 levels. In contrast, a mutant deficient for trans-acting siRNA biogenesis [60], rdr6, did not show 5S LT1 derepression (Figure S5). These data suggest that Pol IV/RDR2- dependent siRNA production, but not RDR6-dependent pathways, contribute to silencing of aberrant transcripts that include 5S rRNA gene sequences (120 bp) but continue at least 90 bp downstream into the IGS. Pol V is functionally and structurally distinct from Pol IV and its largest subunit, NRPE1, is unique [8–10,19]. The nrpe1 mutant showed modest derepression of 5S LT1. Therefore, both plant-specific RNA polymerases are potentially required for suppressing long 5S rDNA transcripts. We did not detect reproducible changes in 5S LT2, the truncated transcript [52]. Multiple DCLs mediate silencing of 5S rDNA transcripts Triple (dcl2/3/4) and quadruple (dcl1/2/3/4) dicer mutants showed slightly more 5S LT1 accumulation than did dcl3 alone (Figure 1C). Thus, alternate DCLs might mediate 5S LT1 silencing, compensating for DCL3 deficiency. To test this hypothesis, we used RNA blot hybridization to analyze genetic requirements for IGS siRNA biogenesis (Figure 1D). We confirmed that siRNAs hybridizing to an siR1003 probe accumulate in wild type but not in nrpd1, nrpd2 or rdr2 mutants [6,7,18]. Comparison to Figure 1C suggests that the absence of siR1003 in these mutants correlates with derepression of 5S LT1 transcripts. Accumulation of 24-nt siR1003-related siRNAs was also decreased in dcl3 mutants, but was accompanied by the appearance of alternate siRNAs in the range 21–23 nt (Figure 1D), presumably due to the action of other dicers. Overall, the siR1003 signal in the 21 to 24-nt range in each dcl mutant roughly correlated with the extent of 5S LT1 silencing (Figure 1C). A longer probe spanning a region of the IGS that includes siR1003 (Figure 1A, labeled 39flank), detected siRNA PLoS ONE | www.plosone.org 2 June 2009 | Volume 4 | Issue 6 | e5932