5S rDNA Silencing by siRNAs Figure 1. Long 5S rDNA-derived transcripts are subject to RNA silencing. A) Map of Arabidopsis small RNAs matching the 5S rDNA unit repeat, based on analysis of leaf datasets from Rajagopalan et al. (2006) and Kasschau et al. (2007). Small RNA 59-end positions are indicated on the x- axis, with sequencing reads tallied on the y-axis. Upward bars are matches to the forward strand; downward bars represent reverse strand matches. Read tallies are stacked in 10-bp bins, with size-class indicated by color. The diagram at bottom shows a 5S rRNA gene (thick black arrow), the surrounding intergenic spacers (IGS, gray), and regions probed by RNA blot hybridization (red lines). RT-PCR products obtained with F and R primers are indicated (black lines). B) Searching small RNA datasets for the siR1003 core sequence (yellow box) yielded a family of 21 to 24-nt siRNAs, which all match 5S LT1 transcripts identified by Vaillant et al. (2006) [52]. C) Analysis of 5S LT1 transcript accumulation: RNA samples from inflorescences of wild type (WT), ddm1 and siRNA biogenesis mutants were analyzed by one-step RT-PCR. Reverse transcription was performed using R primer, and PCR performed using F and R primers (panel A, diagram). Control reactions were performed using ACT2 primers. RT enzyme was omitted from duplicate 5S LT1 and ACT2 reactions (no RT). D) Blot analysis of small RNA isolated from material described in panel C. The membrane was sequentially hybridized with a DNA oligonucleotide (oligo) that detects siR1003, an RNA probe for the 39-flanking IGS region (panel A, diagram), a DNA oligo that detects miR160, and DNA oligos that detect U6 snRNA. Migration of 21-nt and 24-nt RNA oligo size standards is indicated at left. doi:10.1371/journal.pone.0005932.g001 accumulation patterns identical to those detected using the siR1003 probe (Figure 1D). To infer processing functions for individual DCLs, we analyzed siR1003 accumulation in double mutant combinations of three DCLs (DCL2, DCL3 and DCL4). We detected ,22-nt siRNAs in dcl3, but this size-class was absent in the double mutant dcl2/3. Moreover, ,21-nt species detected in dcl2/3 were absent in the triple mutant dcl2/3/4 (Figure 2A). These hierarchical DCL dependencies, previously recognized for other repeat-derived and viral siRNAs [61–64], suggest that 21-nt and 22-nt IGS siRNAs are products of DCL4 and DCL2, respectively. Essentially the same results were obtained using probes for the reverse PLoS ONE | www.plosone.org 3 June 2009 | Volume 4 | Issue 6 | e5932