(A) Pie charts showing the abundance of small RNA species in wild type (WT) and prg-1(n4357) mutants. (B,C) Scatter plots showing the abundance of 22G-RNAs (B) and target mRNAs (C) associated with WAGO (blue) or CSR-1 (red) targets in wild type (WT) compared to the prg-1 mutant. The two dashed diagonal lines indicate two-fold enrichment or depletion. In (B) the solid blue diagonal line indicates eight-fold depletion in the prg-1 mutant, and the percentage of WAGO and CSR-1 targets that exhibit 8-fold, or greater, depletion in 22G-RNA levels is indicated. See also Figure S1.

(A-H) Density of 22G-RNAs within a 100 nt window around predicted 21U-RNA (or control) target sites in the wild type (blue) or prg-1 mutant (red). The plots are centered on the 10^th nucleotide of the small RNA shown schematically in each graph. WAGO targets (A,C,E,F) and CSR-1 targets (B,D,F,H) were analyzed separately. All 21U-RNAs (A,B), the top 20% most-(C,D) and least-abundant (E,F), as well as, binding sites of hypothetical reverse-complement 21U-RNAs (G,H) were analyzed separately (as indicated). (I) Box and whisker plots showing the fold change of 22G RNA levels for each category of target/small RNA interaction as indicated. The bottom and top of each box represents the value of the 25^th and 75^th percentile, the horizontal line inside the box represent the median value. The p-value was calculated using a one-sided t-test. (J) Density of 22G-RNAs at 21U-RNA predicted target sites with good or poor seed matches. (K) Distribution of 22G-RNAs at two predicted 21U-RNA targets in a prg-1 mutant and in wild type. The bars indicate the position of the first nucleotide, and the relative abundance of each 22G-RNA species. The positions of predicted 21U-RNA pairing sites are highlighted (red). Base-pairing alignments for the boxed regions are shown at single-nucleotide resolution below each diagram. See also Figure S2.

(A) Western blots of protein isolated from WT and prg-1 mutant transgenic strains (as indicated), probed with anti-FLAG antibody (left panel), anti-PRG-1 polyclonal antibody (right panel), and anti-tubulin antibody as a loading control. (B) Graphic representation of the brood size (at 25°C) observed for the wild type (WT), prg-1 mutant or prg-1 mutant rescued with WT or Slicer-deficient flag::prg-1 transgenes (as indicated). Error bars represent the standard deviation of the mean. (C) Graphic representation of 22G-RNA levels within a 100 nt window near predicted 21U-RNA target sites in the indicated strains. The base-paring parameters were as in Figure 2A-H). (D) Two examples of predicted 21U-RNA targets with the positions of 21U-RNAs highlighted (red) below the gene diagrams. The graphs show 22G-RNA levels in the WT, prg-1 mutant and rescued strains as indicated. (E) Quantitative RT-PCR analysis of three different predicted targets in the indicated strains. Error bars represent the standard deviation of the mean. See also Figure S3.

(A) Fluorescence micrographs showing GFP::H2B expression in transgenic animals carrying a control reporter (left) or a 21U-RNA reporter (right). (B) Schematic of 21U-RNA reporter #1 showing the distribution of 22G-RNAs identified in a silent transgenic strain. The bars indicate reads in the antisense (pink) or sense (blue) orientation. (C) Genetics of reporter silencing. The reporters were either directly injected into the indicated strain or (as indicated by the star) were crossed into the strains after establishment in a wild-type background. “mm10” indicates mismatch mutations in both target sites at position 10 of the 21U-RNA. (D) Model showing the proposed role of PRG-1 in the initiation of silencing on a 21U-RNA target reporter. See also Figure S4.