Analysis of the Affect of <i>rdr2</i> and <i>dcl3</i> on the Expression of Cluster-Proximal Genes

<div><p>(A and B) Fold-change in <i>rdr2</i> (upper) and <i>dcl3</i> (lower) versus Col-0 inflorescence plotted the same way as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0050057#pbio-0050057-g006" target="_blank">Figure 6</a>A for cluster-proximal genes (A) and the random gene set (B). Red lines in (A and B) represent mean expression of 1,000 random sets.</p> <p>(C and D) Natural log (ln) of fold-change in <i>dcl3</i> plotted versus ln of fold-change in <i>rdr2</i> for all genes on the ATH1 array (C) or cluster-proximal genes (D). <i>R</i>² is the square of the Pearson correlation and is the percent variation in <i>rdr2</i> that is explained by variation in <i>dcl3.</i> Red lines are the best-fit lines.</p> <p>(E–J) Venn diagram analysis of genes significantly upregulated at least 1.5-fold (SAM false discovery rate = 0.01) in <i>rdr2</i> or <i>dcl3</i> and the cluster-proximal gene set (E), <i>dcl1-</i>7 (F), <i>rdr6–15</i> (G), or all genes with a 24-nt small RNA within 200 nt of the 5′ end (H), within the gene (I), or within 200 nt of the 3′ end (J).</p></div

Analysis of Small RNA Clusters in or around Genes

<div><p>(A) Raw expression values in Col-0 inflorescence tissues for genes with clusters up to 1,000 nt upstream (5′) of the transcription start site, within the gene, and 1,000 nt downstream of the 3′ end. Genes are represented as points at positions corresponding to distance from a cluster.</p> <p>(B) Three random gene sets, containing the same numbers of genes <i>(n)</i> as in the 5′, 3′, and internal cluster sets, were randomly distributed within the respective zones. Red lines in (A and B) represent the mean expression of 1,000 random sets.</p> <p>(C) Scrolling-window counts of clusters (top), and <i>Z</i>-score showing over- or underrepresentation of clusters at positions relative to 5′, internal, and 3′ sites (bottom). Cluster counts and <i>Z</i>-score values were determined in 20-nt windows with a 10-nt scroll for observed clusters (blue), and for 1,000 sets of randomly distributed clusters that were averaged at each position (red). Figures below each panel indicate the gene region plotted along the x-axis, with arrows indicating the transcription start sites. Gray boxes mark the intragenic regions plotted on a relative scale (0–100). For each graph, the three independent analyses (5′, 3′, and transcribed regions) were merged in the same plots.</p></div

Small RNA Loci in Protein-Coding Genes and Pseudogenes

<p>The percentage of genes (orange) and pseudogenes (blue) with 0, 1–5, 6–10, etc., small RNA loci was plotted.</p

Small RNA Sequencing and the Distribution of Small RNA Loci in Feature Classes

<p>(A) Flowchart for high-throughput sequencing and analysis of small RNAs. (B–G) Distribution of small RNAs from wt Col-0 plants and <i>rdr6–15, rdr1–1, rdr2–1, dcl1–7, dcl2–1, dcl3–1,</i> and <i>dcl4–2</i> mutants in the genome (B), transposons and retroelements (C), genes (D), pseudogenes (E), miRNAs (F), and tasiRNAs (G). In (B), the percentages of small RNAs in each of four size classes within each library are presented. In all other panels, normalized small RNA levels in each feature class are presented.</p

Small RNAs from Segments of Selected Transposons, Retroelements, and Pseudogenes

<p>Each unique small RNA is indicated and color-coded based on size.</p

Distribution of Small RNA-Generating Loci from Each Chromosome

<div><p>(A) Scrolling-window analysis (50,000-nt window and 10,000-nt scroll) of small RNA loci. Total, repeat-normalized, and unique small RNA loci, as well as transposon/retroelement loci, are shown. Abundance of repeat-normalized, library-size-normalized counts (individual sequencing reads) are also shown.</p> <p>(B) Scrolling-window analysis of repeat-normalized, library-size-normalized small RNA abundance in Col-0, <i>rdr2,</i> and <i>dcl3.</i> The summed, 21- and 22-nt size classes (blue, above x-axis) and 24-nt size class (red, below x-axis) were plotted independently. Note that in both (A and B), maximum values plotted were capped at the value corresponding to the maximum y-axis value.</p> <p>(C) Scrolling-window analysis of relative increase (red) or decrease (green) in repeat-normalized, library-size-normalized small RNA abundance in each mutant. Col-0 inflorescence was used as the reference library.</p></div

Analysis of Small RNAs Proximal to Transposons and Retroelements (T/R)

<div><p>(A) Method to analyze over- or underrepresentation of small RNAs in T/R bins.</p> <p>(B and C) <i>Z</i>-score plots showing overrepresentation (positive) or underrepresentation (negative) of small RNA loci in T/R bins from wt Col-0 and mutant plants. Independent analyses were done for each size class from total (B) and unique (C) small RNA loci.</p></div

Small RNA from protein-coding gene and pseudogene loci in <i>P. infestans</i>.

<p>(A) Heatmap-based size profile of 18-30-nucleotide small RNA reads mapping to 1,166 <i>P. infestans</i> genes that overlapped at least 100 total small RNA reads. Gene annotation groups with at least four genes are labeled with alternating black and gray boxes. (B and C) Regional metaplots with average 18-27-nucleotide small RNA reads per position. X-axis positions are relative scale (0–100%) for each region. Genes from (A) with more 24-30- than 18-23-nucleotide small RNA reads, LTR retrotransposons and transposons are shown (B). Genes from (A) with more 18-23- than 24-30-nucleotide small RNA reads, crinklers and type III fibronectins are shown (C).</p

Figure 4

<p>Identification of <i>MIRNA</i> foldbacks with similarity to protein-coding genes. (A) Flowchart for identification of <i>MIRNA</i> foldbacks with similarity, extending beyond the miRNA target site, to protein-coding genes. (B) <i>Arabidopsis</i> gene or transcript hits in FASTA searches using foldback sequences for all conserved and non-conserved <i>MIRNA</i> loci (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000219#pone-0000219-t001" target="_blank">Tables 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000219#pone-0000219-t002" target="_blank">2</a>). The top four hits based on E-values are shown. (C) Z-scores for the Needleman-Wunche alignment values from <i>MIRNA</i> foldback arms with top four gene or transcript FASTA hits. Alignments were done with intact foldback arms (I), and with foldback arms in which miRNA or miRNA-complementary sequences were deleted (D). Z-scores were derived from standard deviation values for alignments of randomized sequences. In (B) and (C), a red symbol represents an experimentally validated target, a pink symbol indicates a gene from a validated target family, and an open symbol indicates a gene that is distinct from either the validated or predicted target family.</p

Distribution of small RNA in the <i>P. infestans</i> genome.

<p>(A–D) Two-dimensional binning of <i>P. infestans</i> genes based on the length of 5′ and 3′ flanking intergenic regions. Plots are divided into quadrants with dashed lines: gene-dense regions (GDRs), gene-sparse regions (GSRs), and border regions (BRs). (A) Heatmap color scale represents the number of genes per bin for all genes (left) and genes without transposable elements in flanking intergenic regions (Data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077181#pone.0077181-Haas1" target="_blank">[9]</a>). (B) Genes were binned as in (A), but the heatmap color scale represents the percentage of base pairs in both the 5′ and 3′ flanking intergenic regions that were occupied by transposable elements. (C and D) Genes were binned as in (A), but the heatmap color scale represents the repeat-normalized small RNA reads per base pair in either both the 5′ and 3′ flanking intergenic regions (C) or the gene body (D).</p