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

Figure 1

<p>Identification and analysis of <i>Arabidopsis</i> miRNAs and targets. (A) Flowchart for the prediction of miRNAs and their targets. (B) Validation of predicted targets for 13 non-conserved miRNAs. Positions of dominant 5′ RACE products (no. 5′ ends at position/total no. 5′ ends sequenced) are indicated by vertical arrows in the expanded regions. Predicted cleavage sites are indicated by a bolded nucleotide at position ten relative to the 5′ end of the miRNA or miRNA-variant. Positions of gene-specific primers are indicated with horizontal arrows above gene diagrams.</p

Figure 6

<p>Targeting specificity of recently evolved <i>MIRNA</i>s. Two target prediction scores are shown for each of 16 miRNAs: best overall predicted target score (blue) and target scores calculated for <i>MIRNA</i> foldback-similar genes (grey). Left column indicates whether or not the best overall predicted target gene is in the same family as the foldback-similar gene. A dot indicates that the predicted gene is in an experimentally validated target family. Two calculations corresponding to the two major populations from the <i>MIR161</i> locus (miR161.1 and miR161.2) are shown. The identities of targets are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000219#pone.0000219.s003" target="_blank">Supplemental Table S3</a>. The plot is centered on a target prediction score of 4, as this corresponds to the upper limit of a reasonable prediction.</p

Figure 3

<p>Expression profiling of <i>MIRNA</i> families using high-throughput pyrosequencing. (A) Comparison of most-abundant miRNA families between biological replicates of Col-0 inflorescence (inf.) tissue. Normalized reads for each miRNA family member were consolidated. Note that <i>MIR159</i> and <i>MIR319</i> derived members were counted separately, even though they are frequently assigned to the same family <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000219#pone.0000219-Xie1" target="_blank">[31]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000219#pone.0000219-Palatnik1" target="_blank">[57]</a>. (B) Fold-change of miRNAs in <i>dcl1-7</i> inflorescence versus Col-0 inflorescence (left axis, bars). Total number of reads for each family is indicated (right axis, green line). As the <i>dcl1-7</i> mutant contained no reads for many miRNA families, fold-change was calculated using normalized reads+1. This had the effect of dampening fold-change values for low-abundance families. (C) Fold-change of miRNA family reads in leaves at 1 hr and 3 hr post-inoculation with <i>P. syringae</i> (DC3000<i>hrcC</i>) (left axis, bars). Fold-change relative to uninoculated leaves was calculated based on normalized reads as described in panel (B). Total number of reads in the control and inoculated samples is shown (right axis, green line). Grey dashed lines indicate the <i>p</i> = 0.05 upper and lower thresholds.</p

Figure 5

<p>Similarity between <i>MIRNA</i> foldback arms and protein-coding genes. Each alignment contains the coding strand for 1–3 genes, the miRNA* arm, and the miRNA arm. Orientation of the foldback arms is indicated by (+) for authentic polarity and (−) for the reverse complement polarity. Two alignments are given for <i>MIR824</i> because the two arms are each most similar to distinct, duplicated regions within the <i>AGL16</i> gene (At3g57230). Alignments were generated using T-COFFEE. Colors indicate alignment quality in a regional context.</p

Figure 2

<p>Comparison of conserved and non-conserved <i>MIRNA</i> families. (A) Effect of <i>dcl1-7</i> and <i>hen1-1</i> mutations on levels of target transcripts for conserved (black) and non-conserved (red) miRNAs. Expression data are shown for two validated or high-confidence predicted targets, if available, for each family. Arrows indicate targets for miR824 (<i>AGL16</i>), miR858 (<i>MYB12</i>) and miR161.1 (At1g63130, a <i>PPR</i> gene). (B) Numbers of gene family members for conserved and non-conserved <i>MIRNA</i>s (<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>). (C) Relative numbers of miRNA target family functions for conserved and non-conserved miRNAs (<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>). Only target classes that have been validated experimentally are included. Note that <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000219#pone-0000219-t002" target="_blank">Table 2</a> shows many <i>MIRNA</i> families with weak or no predicted targets, and these are not represented in the chart.</p

Reference set of <i>Arabidopsis</i><i>MIRNA</i> families.

a<p>Reviewed in Jones-Rhoades et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000219#pone.0000219-JonesRhoades1" target="_blank">[11]</a>.</p>b<p>Conserved between <i>A. thaliana</i> and <i>P. trichocarpa</i>.</p>c<p>Number of reads are from all libraries in the ASRP database (<a href="http://asrp.cgrb.oregonstate.edu/db" target="_blank">http://asrp.cgrb.oregonstate.edu/db</a>).</p><p>Reads for each locus encompass the defined miRNA sequence ±4 nts on each side.</p

New or recently identified miRNAs

a<p>Conserved between <i>A. thaliana</i> and <i>P. trichocarpa</i>.</p>b<p>Number of reads are from all libraries in the ASRP (<a href="http://asrp.cgrb.oregonstate.edu/db" target="_blank">http://asrp.cgrb.oregonstate.edu/db</a>) and MPSS Plus (<a href="http://mpss.udel.edu/at/" target="_blank">http://mpss.udel.edu/at/</a>) databases. Reads for each locus encompass the defined miRNA/miRNA* sequence ±4 nts on each side. ASRP (MPSS Plus).</p>c<p>Top three predicted targets with a score of 3.5 or less are listed with their score in parentheses. Targets validated by 5′ RACE are in bold. Remaining number of targets predicted with a score of 3.5 or less are listed in square brackets (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000219#pone.0000219.s001" target="_blank">Table S1</a>). Dashes indicate no predicted targets with a score of 3.5 or less.</p>d<p>Targets validated by Lu et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000219#pone.0000219-Lu1" target="_blank">[23]</a>.</p>e<p>Target tested but failed in 5′RACE validation assays.</p>f<p>Seventeen nt MPSS Plus signature was extended 4 nts on the 3′ end.</p