Sma-miR-277 family predicted targets downregulated in developing female parasites.

<p>Fold change expression (Log₂) of high confidence targets of sma-miR-277 family during the development of male and female worms in two conditions: paired (solid line red) and unpaired (dashed green). Black lines represent the mean expression of genes in paired (solid black line) and unpaired (dashed black line) worms.</p

Sma-miR-4989 is significantly up-regulated during male and female maturation.

<p>Fold change expression of sma-miR-4989 during development of juvenile to adult worms in male (blue bars) and females (red bars) as measured by RT-qPCR. Samples were collected at the time points (days post infection) indicated in the x-axis from murine hosts infected with pooled (mixed sex) cercariae. Each barplot represents the mean of three biological replicates. T-tests were performed between 49 d.p.i. and 28 d.p.i. and were both significant with p-value ≤ 0.01. Error bars show the standard error of the mean, based on three biological replicates.</p

Sma-miR-277 and sma-miR-4989 belong to a gene cluster.

<p>(A) The genomic locus in Chromosome 4 of sma-miR-277 and sma-miR-4989 suggests they belong to a gene cluster. The average distance between genes (represented by coloured boxes) is 109 bases. Here the mature miRNAs (sma-miR2-277 and sma-miR-4989) and passenger miRNAs are represented with coverage plot and aligned reads from one of the small RNA libraries. (B) Predicted stem-loop structures for sma-miR-277 and sma-miR-4989 –individual cases. Mature miRNAs are located in the 3’-end of the hairpin. (C) Due to the cluster organisation of sma-miR-277/sma-miR-4989, it is likely that they are transcribed as one precursor RNA molecule. This figure represents the predicted stem-loop structure for sma-miR-277/sma-miR-4989 when arising from a larger transcript.</p

High confidence targets of the sma-miR-277 family identified with a combined approach (Sylamer, miRanda and TargetScan with conservation).

<p>F = female; M = male.</p

MiRNA target prediction based on both miRNA-unaware and miRNA-guided approaches.

<p>(A) Sylamer enrichment landscape plots for 7mers in male (top) and female (bottom) expression data. The x-axis represents a list of transcripts, ranked from more expressed in juveniles to more expressed in adults. The y-axis represents the significance values acquired for each 7mer at each position in the ranked list of transcripts. Coloured boxes represent the fraction of transcripts significantly (adjusted p-value < 0.01) differentially expressed between juvenile and adult worm as found using DESeq2. These transcripts were subsequently filtered based on the presence of the 7mers TGCATTT or GCATTTA as found by Sylamer. The resulting sets are referred to as Male and Female Sylamer genes. (B) Venn Diagram showing the intersection of Male and Female Sylamer genes with schistosome-conserved miRNA targets as found using TargetScan with conservation + miRanda. The overlap represents transcripts with highly conserved sma-miR-277 target sites across the three <i>Schistosome</i> spp (S. <i>mansoni</i>, <i>S</i>. <i>haematobium</i> and S. <i>japonicum</i>) that are also significantly down regulated during worm development.</p

Sma-miR-4989 is expressed in the cells surrounding the oesophagus and cells of the tegument in adult worms.

<p>Whole mount <i>in situ</i> hybridisation for (A) <i>cathepsin B</i>, (B) sma-miR-124a-3p (<i>124a</i>), and (C) sma-miR-4989. (D) Fluorescence <i>in situ</i> hybridisation showing the colocalization of sma-miR-4989 with four co-expressed tegument-specific mRNAs (<i>calpain</i>, <i>npp-5</i>, <i>annexin</i> and <i>gtp-4</i>). Nuclei are stained with DAPI and shown in blue. Anterior of worms is to the left in A-C. Scale Bars: A-C 100 μm; D 10 μm.</p

Features of miRNAs used in this study.

<p>Seed sequences highlighted in bold represent seeds common to multiple miRNAs. The seed highlighted in italics possesses a 7mer(3) seed equivalent to the 7mer(2) seed of the bold seed sequences.</p>†<p>iPS cell promoting miRNAs:</p>1<p>D. Lu <i>et al.</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041762#pone.0041762-Lu1" target="_blank">[49]</a>,</p>2<p>R. Sridharan <i>et al.</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041762#pone.0041762-Sridharan1" target="_blank">[62]</a>.</p>§<p>miRNAs that inhibit iPS cell generation:</p>3<p>C.-S. Yang <i>et al.</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041762#pone.0041762-Yang1" target="_blank">[63]</a>.</p

Influence of the targets of individual miRNAs on the mouse molecular interaction network.

<p>Rows represent individual protein clusters derived from the Pathway Commons interaction network. Each cluster was functionally annotated using Gene Ontology enrichment analysis (Right). The most enriched GO Terms are displayed. Clusters are numbered in descending size order (X#), the number of genes in each cluster are recorded in parentheses. Each column within the heat map represents an individual miRNA target set. Those miRNAs in black represent gene lists that were derived as part of this study, those in red represent gene lists derived from Melton <i>et al. </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041762#pone.0041762-Melton1" target="_blank">[40]</a> (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041762#s4" target="_blank">Materials and Methods</a>). The ‘all’ list is a combined set of targets represented in any of the lists derived as part of this study. For each target list the number of genes represented in the interaction network are recorded in parentheses. The relative influence of the targets of each miRNA upon each cluster is ascribed according to the number of nodes adjacent to miRNA-targeted nodes, which reside within a given cluster (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041762#s4" target="_blank">Materials and Methods</a>). The purple to yellow gradient reflects increasing miRNA influence. The green numbers represent the number of genes targeted by the specified miRNA within the respective protein cluster.</p

Candidate <i>Dgcr8</i>-independent miRNAs.

<p>Presented are the candidate <i>Dgcr8</i>-independent miRNAs alongside those identified as <i>Dgcr8</i>-independent by Babiarz <i>et al.</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041762#pone.0041762-Babiarz1" target="_blank">[25]</a>. Included is the fold change of each miRNA seen in this study between heterozygous and <i>Dgcr8</i>-depleted cell lines (bold italics if they meet our fold change threshold) and the status of each miRNA in miRBase version 18.</p>*<p>Reasons for removal from miRBase (<a href="http://www.mirbase.org/" target="_blank">http://www.mirbase.org/</a>) as given in miRBase, except for miR-2142, which was identified for removal by this study (¹<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041762#pone.0041762-Schopman1" target="_blank">[42]</a>, ²<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041762#pone.0041762-Chiang1" target="_blank">[41]</a>).</p

The use of global expression profiles to determine miRNA-dependent transcriptional effects.

<p><b>A</b>) Sylamer analysis of expression profiles following the transfection of miRNA mimics into <i>Dgcr8</i>^gt1/tm1 cells. Array probes and associated transcripts were ordered according to their log fold change in those cells transfected with a miRNA mimic (miR-302a, miR-106a, and miR-21) when compared to those transfected with a control duplex (cel-miR-239b). These lists were then subjected to the Sylamer analysis (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041762#pone-0041762-g002" target="_blank">Figure 2</a> for a description of Sylamer plots). Transcripts relatively down regulated in the cells transfected with the test miRNAs are to the left, while those relatively down-regulated in the controls are to the right. <b>B</b>) GSEA enrichment plots <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041762#pone.0041762-Subramanian1" target="_blank">[29]</a> depicting the enrichment of the transcripts within the miRNA target lists for miR-302a, miR-106a and miR-21 within regions of a list of transcripts ordered according to expression following the depletion of <i>Dgcr8</i>. The relative expression of transcripts in heterozygous cell lines compared <i>Dgcr8</i>-depleted cell lines is plotted on the x-axis, ordered according to log fold change, with those genes up-regulated in homozygous mutant cell lines to the left. Black lines on the horizontal axis represent the positions of the miRNA targets within the ordered transcript lists. The green line represents the “running” enrichment score at each position progressing through the gene list. If the maximum deviation of the “running” enrichment score from 0 is positive this implies an enrichment of miRNA targets amongst those genes up-regulated in the <i>Dgcr8</i>-depleted cells. Conversely if the maximum deviation is negative the targets are relatively enriched amongst down-regulated genes.</p