Thousands of circRNAs are reproducibly detected in human blood.

<p>(a) Total RNA was extracted from human whole blood samples and rRNA was depleted. cDNA libraries were synthesized using random primer and subjected to sequencing. circRNAs were detected as previously described [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141214#pone.0141214.ref020" target="_blank">20</a>]. Sequencing reads that map continuously to the human reference genome were disregarded. From unmapped reads anchors were extracted and independently mapped. Anchors that align consecutively indicate linear splicing events 1) whereas alignment in reverse orientation indicates head-to-tail splicing as observed for circular RNAs 2). After extensive filtering of linear splicing events and circRNA candidates (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141214#sec006" target="_blank">Methods</a>) the genomic coordinates and additional information such as read count and annotation are documented (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141214#pone.0141214.s010" target="_blank">S1 Table</a>) and are available at the circular RNA database circbase.org [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141214#pone.0141214.ref038" target="_blank">38</a>]. (b) circRNA candidate expression in human whole blood samples from two donors, ECDF = empirical cumulative distribution function. circRNA candidates tested in this study are annotated as numbers. Right panel: mRNA and lncRNA (n = 17,282) expression per gene in two blood samples in transcripts per million (TPM), RNAs with putative circular isoforms (n = 2,523) are highlighted in blue; R-values: Spearman correlation for RNAs found in both samples. (c) ENSEMBL genome annotation for reproducibly detected circRNA candidates (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141214#pone.0141214.s001" target="_blank">S1 Fig</a>). Number of circRNAs with at least one splice site in each category is given. (d) Number of distinct circRNA candidates per gene. y-axis = log₂(circRNA frequency+1). Gene names with the highest numbers are highlighted. (e) Expression level of top 8 circRNA candidates measured with sequencing (left panel) and divergent primer in qPCR (right); Ct = cycle threshold, linear control genes VCL and TFRC were measured with convergent primer.</p

Top expressed blood circRNAs dominate over linear RNA isoforms.

<p>(a) Example for the read coverage of a top expressed blood circRNA produced from the PCNT gene locus (<a href="http://genome.ucsc.edu/" target="_blank">http://genome.ucsc.edu/</a> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141214#pone.0141214.ref036" target="_blank">36</a>]). Data are shown for the human HEK293 cell line [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141214#pone.0141214.ref030" target="_blank">30</a>] and two biologically independent blood RNA preparations. (b) Relative expression and raw Ct values of top expressed blood circRNAs and corresponding linear isoforms in HEK293 cells and whole blood (c).</p

Circular to linear RNA isoform expression is high in blood compared to other tissues.

<p>(a) Comparison of circular to linear RNA isoforms in blood. circRNAs were measured by head-to-tail spanning reads. As a proxy for linear RNA expression median linear splice site spanning reads were counted. Data are shown for one replicate each of blood, cerebellum (b) and liver (c). Relative fraction of circRNA candidates with higher expression than linear isoforms are given as insets (>4x in red, >1x in black in brackets). In (a) eight tested circRNA candidates are indicated by numbers, and circRNAs derived from hemoglobin are marked. (d) mean circular-to-linear RNA expression ratio for the same samples, in two biological independent replicates. Error bars indicate the standard error of the mean, *** denotes P <0.001 permutation test on pooled replicate data (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141214#sec006" target="_blank">Methods</a>). For clarity, panels (a-c) represent expression datasets for one replicate per sample (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141214#pone.0141214.t001" target="_blank">Table 1</a>).</p

circRNAs are highly expressed in blood.

<p>Summary of sequencing results for blood RNA compared to liver and cerebellum samples, for each tissue data from two donors were analyzed (* denotes ENCODE dataset, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141214#pone.0141214.s013" target="_blank">S4 Table</a>.)</p><p>circRNAs are highly expressed in blood.</p

Signal-to-Noise Ratios of the PicTar Single Target Site Predictions

<div><p>For both set 1 and set 2 the predicted number of anchor sites for 46 unique microRNAs, conserved in all flies, and corresponding randomized microRNAs (averaged over five cohorts) and the respective signal-to-noise ratio (indicated above the bars) are shown with and without using free energy filtering of anchor sites for UTRs with either masked and unmasked repeats.</p> <p>(A) Predictions for set 1 with anchor sites conserved in the <i>melanogaster</i> and <i>obscura</i> groups.</p> <p>(B) Predictions for set 1 with anchor sites conserved in all flies.</p> <p>(C) Predictions for set 2 with anchor sites conserved in the <i>melanogaster</i> and <i>obscura</i> groups.</p> <p>(D) Predictions for set 2 with anchor sites conserved in all flies.</p></div

Length Distribution of Repeat Elements in 3′ UTRs of Human and <i>D. melanogaster</i>

<p>Data for set 1 on a logarithmic scale. The distribution peaks strongly for both species at a length of 11 nucleotides and decays exponentially for longer repeat elements in <i>D. melanogaster</i>. Up to a length of roughly 50 nucleotides, both distributions are very similar, while for longer elements the distribution for human no longer decays exponentially, but has a broad tail with another significant peak at a length of approximately 300 nucleotides.</p

Lengths Distribution of 3′ UTRs in Human and <i>D. melanogaster</i>

<p>Data for set 1 and set 2 on a logarithmic scale. The distribution decays exponentially with increasing length in human much slower than in <i>D. melanogaster</i>. The average 3′ UTR lengths in human and <i>D. melanogaster</i> are approximately 900 and approximately 400 nucleotides, respectively.</p

Number of Predicted Target Genes for Homologous microRNAs between Mammals and Flies

<p>Scatter plot for relative numbers of targeted genes predicted for homologous microRNAs in mammals and flies. The ratio of the number of predicted target genes of a microRNA and the average number of putative targeted genes per microRNA are plotted in mammals (<i>y</i>-axis) versus flies (<i>x</i>-axis). Conservation in flies included the <i>melanogaster</i> and <i>obscura</i> groups. Outliers (with a ratio of relative numbers of predicted target genes larger than 3.0 or smaller then 0.33) are circled. The microRNA identifiers refer to microRNAs annotated in <i>D. melanogaster</i>.</p

The CCR4-NOT Complex Mediates Deadenylation and Degradation of Stem Cell mRNAs and Promotes Planarian Stem Cell Differentiation

<div><p>Post-transcriptional regulatory mechanisms are of fundamental importance to form robust genetic networks, but their roles in stem cell pluripotency remain poorly understood. Here, we use freshwater planarians as a model system to investigate this and uncover a role for CCR4-NOT mediated deadenylation of mRNAs in stem cell differentiation. Planarian adult stem cells, the so-called neoblasts, drive the almost unlimited regenerative capabilities of planarians and allow their ongoing homeostatic tissue turnover. While many genes have been demonstrated to be required for these processes, currently almost no mechanistic insight is available into their regulation. We show that knockdown of planarian Not1, the CCR4-NOT deadenylating complex scaffolding subunit, abrogates regeneration and normal homeostasis. This abrogation is primarily due to severe impairment of their differentiation potential. We describe a stem cell specific increase in the mRNA levels of key neoblast genes after <i>Smed-not1</i> knock down, consistent with a role of the CCR4-NOT complex in degradation of neoblast mRNAs upon the onset of differentiation. We also observe a stem cell specific increase in the frequency of longer poly(A) tails in these same mRNAs, showing that stem cells after <i>Smed-not1</i> knock down fail to differentiate as they accumulate populations of transcripts with longer poly(A) tails. As other transcripts are unaffected our data hint at a targeted regulation of these key stem cell mRNAs by post-transcriptional regulators such as RNA-binding proteins or microRNAs. Together, our results show that the CCR4-NOT complex is crucial for stem cell differentiation and controls stem cell-specific degradation of mRNAs, thus providing clear mechanistic insight into this aspect of neoblast biology.</p></div

<i>Smed-not1(RNAi)</i> animals maintain mitotic neoblasts.

<p>(A) Quantification of mitosis by counting of h3p-positive cells in whole mount immunohistochemistry on <i>control(RNAi)</i> and <i>Smed-not1(RNAi)</i> animals 5, 10, 15 and 20 days after RNAi (N = 7 per time point). No significant differences are detected. Representative <i>control(RNAi)</i> (B) and different <i>Smed-not1(RNAi)</i> worms (C–E) 20 days after RNAi, immunostained with the mitotic marker h3p (h3p, green) and counterstained with phospho-tyrosine (p-tyr, red) in order to show head regression defects. <i>Smed-not1(RNAi)</i> animals still display detectable mitotic cells, even as head regression defects occur. The number of mitotic cells detected is smaller in the animals with the most severe head regression defects (D–E). Anterior is to the left. Scale bars: 500 µm. (F) Quantification of FACS sorted X1 cells in <i>control(RNAi)</i> and <i>Smed-not1(RNAi)</i> animals 10 and 15 days after RNAi, and wild type irradiated animals. While no significant differences are observed 10 days after knock down, <i>Smed-not1(RNAi)</i> animals show a reduced but significant decrease in percentage of X1 cells. Error bars represent standard deviation and asterisks represent statistical significance. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004003#pgen.1004003.s004" target="_blank">Figure S4</a>.</p