A map of human circular RNAs in clinically relevant tissues

Abstract Cellular circular RNAs (circRNAs) are generated by head-to-tail splicing and are present in all multicellular organisms studied so far. Recently, circRNAs have emerged as a large class of RNA which can function as post-transcriptional regulators. It has also been shown that many circRNAs are tissue- and stage-specifically expressed. Moreover, the unusual stability and expression specificity make circRNAs important candidates for clinical biomarker research. Here, we present a circRNA expression resource of 20 human tissues highly relevant to disease-related research: vascular smooth muscle cells (VSMCs), human umbilical vein cells (HUVECs), artery endothelial cells (HUAECs), atrium, vena cava, neutrophils, platelets, cerebral cortex, placenta, and samples from mesenchymal stem cell differentiation. In eight different samples from a single donor, we found highly tissue-specific circRNA expression. Circular-to-linear RNA ratios revealed that many circRNAs were expressed higher than their linear host transcripts. Among the 71 validated circRNAs, we noticed potential biomarkers. In adenosine deaminase-deficient, severe combined immunodeficiency (ADA-SCID) patients and in Wiskott-Aldrich-Syndrome (WAS) patients’ samples, we found evidence for differential circRNA expression of genes that are involved in the molecular pathogenesis of both phenotypes. Our findings underscore the need to assess circRNAs in mechanisms of human disease. Key messages circRNA resource catalog of 20 clinically relevant tissues.circRNA expression is highly tissue-specific.circRNA transcripts are often more abundant than their linear host RNAs.circRNAs can be differentially expressed in disease-associated genes. Electronic supplementary material The online version of this article (10.1007/s00109-017-1582-9) contains supplementary material, which is available to authorized users

In vivo partial cellular reprogramming enhances liver plasticity and regeneration.

Mammals have limited regenerative capacity, whereas some vertebrates, like fish and salamanders, are able to regenerate their organs efficiently. The regeneration in these species depends on cell dedifferentiation followed by proliferation. We generate a mouse model that enables the inducible expression of the four Yamanaka factors (Oct-3/4, Sox2, Klf4, and c-Myc, or 4F) specifically in hepatocytes. Transient in vivo 4F expression induces partial reprogramming of adult hepatocytes to a progenitor state and concomitantly increases cell proliferation. This is indicated by reduced expression of differentiated hepatic-lineage markers, an increase in markers of proliferation and chromatin modifiers, global changes in DNA accessibility, and an acquisition of liver stem and progenitor cell markers. Functionally, short-term expression of 4F enhances liver regenerative capacity through topoisomerase2-mediated partial reprogramming. Our results reveal that liver-specific 4F expression in vivo induces cellular plasticity and counteracts liver failure, suggesting that partial reprogramming may represent an avenue for enhancing tissue regeneration

Identification and Characterization of Circular RNAs As a New Class of Putative Biomarkers in Human Blood.

Covalently closed circular RNA molecules (circRNAs) have recently emerged as a class of RNA isoforms with widespread and tissue specific expression across animals, oftentimes independent of the corresponding linear mRNAs. circRNAs are remarkably stable and sometimes highly expressed molecules. Here, we sequenced RNA in human peripheral whole blood to determine the potential of circRNAs as biomarkers in an easily accessible body fluid. We report the reproducible detection of thousands of circRNAs. Importantly, we observed that hundreds of circRNAs are much higher expressed than corresponding linear mRNAs. Thus, circRNA expression in human blood reveals and quantifies the activity of hundreds of coding genes not accessible by classical mRNA specific assays. Our findings suggest that circRNAs could be used as biomarker molecules in standard clinical blood samples

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

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

Analysis of Intron Sequences Reveals Hallmarks of Circular RNA Biogenesis in Animals

Circular RNAs (circRNAs) are a large class of animal RNAs. To investigate possible circRNA functions, it is important to understand circRNA biogenesis. Besides human ALU repeats, sequence features that promote exon circularization are largely unknown. We experimentally identified circRNAs in C. elegans. Reverse complementary sequences between introns bracketing circRNAs were significantly enriched in comparison to linear controls. By scoring the presence of reverse complementary sequences in human introns, we predicted and experimentally validated circRNAs. We show that introns bracketing circRNAs are highly enriched in RNA editing or hyperediting events. Knockdown of the double-strand RNA-editing enzyme ADAR1 significantly and specifically upregulated circRNA expression. Together, our data support a model of animal circRNA biogenesis in which competing RNA-RNA interactions of introns form larger structures that promote circularization of embedded exons, whereas ADAR1 antagonizes circRNA expression by melting stems within these interactions