Expanded identification and characterization of mammalian circular RNAs
ABSTRACT: The recent reports of two circular RNAs (circRNAs) with strong potential to act as microRNA (miRNA) sponges suggest that circRNAs might play important roles in regulating gene expression. However, the global properties of circRNAs are not well understood. We developed a computational pipeline to identify circRNAs and quantify their relative abundance from RNA-seq data. Applying this pipeline to a large set of non-poly(A)-selected RNA-seq data from the ENCODE project, we annotated 7,112 human circRNAs that were estimated to comprise at least 10% of the transcripts accumulating from their loci. Most circRNAs are expressed in only a few cell types and at low abundance, but they are no more cell-type–specific than are mRNAs with similar overall expression levels. Although most circRNAs overlap protein-coding sequences, ribosome profiling provides no evidence for their translation. We also annotated 635 mouse circRNAs, and although 20% of them are orthologous to human circRNAs, the sequence conservation of these circRNA orthologs is no higher than that of their flanking linear exons. The previously proposed miR-7 sponge, CDR1as, is one of only two circRNAs with more miRNA sites than expected by chance, with the next best miRNA-sponge candidate deriving from a primate-specific zinc-finger gene, ZNF91. These results provide a new framework for future investigation of this intriguing topological isoform while raising doubts regarding a biological function of most circRNAs. Examination of 9 samples in 1 cell type Note: The ENCODE data we used are under GEO SuperSeries GSE26284 (all samples labeled "_cell_total"). But they were not used in the processing of the U2OS data.
Project description:We determined circRNA abundance in fly Heads and S2 cells by generating and analyzing high-throughput RNA-sequencing libraries prepared from rRNA-depleted RNA. In order to determine whether the observed sequencing reads are due to bona fide circRNAs, we pre-treated the RNA with RNAse-R before the rRNA-depletion procedure. Indeed, most of the identified circRNAs were more enriched in comparison to the canonical mRNA isoforms following the RNAse-R treatment. We compare circRNA levels in wt (Canton S) flies with flies carrying the C4 ("slow polymerase") mutation. 4 samples of Drosophila Canton S and 4 samples of flies carrying the C4 ("slow polymerase") mutation. For each sample, one library was prepared from RNA after RNaseR treatment and the second from RNA with without treatment (mock). RNA library from one Canton Sample was used for stranded libray preprepation.
Project description:Circular RNAs (circRNAs) are widespread circular forms of non-coding RNAs with largely unknown function. Because stimulation of mammary cells with the epidermal growth factor (EGF) leads to dynamic changes in the abundance of both coding and non-coding RNA molecules, and culminates in the acquisition of a robust migratory phenotype, this cellular model might disclose functions of circRNAs. Here we show that circRNAs of EGF-stimulated mammary cells are stably expressed, while mRNAs and micro-RNAs change within minutes. In general, the circRNAs we detected are relatively long-lived and weakly expressed. Interestingly, they are almost ubiquitously co-expressed with the corresponding linear transcripts, and the respective, shared promoter regions are more active compared to genes producing linear isoforms only. These findings imply that altered abundance of circRNAs, unlike changes in the levels of other RNAs, might not play critical roles in signaling cascades and downstream transcriptional networks that rapidly commit cells to specific outcomes. Detection of circRNAs from RNA-Seq – triplicate
Project description:Circular RNAs (circRNAs) are an endogenous class of animal RNAs. Despite their abundance, their function and expression in the nervous system are unknown. Therefore, we sequenced RNA from different brain regions, primary neurons, isolated synapses, as well as during neuronal differentiation. Using these and other available data, we discovered and analyzed thousands of neuronal human and mouse circRNAs. circRNAs were extraordinarily enriched in the mammalian brain, well conserved in sequence, often expressed as circRNAs in both human and mouse, and sometimes even detected in Drosophila brains. circRNAs were overall upregulated during neuronal differentiation, highly enriched in synapses, and often differentially expressed compared to their mRNA isoforms. circRNA expression correlated negatively with expression of the RNA-editing enzyme ADAR1. Knockdown of ADAR1 induced elevated circRNA expression. Together, we provide a circRNA brain expression atlas and evidence for important circRNA functions and values as biomarkers. To assess circRNA expression in mammalian brain, we sequenced and analyzed mouse brain regions (hippocampus, cerebellum, prefrontal cortex and olfactory bulb), various neuronal differentiation (mouse P19 and human SH-SY5Y cells) and maturation (mouse cortical neurons) stages, and subcellular compartments in mouse (synaptoneurosomal fraction, cytoplasmic fraction, whole brain lysate).
Project description:Circular RNAs (circRNAs) in animals are an enigmatic class of RNAs with unknown function. To systematically explore circRNAs, we sequenced and computationally analyzed human, mouse and nematode RNA. We detected thousands of well-expressed, stable circRNAs, with oftentimes tissue/developmental stage specific expression. Sequence analysis suggested important regulatory functions for circRNAs. Indeed, we discovered that human circRNA CDR1as is densely bound by miRNA effector complexes and harbors 63 conserved binding sites for the ancient miRNA miR-7. Further analyses indicated that CDR1as functions to bind miR-7 in neuronal tissues. Human CDR1as expression in zebra fish impaired midbrain development similar to knocking down miR-7, suggesting that CDR1as is a miRNA antagonist with a miRNA binding capacity ten times higher than any other known transcript. Together, our data provide evidence that circRNAs form a large class of post-transcriptional regulators. Numerous circRNAs form by head-to-tail splicing of exons, indicating previously unrecognized regulatory potential of coding sequences. 1 Sample
Project description: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. Sequencing of blood RNA from five healthy individuals (biological replicates) plus technical replicate of one sample and detection of circRNAs.
Project description:Because maturing oocytes and early embryos lack transcription, posttranscriptional regulatory processes must control their development. To better understand this control, we profiled translational efficiencies and poly(A)-tail lengths throughout Drosophila oocyte maturation and early embryonic development. The correspondence between translational-efficiency changes and tail-length changes indicated that tail-length changes broadly reshape translational activity until gastrulation, when this coupling disappears. Relative changes were largely retained in the absence of poly(A)-tail lengthening, which indicated that selective poly(A)-tail shortening primarily specifies the changes. Many translational changes depended on PAN GU and Smaug, and both acted primarily through tail-length changes. Our results also revealed tail-length–independent mechanisms of translational control that repressed translation regardless of tail-length changes during oocyte maturation, maintained translation despite tail-length shortening during oocyte maturation, and prevented detectable translation of bicoid and several other mRNAs before egg activation. In addition to these fundamental insights, our results provide valuable resources for future studies. 42 samples analyzed using RNA-seq, ribosome footprint profiling, and PAL-seq.
Project description:We report HERV-K rec iCLIP-seq binding data, ribosome profiling data, and RNA-seq from ELF1 naïve hESC and RNA-seq from NCCIT cells. HERV-K Rec iCLIP-seq: 2 replicates in NCCIT. Ribosome profiling: 4 replicates each of Rec-overexpressing NCCIT vs. control NCCIT; RNAseq: 3 replicates each of HERV-K Rec siRNA vs. control siRNA in NCCIT; RNA-seq: 3 replicates each of ELF1 naïve hESC vs. primed hESC.
Project description:In platelets, splicing and translation occur in the absence of a nucleus. However, the integrity and stability of mRNAs derived from megakaryocyte progenitor cells remain poorly quantified on a transcriptome-wide level. As circular RNAs (circRNAs) are resistant to degradation by exonucleases, their abundance relative to linear RNAs can be used as a surrogate marker for mRNA stability in the absence of transcription. Here we show that circRNAs are enriched in human platelets 17-188 fold relative to nucleated tissues, and 14-26 fold relative to samples digested with RNAseR to selectively remove linear RNA. We compare RNAseq read depths inside and outside circRNAs to provide in silico evidence of transcript circularity, show that exons within circRNAs are enriched ~13X in platelets relative to nucleated tissues, and identify 3162 genes significantly enriched for circRNAs including some where all RNAs appear to be derived from circular molecules. We also confirm that this is a feature of other anucleate cells through transcriptome sequencing of mature erythrocytes, demonstrate that circRNAs are not enriched in megakaryocytes, and that linear RNAs decay more rapidly than circRNAs in platelet preparations. Collectively, these results suggest that circulating platelets have lost on aveage over 90% of their progenitor mRNAs, and that translation in platelets occurrs against the backdrop of a highly degraded transcriptome. Finally, we find that transcripts classified as products of reverse transcriptase template switching are both enriched in platelets and resistant to decay, countering the recent suggestion that up to 50% of rearranged RNAs are artefacts. A single rRNA depleted total RNA sample was sequenced. This together with 25 publicly available rRNA depleted total RNA samples (including 3 from platelets) were analysed using PTESFinder v 1 (http://sourceforge.net/projects/ptesfinder-v1/) to identify back-splice junctions, characteristic of circRNA transcripts. The contribution of circRNA producing exons was analysed on a gene by gene basis as follows: All circRNA transcripts identified in any sample were first pooled to define exons which can contribute to circRNA generation using custom scripts (available on request). For each sample, expression estimates (RPKMI) across all circRNA producing exons were computed for each locus using the total size of exons (in bp) and the read counts mapping to them. Similarly, total size and exonic read counts for exons for which no circRNA were detected in any sample were used to compute expression estimates (RPKME) for non-circRNA producing exons for each locus. Abundance ratios (RPKMI/RPKME and RPKMI/RPKMI+RPKME) were calculated and compared between Platelets and human tissues using Wilcoxon signed-rank test. Please note that the '25sample_info_accn_no.txt' contains the accession numbers and tissue/cell type information for 25 samples analyzed together.
Project description:To explore the potential involvement of circular RNAs (circRNAs) in pancreatic ductal adenocarcinoma (PDAC) oncogenesis, we conducted circRNA profiling in six pairs of human PDAC and adjacent normal tissue by microarray. Our results showed that clusters of circRNAs were aberrantly expressed in PDAC compared with normal samples, and provided potential targets for future treatment of PDAC and novel insights into PDAC biology. Analyze circular RNA expression in pancreatic ductal adenocarcinoma (PDAC) by microarray platform.
Project description:Circular RNAs (circRNAs) are highly expressed in mammalian brains, with oftentimes conserved expression patterns. However, so far no circRNAs loss-of-function has been reported. The circRNA CDR1as has been proposed to function as a sponge for microRNA miR-7. We removed the CDR1as locus from the mouse genome, and performed a series of high-throughput experiments to characterize the gene expression in wild type and knockout mice. Our data reveal the first in vivo loss-of-function phenotype for a circRNA and show that interactions between circRNAs and miRNAs are important for healthy brain function. Overall design: We performed miRNA sequencing for three knockout and three wild type replicates in four brain regins - cerebellum, cortex, hippocampus (2 KO replicates) and olfactory bulb.