Circular RNAs are abundant, conserved, and associated with ALU repeats
TL;DR: High-throughput sequencing of libraries prepared from ribosome-depleted RNA with or without digestion with the RNA exonuclease showed that ecircRNAs are abundant, stable, conserved and nonrandom products of RNA splicing that could be involved in control of gene expression.
Abstract: Circular RNAs composed of exonic sequence have been described in a small number of genes. Thought to result from splicing errors, circular RNA species possess no known function. To delineate the universe of endogenous circular RNAs, we performed high-throughput sequencing (RNA-seq) of libraries prepared from ribosome-depleted RNA with or without digestion with the RNA exonuclease, RNase R. We identified >25,000 distinct RNA species in human fibroblasts that contained non-colinear exons (a "backsplice") and were reproducibly enriched by exonuclease degradation of linear RNA. These RNAs were validated as circular RNA (ecircRNA), rather than linear RNA, and were more stable than associated linear mRNAs in vivo. In some cases, the abundance of circular molecules exceeded that of associated linear mRNA by >10-fold. By conservative estimate, we identified ecircRNAs from 14.4% of actively transcribed genes in human fibroblasts. Application of this method to murine testis RNA identified 69 ecircRNAs in precisely orthologous locations to human circular RNAs. Of note, paralogous kinases HIPK2 and HIPK3 produce abundant ecircRNA from their second exon in both humans and mice. Though HIPK3 circular RNAs contain an AUG translation start, it and other ecircRNAs were not bound to ribosomes. Circular RNAs could be degraded by siRNAs and, therefore, may act as competing endogenous RNAs. Bioinformatic analysis revealed shared features of circularized exons, including long bordering introns that contained complementary ALU repeats. These data show that ecircRNAs are abundant, stable, conserved and nonrandom products of RNA splicing that could be involved in control of gene expression.
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TL;DR: It is found that a human circRNA, antisense to the cerebellar degeneration-related protein 1 transcript (CDR1as), is densely bound by microRNA (miRNA) effector complexes and harbours 63 conserved binding sites for the ancient miRNA miR-7.
Abstract: Circular RNAs (circRNAs) in animals are an enigmatic class of RNA with unknown function. To explore circRNAs systematically, we sequenced and computationally analysed human, mouse and nematode RNA. We detected thousands of well-expressed, stable circRNAs, often showing tissue/developmental-stage-specific expression. Sequence analysis indicated important regulatory functions for circRNAs. We found that a human circRNA, antisense to the cerebellar degeneration-related protein 1 transcript (CDR1as), is densely bound by microRNA (miRNA) effector complexes and harbours 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 zebrafish 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, suggesting previously unrecognized regulatory potential of coding sequences.
5,922 citations
TL;DR: Understanding this novel RNA crosstalk will lead to significant insight into gene regulatory networks and have implications in human development and disease.
Abstract: Recent reports have described an intricate interplay among diverse RNA species, including protein-coding messenger RNAs and non-coding RNAs such as long non-coding RNAs, pseudogenes and circular RNAs. These RNA transcripts act as competing endogenous RNAs (ceRNAs) or natural microRNA sponges — they communicate with and co-regulate each other by competing for binding to shared microRNAs, a family of small non-coding RNAs that are important post-transcriptional regulators of gene expression. Understanding this novel RNA crosstalk will lead to significant insight into gene regulatory networks and have implications in human development and disease.
2,869 citations
TL;DR: This Review describes special events in the lifetimes of lncRNAs — before, during and after transcription — and discusses how these events ultimately shape the unique characteristics and functional roles of lNCRNAs.
Abstract: Long non-coding RNAs (lncRNAs) are a diverse class of RNAs that engage in numerous biological processes across every branch of life. Although initially discovered as mRNA-like transcripts that do not encode proteins, recent studies have revealed features of lncRNAs that further distinguish them from mRNAs. In this Review, we describe special events in the lifetimes of lncRNAs - before, during and after transcription - and discuss how these events ultimately shape the unique characteristics and functional roles of lncRNAs.
2,568 citations
TL;DR: Advances in high-throughput RNA sequencing and circRNA-specific computational tools have driven the development of state-of-the-art approaches for their identification, and novel approaches to functional characterization are emerging.
Abstract: Circular RNAs (circRNAs) are covalently closed, endogenous biomolecules in eukaryotes with tissue-specific and cell-specific expression patterns, whose biogenesis is regulated by specific cis-acting elements and trans-acting factors. Some circRNAs are abundant and evolutionarily conserved, and many circRNAs exert important biological functions by acting as microRNA or protein inhibitors ('sponges'), by regulating protein function or by being translated themselves. Furthermore, circRNAs have been implicated in diseases such as diabetes mellitus, neurological disorders, cardiovascular diseases and cancer. Although the circular nature of these transcripts makes their detection, quantification and functional characterization challenging, recent advances in high-throughput RNA sequencing and circRNA-specific computational tools have driven the development of state-of-the-art approaches for their identification, and novel approaches to functional characterization are emerging.
2,372 citations
TL;DR: Evidence that animal circRNAs are generated cotranscriptionally and that their production rate is mainly determined by intronic sequences is provided and it is demonstrated that circularization and splicing compete against each other.
2,225 citations
Cites background from "Circular RNAs are abundant, conserv..."
...As previously reported for other circRNAs (Jeck et al., 2013;Memczak et al., 2013), circMbl is unusually stable and its level did not decrease in theassayed time frame....
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...…asmiRNA sponges (Hansen et al., 2013; Memczak et al., 2013), but no function is known for the other thousands of circRNAs that have been found across the animal kingdom (Danan et al., 2012; Hansen et al., 2013; Jeck et al., 2013; Memczak et al., 2013; Salzman et al., 2012, 2013; Wang et al., 2014)....
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..., 2013), but no function is known for the other thousands of circRNAs that have been found across the animal kingdom (Danan et al., 2012; Hansen et al., 2013; Jeck et al., 2013; Memczak et al., 2013; Salzman et al., 2012, 2013; Wang et al., 2014)....
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...Moreover, these introns in humans are enriched in ALU repeats (Jeck et al., 2013), and in the single case of the Sry circRNA it has been shown that intronic elements can be sufficient to promote circularization (Dubin et al....
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...This is likely due to the fact that circRNAs are flanked by considerably longer introns (see Jeck et al., 2013 and Figure S3B), which are less efficiently spliced cotranscriptionally (Khodor et al., 2012)....
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References
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TL;DR: A new method of total RNA isolation by a single extraction with an acid guanidinium thiocyanate-phenol-chloroform mixture is described, providing a pure preparation of undegraded RNA in high yield and can be completed within 4 h.
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4,016 citations
"Circular RNAs are abundant, conserv..." refers background in this paper
...Though the bulk of these noncoding RNAs consist of the rRNA and tRNA apparatus required for translation, it has been increasingly appreciated that noncoding products comprise a diverse set of species relevant in core biological processes and disease (Esteller 2011)....
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