Detecting and characterizing circular RNAs
TL;DR: Evidence is emerging that some circRNAs might regulate microRNA (miRNA) function, and roles in transcriptional control have also been suggested.
Abstract: Circular RNA transcripts were first identified in the early 1990s but knowledge of these species has remained limited, as their study through traditional methods of RNA analysis has been difficult Now, novel bioinformatic approaches coupled with biochemical enrichment strategies and deep sequencing have allowed comprehensive studies of circular RNA species Recent studies have revealed thousands of endogenous circular RNAs in mammalian cells, some of which are highly abundant and evolutionarily conserved Evidence is emerging that some circRNAs might regulate microRNA (miRNA) function, and roles in transcriptional control have also been suggested Therefore, study of this class of noncoding RNAs has potential implications for therapeutic and research applications We believe the key future challenge for the field will be to understand the regulation and function of these unusual molecules
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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: The emerging functions and association of lncRNAs in different types of cancer and their potential implications in cancer diagnosis and therapy are reviewed.
Abstract: In addition to mutations or aberrant expression in the protein-coding genes, mutations and misregulation of noncoding RNAs, in particular long noncoding RNAs (lncRNA), appear to play major roles in cancer. Genome-wide association studies of tumor samples have identified a large number of lncRNAs associated with various types of cancer. Alterations in lncRNA expression and their mutations promote tumorigenesis and metastasis. LncRNAs may exhibit tumor-suppressive and -promoting (oncogenic) functions. Because of their genome-wide expression patterns in a variety of tissues and their tissue-specific expression characteristics, lncRNAs hold strong promise as novel biomarkers and therapeutic targets for cancer. In this article, we have reviewed the emerging functions and association of lncRNAs in different types of cancer and discussed their potential implications in cancer diagnosis and therapy. Cancer Res; 77(15); 3965-81. ©2017 AACR.
1,800 citations
TL;DR: A circRNA brain expression atlas and evidence for important circRNA functions and values as biomarkers are provided and discovered and analyzed thousands of neuronal human and mouse circRNAs.
1,759 citations
Cites background from "Detecting and characterizing circul..."
...Circular RNAs (circRNAs) have recently emerged as a large class of animal RNAs (Hansen et al., 2013; Jeck and Sharpless, 2014; Jeck et al., 2013; Lasda and Parker, 2014; Memczak et al., 2013; Salzman et al., 2012, 2013) with complex tissue- and stage-specific expression patterns....
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...Circular RNAs (circRNAs) have recently emerged as a large class of animal RNAs (Hansen et al., 2013; Jeck and Sharpless, 2014; Jeck et al., 2013; Lasda and Parker, 2014; Memczak et al., 2013; Salzman et al., 2012, 2013) with complex tissue- and stage-specific expression patterns. circRNAs are much…...
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TL;DR: The results provide evidence that circular RNA produced from precursor mRNA may have a regulatory role in human cells and characterize one abundant circRNA derived from Exon2 of the HIPK3 gene, termed circHIPK3.
Abstract: Circular RNAs (circRNAs) represent a class of widespread and diverse endogenous RNAs that may regulate gene expression in eukaryotes. However, the regulation and function of human circRNAs remain largely unknown. Here we generate ribosomal-depleted RNA sequencing data from six normal tissues and seven cancers, and detect at least 27,000 circRNA candidates. Many of these circRNAs are differently expressed between the normal and cancerous tissues. We further characterize one abundant circRNA derived from Exon2 of the HIPK3 gene, termed circHIPK3. The silencing of circHIPK3 but not HIPK3 mRNA significantly inhibits human cell growth. Via a luciferase screening assay, circHIPK3 is observed to sponge to 9 miRNAs with 18 potential binding sites. Specifically, we show that circHIPK3 directly binds to miR-124 and inhibits miR-124 activity. Our results provide evidence that circular RNA produced from precursor mRNA may have a regulatory role in human cells.
1,537 citations
TL;DR: It is shown that hundreds of circRNAs are regulated during human epithelial-mesenchymal transition (EMT) and that the production of over one-third of abundant circ RNAs is dynamically regulated by the alternative splicing factor, Quaking (QKI), which itself is regulated during EMT.
1,531 citations
Cites background from "Detecting and characterizing circul..."
...However, the advent of next generation sequencing has illuminated circRNAs as an entire class of abundant, non-coding RNAs ubiquitous among eukaryotes (Guo et al., 2014; Jeck and Sharpless, 2014; Jeck et al., 2013; Lasda and Parker, 2014; Memczak et al., 2013; Salzman et al., 2012, 2013; Wang et al., 2014a; Wilusz and Sharp, 2013; Zhang et al., 2014)....
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...Lacking 30 termini, circRNAs are non-polyadenylated and resistant to digestion of the RNAwith RNase R, a highly processive 30 exonuclease that non-specifically degrades linear RNA, but not circRNAs, attributes which are exploited in their sequencing and identification (Jeck and Sharpless, 2014)....
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...Lacking 30 termini, circRNAs are non-polyadenylated and resistant to digestion of the RNAwith RNase R, a highly processive 30 exonuclease that non-specifically degrades linear RNA, but not circRNAs, attributes which are exploited in their sequencing and identification (Jeck and Sharpless, 2014)....
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...…next generation sequencing has illuminated circRNAs as an entire class of abundant, non-coding RNAs ubiquitous among eukaryotes (Guo et al., 2014; Jeck and Sharpless, 2014; Jeck et al., 2013; Lasda and Parker, 2014; Memczak et al., 2013; Salzman et al., 2012, 2013; Wang et al., 2014a; Wilusz and…...
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...However, this intron-pairing phenomenon alone cannot explain how a single abundant transcript common to a multitude of cells can host cell-type specific circRNAs (Jeck and Sharpless, 2014; Salzman et al., 2013)....
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References
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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: This study serves as the first functional analysis of a naturally expressed circular RNA, ciRS-7, which contains more than 70 selectively conserved miRNA target sites, and is highly and widely associated with Argonaute proteins in a miR-7-dependent manner.
Abstract: MicroRNAs (miRNAs) are important post-transcriptional regulators of gene expression that act by direct base pairing to target sites within untranslated regions of messenger RNAs. Recently, miRNA activity has been shown to be affected by the presence of miRNA sponge transcripts, the so-called competing endogenous RNA in humans and target mimicry in plants. We previously identified a highly expressed circular RNA (circRNA) in human and mouse brain. Here we show that this circRNA acts as a miR-7 sponge; we term this circular transcript ciRS-7 (circular RNA sponge for miR-7). ciRS-7 contains more than 70 selectively conserved miRNA target sites, and it is highly and widely associated with Argonaute (AGO) proteins in a miR-7-dependent manner. Although the circRNA is completely resistant to miRNA-mediated target destabilization, it strongly suppresses miR-7 activity, resulting in increased levels of miR-7 targets. In the mouse brain, we observe overlapping co-expression of ciRS-7 and miR-7, particularly in neocortical and hippocampal neurons, suggesting a high degree of endogenous interaction. We further show that the testis-specific circRNA, sex-determining region Y (Sry), serves as a miR-138 sponge, suggesting that miRNA sponge effects achieved by circRNA formation are a general phenomenon. This study serves as the first, to our knowledge, functional analysis of a naturally expressed circRNA.
5,885 citations
TL;DR: Using a quantitative model, the first genome-scale prediction of synthesis rates of mRNAs and proteins is obtained and it is found that the cellular abundance of proteins is predominantly controlled at the level of translation.
Abstract: Gene expression is a multistep process that involves the transcription, translation and turnover of messenger RNAs and proteins. Although it is one of the most fundamental processes of life, the entire cascade has never been quantified on a genome-wide scale. Here we simultaneously measured absolute mRNA and protein abundance and turnover by parallel metabolic pulse labelling for more than 5,000 genes in mammalian cells. Whereas mRNA and protein levels correlated better than previously thought, corresponding half-lives showed no correlation. Using a quantitative model we have obtained the first genome-scale prediction of synthesis rates of mRNAs and proteins. We find that the cellular abundance of proteins is predominantly controlled at the level of translation. Genes with similar combinations of mRNA and protein stability shared functional properties, indicating that half-lives evolved under energetic and dynamic constraints. Quantitative information about all stages of gene expression provides a rich resource and helps to provide a greater understanding of the underlying design principles.
5,635 citations
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.
3,310 citations
TL;DR: It is found that PTENP1 is biologically active as it can regulate cellular levels of PTEN and exert a growth-suppressive role, and this analysis extended to other cancer-related genes that possess pseudogenes, and revealed a non-coding function for mRNAs.
Abstract: The canonical role of messenger RNA (mRNA) is to deliver protein-coding information to sites of protein synthesis. However, given that microRNAs bind to RNAs, we hypothesized that RNAs could possess a regulatory role that relies on their ability to compete for microRNA binding, independently of their protein-coding function. As a model for the protein-coding-independent role of RNAs, we describe the functional relationship between the mRNAs produced by the PTEN tumour suppressor gene and its pseudogene PTENP1 and the critical consequences of this interaction. We find that PTENP1 is biologically active as it can regulate cellular levels of PTEN and exert a growth-suppressive role. We also show that the PTENP1 locus is selectively lost in human cancer. We extended our analysis to other cancer-related genes that possess pseudogenes, such as oncogenic KRAS. We also demonstrate that the transcripts of protein-coding genes such as PTEN are biologically active. These findings attribute a novel biological role to expressed pseudogenes, as they can regulate coding gene expression, and reveal a non-coding function for mRNAs.
2,107 citations