Trends in Cell Biology
Volume 30, Issue 3, March 2020, Pages 226-240
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Review
Biogenesis and Functions of Circular RNAs Come into Focus

https://doi.org/10.1016/j.tcb.2019.12.004Get rights and content

Highlights

  • Circular RNAs are generated from many eukaryotic protein-coding genes when the pre-mRNA splicing machinery 'backsplices' and joins a downstream 5' splice site to an upstream 3' splice site.

  • Circular RNA biogenesis is often facilitated by base pairing between intronic repeat elements, and the expression of these transcripts is further controlled by the combinatorial action of RNA-binding proteins, the levels of core spliceosome components, and exon-skipping events.

  • Once generated, most circular RNAs are highly stable and accumulate in the cytoplasm after being exported from the nucleus in a length-dependent manner.

  • The biological functions of most circular RNAs remain unknown, but it is becoming increasingly clear that specific circular RNAs may modulate the activity of microRNAs or RNA-binding proteins, be translated to yield protein products, or regulate innate immune responses.

  • Circular RNAs are most abundant in neuronal tissues, accumulate with aging, and have functional roles in human diseases including cancer.

Many eukaryotic protein-coding genes are able to generate exonic circular RNAs. Most of these covalently linked transcripts are expressed at low levels, but some accumulate to higher levels than their associated linear mRNAs. We highlight several methodologies that have been developed in recent years to identify and characterize these transcripts, and which have revealed an increasingly detailed view of how circular RNAs can be generated and function. It is now clear that modulation of circular RNA levels can result in a variety of molecular and physiological phenotypes, including effects on the nervous system, innate immunity, microRNAs, and many disease-relevant pathways.

Section snippets

Circular RNAs are Generated from Many Eukaryotic Protein-Coding genes

Most eukaryotic genes are disrupted by intronic sequences that must be removed from nascent precursor mRNA (pre-mRNAs) by the spliceosome (reviewed in [1]). It had long been thought that most introns are removed sequentially and rapidly once they have been transcribed, thereby allowing each exon to be covalently linked to the next and a functional linear mRNA to be produced (Figure 1A, top). Nevertheless, splicing decisions are highly regulated, and many genes generate a variety of mature

Improved Methods for Genome-Wide Detection of Circular RNAs

Circular RNAs are most commonly detected using RNA-seq followed by identification of spliced reads that span backsplicing junctions (reviewed in [15]). Several computational algorithms to identify such reads have been developed, including algorithms that can reconstruct full-length circular RNAs [16], but there are striking differences in their sensitivity and precision [17,18]. For example, when the same RNA-seq dataset was analyzed using five different algorithms, ~40% of putative circular

Both Sequence Features and Trans-Acting Factors Dictate the Efficiency of Circular RNA Biogenesis

Early studies of the mouse Sry circular RNA revealed that ~50 kb of near-perfectly complementary intronic sequences flank this exon [6], and that inclusion of ~400 nt of the flanking repeats was sufficient for production of this circular RNA from the intervening exon [49]. It is now recognized that most circular RNAs are similarly flanked by longer than average introns that have complementary repeats, often Alu elements in human cells [8,32,41,50]. Base pairing between these intronic repeats is

Once Generated, Most Circular RNAs Are Stable and Traffic to the Cytoplasm

Owing to their covalently closed structure, circular RNAs are naturally resistant to digestion by RNA exonucleases and generally have much longer half-lives than linear RNAs (often 24 h or longer) [40]. The CDR1as/ciRS-7 circular RNA contains a near-perfect target site for the miR-671 microRNA and can be cleaved by Argonaute-2 (Ago2) to trigger transcript degradation [53,67], but this regulatory strategy appears to be unique to this locus. It is thus still largely unclear how circular RNAs are

Methods for Modulating Circular RNA Levels Have Revealed the First Phenotypes in Animal Models

The molecular and physiological functions of most circular RNAs remain unknown, but there has been increasing progress in this area as a result of the development of methods to overexpress and knock-down/out specific circular RNAs. Circular RNAs can be generated in vitro using self-splicing introns [75., 76., 77., 78.] or splint ligation approaches [79], and subsequently added to cells. There are also now plasmid- and virus-based methods for expressing circular RNAs of interest using the

Molecular Functions of Circular RNAs: Moving beyond Simple MicroRNA Sponging Models

To date the CDR1as/ciRS-7 circular RNA has been studied in most detail, in part because it harbors >60 evolutionarily conserved miR-7 binding sites [9,53]. This circular RNA is highly expressed in the brain, especially in excitatory neurons [83], and localizes to the cytoplasm and neuronal cell extensions [87]. Given its many binding sites for miR-7, it has long been assumed that CDR1as/ciRS-7 sequesters this microRNA and prevents it from regulating the expression of its target mRNAs (Figure 5A

Emerging Connections between Circular RNAs and the Immune System

The first identified circular RNAs were pathogenic plant viroids [100], and recent years have revealed an increasing number of DNA viruses that generate circular RNAs [101., 102., 103., 104., 105.]. It thus makes sense that eukaryotic immune systems may have evolved mechanisms to sense foreign circular RNAs. However, there are currently conflicting data on whether these transcripts are, in fact, immunogenic. Chen and colleagues have shown that addition of non-self circular RNAs (e.g.,

Functions for Circular RNAs in Cancer

Compared to normal cells, there is a general decrease in circular RNA levels in most cancer types, suggesting a connection between cellular proliferation and the steady-state levels of these transcripts [22,109]. Indeed, treating LNCaP prostate cancer cells with dinaciclib, a kinase inhibitor that decreases cellular proliferation, led to an overall increase (~50%) in circular RNA levels that was independent of changes in parent gene expression [22]. This suggests that circular RNAs may be

Concluding Remarks

In summary, recent work has continued to make it clear that circular RNAs are not simply rare oddities or errors of pre-mRNA splicing, but instead are tightly regulated transcripts that, at least in some cases, can perform important biological functions. Mechanisms that regulate the levels of many circular RNAs have been discovered, but many of the molecular details are still poorly understood and several key questions remain unanswered (see Outstanding Questions). It is important to keep in

Acknowledgments

We thank all members of the laboratory of J.E.W. for helpful discussions and suggestions. Research on circular RNAs in our laboratory is supported by National Institutes of Health (NIH) grants R35-GM119735 and R01-NS099371. J.E.W. is a Rita Allen Foundation Scholar.

Glossary

Alternative splicing
the process by which exons and introns are selectively included or excluded from the mature mRNA, thereby allowing genes to generate multiple distinct transcript isoforms that may have unique functions.
Backsplicing
the joining of a 5′ splice site to an upstream 3′ splice site by the spliceosome, thereby producing a mature circular RNA that has covalently linked ends.
Competing endogenous RNA (ceRNA)
an RNA that binds to a microRNA, thereby competitively limiting the ability of

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