The biogenesis, biology and characterization of circular RNAs

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.

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Fig. 1: The biogenesis of circRNAs.
Fig. 2: Methodologies used to detect and quantify the expression of circRNAs.
Fig. 3: General mechanisms of circRNA functions.
Fig. 4: circRNAs acting as miRNA or protein sponges.
Fig. 5: Methodologies for the functional characterization of circRNAs.

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Acknowledgements

This work was supported by a grant to L.S.K. from the Carlsberg Foundation (CF16-0087).

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L.S.K. researched data for the article. All of the authors wrote and edited the manuscript before submission.

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Correspondence to Lasse S. Kristensen.

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Glossary

Viroids

Small, circular, single-stranded RNA molecules (246–401 nucleotides) that are uncoated and do not encode proteins. They are pathogenic to higher plants.

Alternative splicing

A mechanism by which different forms of a mature RNA can be generated from the same primary RNA by the use of different splice sites.

Alu elements

The most abundant primate-specific DNA transposable elements. These are highly repetitive and composed of ~300 bases.

Innate immune system

The first-line host defence to confine and combat infection.

Lariat formation

Splicing intermediates formed when the 5′ end of the intron being removed is joined to the branch-point adenosine with a 2′,5′-phosphodiester linkage, creating a lasso-shaped molecule.

Debranching

The hydrolysis of 2′,5′-phosphodiester bonds in intron lariats by the lariat debranching enzyme, encoded by DBR1. This hydrolysis converts the intron lariat into a linearized intron, which is subsequently degraded.

Backsplice junction (BSJ) region

The only region of a circular RNA (circRNA) that is distinct from the corresponding linear RNA at the primary sequence level. It is generated through the backsplicing event that generates the circRNA and is composed of a canonical 5′ splice site sequence joined to an upstream 3′ splice site sequence.

Droplet digital PCR

A quantitative PCR method that uses microfluidics (oil–water separation) to amplify individual nucleic acids within individual droplets in the same tube. By measuring the fluorescence signal in each droplet, the copy number of the target molecule can be determined.

Long-term haematopoietic stem cells

Haematopoietic stem cells that are defined by specific surface markers and can self-renew infinitely and differentiate to all cell types within the blood and immune system.

Pattern recognition receptor

Host receptors that recognize molecules typical for pathogens. Upon recognition of pathogen-associated patterns, the innate immune system is activated.

Internal ribosome entry sites

Structural RNA elements that enable the initiation of a cap-independent translation.

Argonaute-crosslinking and immunoprecipitation

A method to identify and map microRNAs bound to AGO proteins and the target transcripts associated with them.

Polysome profiling

A technique to study the translatome based on a sucrose-gradient separation of untranslated and translated RNA transcripts; translated RNA transcripts are associated with polysomes.

Ribosome footprinting

A technique to measure translation by the high-throughput sequencing of ribosome-protected RNA fragments, which determines the position of ribosomes at codon resolution.

Locked nucleic acids

A modified RNA nucleotide in which the ribose moiety is modified with a methylene bridge connecting the 2′ oxygen and 4′ carbon. It has an increased affinity for its complementary nucleotide relative to traditional DNA or RNA oligonucleotides.

Unlocked nucleic acids

An acyclic RNA nucleotide that lacks the C2′–C3′ bond of the ribose moiety found in traditional RNA. It has a decreased affinity for its complementary nucleotide relative to traditional DNA or RNA oligonucleotides.

Passenger disabled siRNA

A small interfering RNA (siRNA) in which an intact antisense strand is complemented with a fragmented sense strand. These siRNAs, which are known as small internally segmented interfering RNAs, eliminate off-target effects by only allowing the functional incorporation of the antisense strand into the RNA-induced silencing complex (RISC).

AgoshRNAs

Short hairpin RNAs that are characterized by a relatively short base-paired stem, which allows them to avoid cleavage by Dicer. Instead, they are processed by the slicer activity of Ago2, which creates a single guide RNA strand that targets a specific RNA for degradation and has less off-target effects as no passenger strand is created.

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Kristensen, L.S., Andersen, M.S., Stagsted, L.V.W. et al. The biogenesis, biology and characterization of circular RNAs. Nat Rev Genet 20, 675–691 (2019). https://doi.org/10.1038/s41576-019-0158-7

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