Circular RNAs (circRNAs): Structure, Biogenesis, Functions, Disease Mechanisms, Biomarkers, and Therapeutic Applications

Table of Contents

• Introduction to Circular RNAs (circRNAs)
• Biogenesis via Back-Splicing
• Structural Properties and Stability of Circular RNAs (circRNAs)
• Mechanisms of Action of Circular RNAs (circRNAs)
• The miRNA Sponge Effect
• Protein Translation Potential
• Methods of Identification of Circular RNAs (circRNAs)
• Role of Circular RNAs (circRNAs) in Disease Pathogenesis
• Diagnostic Biomarkers of circRNAs
• Delivery Platforms
• Conclusions
• References

Introduction to Circular RNAs (circRNAs)

• Circular RNAs (circRNAs) are a class of endogenous RNA molecules with a covalently closed-loop structure, meaning they lack the free 5′ and 3′ ends found in linear RNAs.
• They were first discovered in the 1970s in plant viroids and viruses.
• For many years, circRNAs were considered splicing byproducts or non-functional "junk" RNAs produced due to errors during messenger RNA (mRNA) processing.
• Advances in high-throughput RNA sequencing technologies have demonstrated that circRNAs are widely expressed across eukaryotic organisms, including:
• Metazoans (animals)
• Plants
• Fungi
• More than 150,000 unique human circRNAs have been identified, and many are highly conserved across different species, suggesting important biological functions.
• Compared with their linear RNA counterparts, circRNAs are generally:
• Expressed at lower overall levels
• Highly tissue-specific
• Cell-type-specific in their expression patterns
• CircRNAs are especially abundant in the mammalian brain, where:
• Their expression levels are significantly higher than in most other tissues.
• They are frequently enriched in synaptosomes, indicating potential roles in neuronal communication and brain function.

Biogenesis via Back-Splicing

• The majority of circular RNAs (circRNAs) are generated from precursor messenger RNA (pre-mRNA) through a unique process called back-splicing.
• In back-splicing, a downstream 5′ splice donor site is covalently linked to an upstream 3′ splice acceptor site, forming a closed circular RNA molecule instead of a linear transcript.
• Unlike conventional splicing, back-splicing creates a covalently closed RNA loop with no free 5′ or 3′ ends.

Role of the Spliceosome

• Back-splicing is catalyzed by the canonical spliceosomal machinery, the same molecular complex responsible for conventional RNA splicing.
• This process occurs in competition with canonical linear splicing, meaning a pre-mRNA transcript can produce either:
• A linear mRNA, or
• A circular RNA (circRNA)

Regulatory Elements Controlling circRNA Biogenesis

Cis-Regulatory Elements

• Cis-elements are regulatory sequences located within the same RNA molecule that facilitate circularization.
• CircRNA formation is commonly promoted by intronic complementary sequences (ICSs) located in the introns flanking the circularized exons.
• In humans, Alu repetitive elements are the most well-known ICSs.
• Complementary pairing between these intronic sequences brings distant splice donor and acceptor sites into close proximity, promoting efficient back-splicing.

Trans-Acting Factors

• Trans-acting factors are proteins that regulate circRNA formation by interacting with pre-mRNA.
• Several RNA-binding proteins (RBPs) promote circularization by binding specific sequence motifs within flanking introns and bringing splice sites together through protein dimerization.
• Important RBPs involved in circRNA biogenesis include:
• Quaking (QKI)
• Muscleblind (MBL)
• NOVA2

Other Mechanisms of circRNA Formation

Although back-splicing is the predominant mechanism of circRNA biogenesis, alternative pathways also contribute to circRNA production:

Intron lariat-driven circularization

• Occurs during exon skipping, where skipped exons are enclosed within a lariat structure.
• If the lariat escapes normal processing, the exons can be circularized to form a circRNA.

Formation of Circular Intronic RNAs (ciRNAs)

• Generated from intron lariat structures that escape the normal debranching process.
• Instead of being degraded, these intronic lariats remain circular and function as circular intronic RNAs (ciRNAs).

Structural Properties and Stability of Circular RNAs (circRNAs)

• The defining feature of circular RNAs (circRNAs) is their covalently closed-loop structure, in which the RNA ends are joined by a 3′–5′ phosphodiester bond to form a continuous circular molecule.
• Unlike linear messenger RNAs (mRNAs), circRNAs lack both the 5′ 7-methylguanosine (m⁷G) cap and the 3′ polyadenylated [poly(A)] tail, as they do not have free 5′ or 3′ termini.
• The absence of free RNA ends makes circRNAs highly resistant to exonuclease-mediated degradation, including digestion by RNase R, which readily degrades most linear RNAs.
• Due to their circular structure, circRNAs exhibit exceptional stability and have significantly longer half-lives than linear RNAs.
• In mammary cells, the median half-life of circRNAs is approximately 18.8–23.7 hours, compared with only 4.0–7.4 hours for their corresponding linear RNA counterparts.
• The remarkable stability of circRNAs causes them to accumulate progressively with age, particularly in post-mitotic tissues such as neurons.
• Because of their long-term persistence and age-dependent accumulation, circRNAs are considered more reliable molecular markers of aging than linear mRNAs in many biological and biomedical studies.

Mechanisms of Action of Circular RNAs (circRNAs)

• Circular RNAs (circRNAs) regulate gene expression through multiple molecular mechanisms, influencing transcription, splicing, protein interactions, and RNA modifications.
• Transcriptional regulation: Nuclear-retained circRNAs, particularly exon–intron circular RNAs (EIciRNAs), interact with U1 small nuclear ribonucleoproteins (U1 snRNPs) and RNA Polymerase II (Pol II) to enhance the transcription of their parental genes.
• Circular intronic RNAs (ciRNAs) accumulate at transcription sites, where they function as positive regulators of parental gene expression by promoting transcription.
• Splicing interference: The formation of circRNAs competes with canonical linear splicing because both processes use the same precursor mRNA (pre-mRNA). As a result, increased circRNA production often reduces the inclusion of specific exons in linear messenger RNAs (mRNAs), thereby altering gene expression.
• Protein interactions: CircRNAs can function as protein sponges (decoys) by binding RNA-binding proteins (RBPs) and sequestering them in the cytoplasm, preventing their normal localization or biological activity.
• CircRNAs also act as protein scaffolds, bringing multiple proteins together to facilitate the formation of protein complexes and regulate protein–protein interactions.
• RNA modification (m⁶A): Many circRNAs contain N6-methyladenosine (m⁶A) modifications, which regulate their biogenesis, intracellular transport, stability, and translation potential, thereby influencing their biological functions.

The miRNA Sponge Effect of Circular RNAs (circRNAs)

• Many circular RNAs (circRNAs) function as competing endogenous RNAs (ceRNAs) by containing microRNA response elements (MREs) that bind and sequester microRNAs (miRNAs).
• By acting as miRNA sponges, circRNAs prevent miRNAs from binding to their target messenger RNAs (mRNAs), thereby relieving miRNA-mediated gene repression and increasing the expression of target genes.
• Although the miRNA sponge effect is one of the most well-known functions of circRNAs, most endogenous circRNAs are expressed at relatively low levels and contain only a few miRNA-binding sites, making them unlikely to function as highly effective miRNA super-sponges in most biological contexts.
• These findings suggest that other regulatory mechanisms, such as protein interactions and transcriptional regulation, may be more common functions of many circRNAs.

Key Examples

• CDR1as (ciRS-7) is one of the best-characterized circRNAs and contains more than 60 conserved binding sites for miR-7, making it a highly effective miRNA sponge, particularly in the brain, where it plays an important role in regulating neuronal gene expression.
• circSry, a testis-specific circRNA, contains 16 binding sites for miR-138 and regulates gene expression by sequestering this microRNA.
• circFOXO3 also functions as a miRNA sponge by containing microRNA response elements (MREs) that bind miR-149, miR-22, and miR-136, thereby modulating the activity of these microRNAs and influencing downstream gene expression.

Protein Translation Potential of Circular RNAs (circRNAs)

• Although circular RNAs (circRNAs) lack the 5′ cap and 3′ poly(A) tail required for canonical translation, some circRNAs can still be translated into proteins through cap-independent translation mechanisms.
• Cap-independent translation is primarily mediated by Internal Ribosome Entry Sites (IRESs) or N6-methyladenosine (m⁶A)-induced ribosome engagement sites (MIRESs), which enable ribosomes to initiate protein synthesis without a 5′ cap.
• Some circRNAs contain open reading frames (ORFs) along with appropriate start and stop codons, allowing them to encode functional peptides or proteins.
• circZNF609 is a well-characterized protein-coding circRNA that produces a protein involved in regulating myoblast proliferation.
• circMbl can be translated into a peptide in the brain, with translation becoming more prominent under starvation stress.
• circAβ, which is derived from the amyloid precursor protein (APP) gene, can produce an amyloid-beta-containing polypeptide that has been implicated in Alzheimer’s disease.
• Proteins translated from circRNAs may function as competitors, regulators, or functional partners of proteins encoded by their corresponding linear messenger RNAs, thereby contributing to diverse cellular and physiological processes.

Methods of Identification of Circular RNAs (circRNAs)

• The identification of circular RNAs (circRNAs) requires specialized experimental and computational approaches because their circular structure differs from that of linear RNAs.
• During RNA library preparation, circRNA analysis typically involves ribosomal RNA (rRNA) depletion and non-poly(A) RNA selection, since circRNAs lack the 3′ poly(A) tail used in conventional mRNA sequencing.
• A common enrichment step is RNase R treatment, in which the exonuclease RNase R selectively degrades most linear RNAs while preserving circular RNAs, thereby significantly increasing the proportion of circRNAs in the sample.
• Bioinformatic identification of circRNAs relies on detecting back-splice junction (BSJ) reads, which map non-colinearly to the reference genome and serve as the defining feature of circRNAs.
• Several computational tools have been developed for circRNA detection, including find_circ, CIRI2, and STAR-chip.
• Quantitative reverse transcription PCR (qRT-PCR) is considered the gold standard for circRNA validation and uses divergent primers that span the back-splice junction, ensuring amplification of only the circular RNA.
• Northern blotting provides direct evidence of RNA circularity because circRNAs migrate more slowly than linear RNAs of the same length during electrophoresis.
• Sanger sequencing is commonly performed after PCR amplification to verify the exact nucleotide sequence of the back-splice junction (BSJ), confirming the identity of the circRNA.

Role of Circular RNAs (circRNAs) in Disease Pathogenesis

Cardiovascular Diseases (CVD)

• circRNAs play important regulatory roles in the development and progression of cardiovascular diseases by influencing gene expression, cardiac remodeling, and vascular function.
• During cardiac hypertrophy, circSLC8A1 and circMYO9A are upregulated and promote pathological heart enlargement by sponging cardioprotective microRNAs (miRNAs).
• In atherosclerosis, circANRIL functions as a context-dependent regulator by interacting with the ribosome biogenesis factor PES1, thereby influencing vascular smooth muscle cell proliferation, apoptosis, and the stability of atherosclerotic plaques.

Neurological Disorders

• Abnormal circRNA expression has been associated with several neurodegenerative and neuropsychiatric disorders.
• In Alzheimer’s disease (AD), widespread dysregulation of circRNA expression has been reported. circHOMER1 is significantly downregulated, whereas circAβ, derived from the APP gene, may contribute to amyloid-beta production through an alternative pathway.
• In Parkinson’s disease (PD), circSNCA and circPANK1 act as sponges for miR-7, resulting in increased α-synuclein expression and promoting neurotoxic protein aggregation.
• Schizophrenia is associated with a global reduction in circRNA expression in postmortem brain tissue, which may increase miRNA availability and disrupt the regulation of genes involved in synaptic function.

Cancer

• CircRNAs can function as either oncogenes or tumor suppressors, depending on the cancer type and cellular context.
• They regulate key cancer-related processes, including cell proliferation, apoptosis, migration, invasion, angiogenesis, and metastasis.
• circAGFG1 is an example of an oncogenic circRNA that promotes tumor progression.
• circTADA2A functions as a tumor suppressor circRNA, inhibiting cancer development and progression.

Diagnostic Biomarkers of circRNAs

Stability in Biofluids

• CircRNAs are highly resistant to exonuclease degradation, making them exceptionally stable in plasma, blood, saliva, urine, cerebrospinal fluid, and extracellular vesicles such as exosomes.
• Their remarkable stability makes them promising non-invasive biomarkers for disease diagnosis and monitoring.

Tissue Specificity

• Many circRNAs exhibit tissue-specific and cell-type-specific expression patterns, often showing greater specificity than linear RNAs.
• This characteristic enables circRNAs to serve as valuable liquid biopsy biomarkers for detecting organ-specific diseases.

Aging and Environmental Exposure Markers

• Due to their long half-life and gradual accumulation over time, circRNAs are considered superior molecular markers of aging compared with messenger RNAs (mRNAs).
• CircRNA expression profiles can also retain information about previous environmental exposures, such as heat stress, even weeks after the exposure has occurred.

Diagnostic Performance

• Clinical studies have demonstrated that circRNA biomarker panels can provide higher sensitivity and specificity than conventional diagnostic biomarkers.
• For example, the CircPanel developed for hepatocellular carcinoma (HCC) has shown superior diagnostic performance compared with the traditional biomarker alpha-fetoprotein (AFP) in multicenter studies.

Therapeutic Applications of Circular RNAs (circRNAs)

Loss-of-Function (LOF) Strategies

• Loss-of-function (LOF) strategies aim to inhibit or silence disease-associated circRNAs that contribute to pathological conditions.
• RNA interference (RNAi) using small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) can specifically target the back-splice junction (BSJ) sequence of circRNAs, enabling selective degradation without affecting the corresponding linear messenger RNA (mRNA).
• Antisense oligonucleotides (ASOs) are synthetic nucleic acid molecules designed to bind circRNAs, allowing the selective silencing of upregulated circRNAs or blocking their interaction with proteins and other regulatory molecules.
• CRISPR-Cas13, an RNA-targeting CRISPR system, has emerged as a promising therapeutic approach for the highly specific degradation of circRNAs, owing to its low mismatch tolerance and ability to selectively target RNA without altering the genome.

Gain-of-Function (GOF) Strategies

• Gain-of-function (GOF) strategies are designed to increase the expression or activity of beneficial circRNAs for therapeutic purposes.
• Expression plasmids containing inverted intronic repeat sequences are used to promote back-splicing, enabling the efficient overexpression of specific circRNAs in target cells.
• Engineered circular microRNA sponges (circmiRs) are artificially designed circRNAs that contain multiple microRNA response elements (MREs), allowing them to simultaneously bind and inhibit multiple disease-associated microRNAs (miRNAs).
• By targeting several pathogenic miRNAs at once, engineered circmiRs have the potential to regulate multiple disease-related signaling pathways, making them promising candidates for gene therapy and precision medicine.

Delivery Platforms

• Efficient delivery platforms are essential for transporting circRNAs or circRNA-targeting molecules to specific tissues while protecting them from degradation and improving therapeutic efficacy.
• Adeno-associated virus (AAV) vectors are widely used for long-term and tissue-specific expression of therapeutic circRNAs. For example, AAV9 exhibits high tropism for cardiomyocytes, making it a preferred vector for cardiovascular gene therapy.
• Nanoparticles, including lipid nanoparticles (LNPs) and gold nanoparticles (AuNPs), protect RNA cargo from enzymatic degradation, enhance cellular uptake, and generally exhibit low immunogenicity, making them effective delivery systems for circRNA therapeutics.
• Exosomes are naturally occurring extracellular vesicles that serve as biocompatible delivery vehicles for circRNAs and circRNA-targeting agents. Their ability to cross biological barriers, including the blood–brain barrier (BBB), makes them particularly attractive for treating neurological disorders.
• Multiple in vivo therapeutic strategies have been developed to target circRNAs, combining delivery platforms with approaches such as RNA interference (RNAi), antisense oligonucleotides (ASOs), CRISPR-Cas13 systems, expression vectors, and engineered circRNAs to either suppress or enhance circRNA function depending on the therapeutic objective.

Conclusions

• Circular RNAs (circRNAs) are a unique class of covalently closed RNA molecules that are primarily generated through spliceosome-mediated back-splicing, a process often facilitated by intronic complementary sequences (ICSs) and RNA-binding proteins (RBPs).
• Their closed-loop structure provides exceptional stability and resistance to exonuclease degradation, resulting in significantly longer half-lives than linear RNAs.
• Owing to their remarkable stability and presence in blood, saliva, urine, and other biofluids, circRNAs are considered highly promising non-invasive diagnostic biomarkers for a wide range of diseases.
• CircRNAs regulate gene expression through multiple mechanisms, including acting as microRNA (miRNA) sponges, protein scaffolds, transcriptional regulators, and modulators of RNA-binding proteins. Some circRNAs can also encode functional proteins or peptides through cap-independent translation mechanisms.
• The identification of circRNAs relies on specialized bioinformatic pipelines that detect back-splice junctions (BSJs) and is experimentally validated using techniques such as RNase R enrichment, divergent qRT-PCR, Northern blotting, and Sanger sequencing.
• Dysregulated circRNA expression has been implicated in the development and progression of cancers, cardiovascular diseases, neurological disorders, and numerous other human diseases, highlighting their importance in disease pathogenesis.
• Current research is exploring circRNA-based therapeutics, including engineered circular RNA sponges, RNA-targeting technologies, and advanced delivery platforms such as nanoparticles, viral vectors, and exosomes to selectively modulate disease-associated molecular pathways.
• As research continues to advance, circRNAs are expected to become valuable tools in precision medicine, offering new opportunities for disease diagnosis, prognosis, and targeted therapy.

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