Biogenesis of circRNAs
CircRNAs are generated through a non-canonical splicing event known as backsplicing, where a downstream splice donor site is joined to an upstream splice acceptor site, resulting in a covalently closed circular RNA molecule without 5’ caps or 3’ poly(A) tails (see figure 1). This process can involve exonic, intronic, or exon-intron sequences, producing different types of circRNAs such as exonic circRNAs (ecircRNAs), circular intronic RNAs (ciRNAs), and exon-intron circRNAs (EIciRNAs).
Figure 1. Schematic of canonical back-splicing producing circRNAs
CircRNA have shown to be 3-5 times more stable than linear mRNAs under physiological conditions. The circular topology prevents exonucleases from accessing RNA ends, which are the primary sites for degradation in linear RNAs.
Functions of circRNAs
Several functions have been reported for circRNAs, including
- MicroRNA (miRNA) sponging: Many circRNAs contain multiple binding sites for specific miRNAs, acting as competitive endogenous RNAs that sequester miRNAs and prevent them from repressing their target mRNAs. A prominent example is ciRS-7 (CDR1as), which harbours over 70 binding sites for miR-7. By sponging miR-7, ciRS-7 modulates gene expression involved in various cancers such as pancreatic, colorectal, and esophageal squamous cell carcinoma. This interaction leads to upregulation of oncogenes like EGFR, STAT3, and HOXB13, promoting tumour proliferation and metastasis.
- Protein interactions: CircRNAs can bind RNA-binding proteins, influencing protein localization, stability, or function. For instance, circFOXO3 interacts with stress-related proteins such as p21 and CDK2, forming ternary complexes that regulate cell cycle progression and promote cellular senescence under stress conditions. Another example is circFAM120A, which binds to the RBP IGF2BP2, preventing it from binding to the linear FAM120A mRNA, thereby enhancing FAM120A translation and promoting tumor cell proliferation.
- Formation of regulatory complexes: CircRNAs can form complexes with proteins and mRNAs or chromatin. For example, circNSUN2 forms a complex with the RBP IGF2BP2 and HMGA2 mRNA, enhancing HMGA2 mRNA stability and promoting colorectal cancer metastasis.
- Transcriptional regulation: Certain nuclear circRNAs regulate gene transcription by interacting with promoter regions and transcriptional machinery. For example, circZNF827 recruits transcription regulators to modulate neuronal differentiation genes.
- Translation: Although traditionally considered non-coding, some circRNAs can be translated into functional peptides. For instance, circRNAs with internal ribosome entry sites (IRES) or N6-methyladenosine (m6A) modifications can initiate cap-independent translation, producing peptides that may have biological functions. Specific examples include circZNF609 and circFBXW7.
Potential as Biomarkers
Due to their stability and specific expression profiles, circRNAs are promising biomarkers for disease diagnosis and prognosis. They have been studied extensively in gynaecological cancers such as cervical, ovarian, and endometrial cancers. For instance, circ_0067934 and circ_0018289 are upregulated in cervical cancer and correlate with metastasis and poor prognosis. Circulating circRNAs in serum, like circMAN1A2, have shown potential as non-invasive biomarkers for multiple cancers, including ovarian cancer. Their resistance to degradation and presence in body fluids make them attractive candidates for liquid biopsy-based diagnostics.
Techniques to study circRNAs
Next Generation Sequencing (NGS) is the main approach used for circRNA discovery and quantification. Unlike linear RNAs, circRNAs are identified by sequencing reads spanning the unique backsplice junctions.
CircRNAs are expressed at low levels, necessitating enrichment to improve detection. RNase R is a 3’ to 5’ exonuclease that it is commonly used to degrade linear RNAs but spares circular RNAs due to their closed-loop structure. Treated RNA can be then processed using a total RNA-seq stranded workflow such as the NEXTFLEX™ Rapid Directional RNA-seq 2.0 Kit However, RNase R treatment efficiency can vary between samples and species, potentially introducing quantification biases. Recent computational approaches, such as RNase R efficiency correction algorithms, have been developed to adjust for these biases and improve the accuracy of circRNA quantification.
Studies have shown that combining RNase R with other methods such as rRNA depletion (for example with the NEXTFLEX RiboNaut rRNA depletion kit upstream or with CRISPR-based depletion downstream) enhances circRNA enrichment.
Conclusion
CircRNAs are a class of RNAs with regulatory roles in gene expression, cellular function, and disease pathogenesis. Their unique biogenesis and diverse molecular functions, including miRNA sponging and protein interactions, highlight their significance in cell biology. CircRNAs hold great promise as biomarkers due to their stability and specific expression patterns. NGS with RNase R treatment is the most popular method for circRNA detection and quantification. Continued methodological improvements and functional studies will further elucidate circRNA biology and accelerate their translation into clinical applications.
References:
- Karimi et al. Circular RNAs: history, metabolism, mechanisms of function, and regulatory roles at a glance. Ann Med Surg (2025).
- Zhang et al. Mechanisms and therapeutic implications of gene expression regulation by circRNA protein interactions in cancer. Commun Biol (2025).
- Pisignano et al. Going circular: history, present, and future of circRNAs in cancer. Oncogene (2023).
- Huang.L., et al. Circular RNAs Are Promising Biomarkers in Liquid Biopsy for the Diagnosis of Non-small Cell Lung Cancer. Front Mol Biosci (2021).
- Kristensen, L.S. et al. The biogenesis, biology and characterization of circular RNAs. Nature (2019).
- Li, R., et al. CiRS-7 promotes growth and metastasis of esophageal squamous cell carcinoma via regulation of miR-7/HOXB13. Nature (2018).
