Origin of circular RNAs
The earliest discovery of circular RNA molecules occurred in pathogens, such as viroids and viruses, in the 1970s. The analysis of viroids revealed their structure as single-stranded, covalently closed circular RNA molecules. In the early 1990s, endogenous circular RNAs (circRNAs) were discovered by chance in mammalian cells, originating from genes such as "deleted in colon cancer" (DCC) and EST-1, a gene crucial for telomere maintenance. Initially thought to be aberrant splicing products, the widespread presence and diverse functions of circRNAs in eukaryotes, ranging from plants and yeast to worms and humans, have now revealed them as significant regulatory molecules.
Most known circRNAs originate from the exons of protein-coding genes but can also arise from introns or a combination of both. Pre-tRNA splicing can generate tRNA intronic circRNAs (tricRNAs), and mitochondrial-encoded circRNAs (mecciRNAs) also exist.
There are five main origins of circRNAs.
1) Exon-only circular RNA: Exon circRNA (Salzman J., et al. 2012)
2) Intron only source: Circular intronic RNA (Zhang Y., et al. 2013)
3) Back-splicing of upstream exons and intron retention: Exon-intron circular RNA (Zhaoyong et al. 2015)
4) Circular RNA from Fusion Gene: f-circRNA (Guarnerio et al. 2016; Tan et al. 2018)
5) Read-through circular RNA formed by polymerase II: rt-circRNA (Peng et al. 2015)
Biogenesis of circRNAs
Backsplicing is the primary mechanism for circRNA biogenesis in eukaryotes.
Unlike canonical linear splicing, where a 5'-splice donor site is joined to a downstream 3'-splice acceptor site, backsplicing involves a downstream 5'-splice donor joining an upstream 3'-splice acceptor, resulting in a "head-to-tail" or "reverse" splicing event.
Several mechanisms contribute to the precise formation of circRNAs through backsplicing:
Intron Pairing-Driven Circularization according to the direct backsplicing model is the prominent mechanism where complementary sequences within the flanking introns, the introns surrounding the exons that will form the circRNA, base-pair with each other. These inverted complementary sequences, for example Alu repeats in humans, bring the splice sites into close proximity, facilitating the backsplicing event. The intervening intronic sequences are then removed, and the exons are ligated in a circular fashion.
RNA Binding Protein (RBP)-Driven Circularization can facilitate circRNA formation by the binding of RBPs to motifs within the flanking introns or the circularizing exon(s). These RBPs can act as "bridges," bringing the splice sites together to promote backsplicing. For example, the Muscleblind protein (MBL) has been shown to regulate circRNA formation in Drosophila.
Lariat-Driven Circularization according to the Exon Skipping Model can lead to exon skipping, producing a lariat intermediate that still contains the skipped exon. This lariat can then undergo an internal splicing reaction to excise the skipped exon as an exonic circRNA.
Circular Intronic RNA (ciRNA) Biogenesis
Some intronic sequences excised during canonical splicing can evade the normal debranching and degradation pathways. Instead, they can undergo trimming and form stable circular intronic RNAs (ciRNAs). The formation of ciRNAs often depends on specific sequence elements, such as GU-rich sequences near the 5' splice site and C-rich sequences near the branch point. These ciRNAs are typically retained in the nucleus and can regulate the transcription of their parental genes.
Exon-Intron Circular RNA (EIciRNA) Biogenesis
EIciRNA contain both exonic and intronic sequences. Their formation mechanism is thought to be similar to exonic circRNAs, involving backsplicing, but with the retention of specific intronic sequences. EIciRNAs are also primarily nuclear and can regulate gene expression.
Factors influencing circRNA biogenesis:
Cis-regulatory elements: The presence of inverted complementary sequences for example Alu repeats, in flanking introns is a key determinant.
Trans-acting factors: RNA binding proteins play crucial roles in promoting or inhibiting circRNA formation.
Splicing machinery: The spliceosome is generally involved in backsplicing, although the exact mechanisms that favor backsplicing over canonical splicing are still being investigated.
Long flanking introns are often associated with circRNA formation. Additionally, modifications like m6A can influence circRNA biogenesis and stability. The diverse mechanisms and regulatory factors highlight the complexity and fine-tuned control of circRNA production, emphasizing their significant roles in cellular biology.
| Biogenesis of circRNA |
| Pre-mRNA Transcription Linear RNA molecule containing exons and introns transcribed are from DNA. 
|
| Canonical Splicing Exons are joined sequentially to form linear mRNA for protein synthesis. 
|
| Back-Splicing A downstream 5' splice site joins to an upstream 3' splice site, forming a circular RNA molecule. 
|
| Exon Circularization Most common: Exons are spliced head-to-tail to form circRNA. | Intron Circularization Introns are spliced out and circularized, forming circular intronic RNAs. |
| 
Resulting circRNA Stable, covalently closed loop structure. | |
CircRNAs are produced through a circularization process by head-to-tail back splicing. The circularization process involves a canonical spliceosome-mediated precursor mRNA (pre-mRNA) back-splicing mechanism. The back-splicing mechanism connects a downstream splice donor site (3′ splice site) to an upstream acceptor splice site (5′ splice site), modulated by RBPs and intronic complementary sequences.
The biogenesis of circRNAs begins with the transcription of a gene from DNA into a pre-messenger RNA (pre-mRNA) molecule. This pre-mRNA contains both coding regions (exons) and non-coding regions (introns). In most cases, pre-mRNAs undergo canonical splicing. This process involves the precise removal of introns and the joining of exons in a linear, sequential manner to produce mature messenger RNA (mRNA) that can be translated into proteins. This is known as the “canonical” or "default" pathway.
Back-Splicing is the defining step for circRNA genesis. Instead of the typical linear joining, a downstream 5' splice site, the end of an exon, unexpectedly joins to an upstream 3' splice site, the beginning of an exon. This "reverse" splicing event forces the intervening sequence, which can include one or more exons and sometimes introns, to form a circular molecule.
There are two types of circularization,
(i) Exon circularization is the most common. In this mechanism, one or more exons are directly joined head-to-tail, forming an exonic circRNA. These are the most widely studied and functionally characterized circRNAs.
(ii) Intron Circularization is less common, but also observed. It is the circularization of excised introns, known as circular intronic RNAs (ciRNAs) or exon-intron circRNAs (EIciRNAs), depending on whether they contain intervening intronic sequences.
The circularization product is a stable, covalently closed-loop RNA molecule that lacks free 5'- and 3'-ends, making it resistant to exonuclease degradation.
Several factors can influence whether back-splicing occurs over canonical splicing, including: The length and sequence composition of the flanking introns surrounding the circularized exons play a crucial role. Inverse complementary sequences, for example Alu elements, in flanking introns can form base-pairing interactions, bringing the splice sites into proximity and facilitating back-splicing. Specific RBPs can bind to flanking introns or exonic sequences, either promoting or inhibiting back-splicing.The availability and activity of components of the spliceosome can also influence the choice between canonical and back-splicing.
References
Guarnerio J., Bezzi M., Jeong J.C., Paffenholz S.V., Berry K., Naldini M.M., Lo-Coco F., Tay Y., Beck A.H., Pandolfi P.P. Oncogenic role of fusion-circRNAs derived from cancer-associated chromosomal translocations. Cell. 2016;165(2):289–302. [PubMed]
Lamond AI. The spliceosome. Bioessays. 1993 Sep;15(9):595-603. [PubMed]
Liang Y, Liu N, Yang L, Tang J, Wang Y, Mei M. A Brief Review of circRNA Biogenesis, Detection, and Function. Curr Genomics. 2021 Dec 31;22(7):485-495. [PMC]
Peng L., Yuan X.Q., Li G.C. The emerging landscape of circular RNA ciRS-7 in cancer (Review). Oncol. Rep. 2015;33(6):2669–2674. [PubMed]
Salzman J., Gawad C., Wang P.L., Lacayo N., Brown P.O. Circular RNAs are the predominant transcript isoform from hundreds of human genes in diverse cell types. PLoS One. 2012;7(2):e30733. [PMC] [PubMed]
Tan S., Sun D., Pu W., Gou Q., Guo C., Gong Y., Li J., Wei Y.Q., Liu L., Zhao Y., Peng Y. Circular RNA F-circEA-2a derived from EML4-ALK fusion gene promotes cell migration and invasion in non-small cell lung cancer. Mol. Cancer. 2018;17(1):138. [PMC] [PubMed]
Zhang Y., Zhang X-O., Chen T., Xiang J.F., Yin Q.F., Xing Y.H., Zhu S., Yang L., Chen L.L. Circular intronic long noncoding RNAs. Mol. Cell. 2013;51(6):792–806. [PubMed]
Zhaoyong, L.; Chuan, H. Exon-intron circular RNAs regulate transcription in the nucleus. Nat. Struct. Mol. Biol. 2015;22(3):256–264. [PubMed]
---...---
Bio-Synthesis provides a full spectrum of oligonucleotide and peptide synthesis including bio-conjugation services as well as high quality custom oligonucleotide modification services, back-bone modifications, conjugation to fatty acids and lipids, cholesterol, tocopherol, peptides as well as biotinylation by direct solid-phase chemical synthesis or enzyme-assisted approaches to obtain artificially modified oligonucleotides, such as BNA antisense oligonucleotides, mRNAs or siRNAs, containing a natural or modified backbone, as well as base, sugar and internucleotide linkages.
Bio-Synthesis also provides biotinylated mRNA and long circular oligonucleotides.
---...---