CircRNAs have been proven to be involved in various biological processes and mechanistically have been widely characterized to conduct their functions by several major mechanisms:
-
One of the most investigated functions of circRNAs is their ability to indirectly control gene expression through sponging of microRNAs (miRNAs)
[6] (
Figure 1D). CircRNAs often contain miRNA response elements (MREs) which can bind miRNAs, thus preventing them from silencing their gene targets
[11,12][11][12]. By such mechanism, these circRNAs are classified as competing endogenous RNAs (ceRNAs)
[11]. Among the most prominent of them is ciRS-7 (CDR1as), which was discovered to possess up to 70 target sites for miR-7
[6]. Therefore, a vast number of studies has been focused on investigating ceRNA mode of action of circRNAs. However, large-scale screening studies indicate that usually circRNAs contain a limited number of MREs, indicative that this is not the only bona fide mechanism.
-
CircRNAs can associate with RNA binding proteins (RBPs) and thus mediate different aspects of their functionality (
Figure 1E). For instance, circRNAs can act as scaffolds facilitating the formation of protein complexes. For example, circ-FOXO3 was shown to interact with both MDM2 and p53, which promoted MDM2-dependent ubiquitination and degradation of p53
[13]. The same circRNA was also shown to form ternary complex with CDK2 and p21, preventing the association of the former into functional complexes with cyclins A and E, thus acting by decoy mechanism
[14].
-
CircRNAs that retain the intronic sequences of their parental genes, such as ciRNAs and EIciRNAs, are predominantly localized in the nucleus. As was shown by Pol II CLIP, EIciRNAs such as circEIF3J and circPAIP2 could associate with RNA polymerase II and enhance their parental gene expression in cis in a U1 snRNP-dependent manner
[15] (
Figure 1F). The broad-scale effects of circRNAs on pre-mRNA splicing are not widely characterized as yet; however, it has been known that circularization and linear splicing compete with each other on a co-transcriptional level (
Figure 1G). Such regulatory mechanism was demonstrated for MBL/MBNL1 gene encoding muscleblind (MBL) splicing factor. The protein product of this gene was shown to directly promote circularization of the exon 2 of its host gene by direct binding to the flanking sequences in the adjacent introns to produce circMBL, concomitantly reducing linear splicing
[5]. Finally, in addition to regulating mRNAs at transcriptional and splicing levels, circRNAs were also shown to regulate the stability of mRNAs. For example, the degraded fragments of CDR1, as circRNA could downregulate CDR1 by promoting degradation via antisense pairing
[16]. On the contrary, circRasGEF1B could enhance the stability of its target ICAM1 mRNA
[17].
-
Although circRNAs have traditionally been regarded as noncoding RNAs given the lack of the 5′ cap that is normally required for the initiation of translation, the discovery of internal ribosome entry sites (IRES) within multiple circRNAs suggests that they may be extensively translated into peptides or proteins in 5′ cap-independent manner
[18] (
Figure 1H). One prominent example of such circRNAs is circ-ZNF609, whose IRES-dependent translation was shown to play an important role in myogenesis
[19]. In addition, it was shown that initiation of translation in circRNAs can be driven by m6A methylation. CircRNAs were found to be enriched in consensus m6A motifs, whose methylation by METTL3/14 methyl transferase complex promoted the translation, and demethylation by FTO demethylase inhibited the translation
[20]. In such a mechanism, m6A deposited on circRNAs could be recognized by YTHDF3 m6A reader protein, which in turn recruited eIF4G2 initiation factor
[20].
43. CircRNA as Therapeutics Molecular Pathway Targets in Cancer
RNA therapeutic technology provides the opportunity of targeting every coding and noncoding gene in biological systems. Interestingly, RNA therapeutics efficacy has been proven to be workable by few approved RNA drugs for liver, muscle, and rare genetic diseases
[132][21]. Considering that many circRNAs were confirmed to promote cancer progression, targeting them offers a great potential therapeutic strategy for combating various cancers
[27][22].
Overall, circRNA studies focus more on circRNA differential expression in cancers based on the premises that circRNAs can serve as more reliable and accessible liquid biomarkers for cancer detection
[133][23]. Other than the diagnostic value, targeting circRNA-mediated molecular pathways for therapeutic purposes is also conceivable if the full molecular mechanisms of action of the most dysregulated circRNAs implicated in cancers are known. Currently, this perspective still remains unrealized in clinical practice
[84][24]. Therefore, gain-of-function and loss-of-function strategies are deployed in circRNA research in order to harness dysregulated circRNAs for cancer therapy
[84][24]. Precisely, tumor suppressor circRNAs are ectopically expressed in order to replenish the downregulated tumor suppressor circRNAs in cancers
[134][25]. For example, several transient circRNA overexpression DNA vectors harboring the Alu repeats that facilitate circularization have been developed
[4,43,135][4][26][27]. Likewise, lentivirus and AAV delivery systems have also been used to generate stable circRNA ectopic expression in vitro and in vivo
[136][28].
In contrast, oncogenic circRNAs are normally targeted for degradation using several techniques. For instance, RNA interference (RNAi) methods represent the most prominent circRNA degradation strategy currently available. Researchers have used both the conventional short interference RNA (siRNA)
[24,137][29][30] and the more lasting short hairpin RNA (shRNA) techniques to degrade copious oncogenic circRNAs
[138,139][31][32].
In addition to siRNAs and shRNAs, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) systems represent an effective and promising approach for targeting circRNAs. Whereas CRISPR/Cas9 is normally used to edit DNA
[140][33], CRISPR/Cas13 is a recently discovered system that has been utilized to target RNA
[141][34]. At present, both systems have been used to modulate the expressions of circRNAs
[134,142][25][35]. In one of the studies, CRISPR/Cas9 was used to delete the complementary Alu repeat in HIPK3 gene, thus preventing the expression of circHIPK3
[72][36]. Similarly, circRNA CDR1as was also downregulated using CRISPR/Cas9 and gRNA targeting CDR1as locus
[35][37]. This evidence proved the possibility of therapeutic targeting of circRNAs using the CRISPR/Cas9 system; however, the concerns are raised about affecting the linear counterparts of circRNAs which can lead to off-target effects
[84][24]. Therefore, various CRISPR/Cas13 orthologs have been investigated for circRNA targeting.
In a recent study, CRISPR/Cas13d was used to downregulate oncogenic circFAM120A
[84][24]. Similarly, circZKSCAN1 was also effectively downregulated using CRISPR/Cas13d system
[143][38]. More detailed information on CRISPR/Cas system-mediated targeting of circRNAs can be found in recent reviews
[84,134][24][25].