Circular RNA (circRNA)

1.2.1 Characteristics of circRNAs

CircRNAs are a large class of non-coding RNAs produced by thousands of protein-coding genes, which are circularized by non-canonical “backsplicing” of pre-mRNAs^83,84. CircRNAs are single-strand circular molecules without 5’ cap or 3’ poly(A) tail (Fig. 2). Owing to this structure, circRNAs are resistant to degradation by exonucleases RNase R^85-87 and relatively stable than their corresponding co-linear mRNA isoforms^86-89. In 1970s, circRNAs were firstly discovered in RNA viruses by electron microscopy⁹⁰ and later in eukaryotic cells in 1979⁹¹. Until 1991, circRNAs

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product of pre-RNA splicing. These recent years, increasing evidence shows the roles of circRNAs in the development and diseases.

Figure 2. Schematic representation of the biogenesis of circRNAs.

1.2.2 Biogenesis of circRNAs

According to biogenesis from different genomic regions, circRNAs can be classified into three subtypes: exonic circular RNAs (ecircRNAs), exon-intron circular RNAs (EIciRNAs), and circular intronic RNAs (ciRNAs) (Fig. 2). More than 80% of circRNAs are derived from exons of protein-coding genes without introns, constituting ecircRNAs⁸⁶. Approximately 20% of circRNAs are derived from both exonic and intronic regions, constituting exon-intron circular RNAs (EIciRNAs)⁹³. A small fraction of circRNAs contain only introns regions, termed circular intronic RNAs (ciRNAs), which stem from spliced out lariats⁹⁴.

CircRNAs formation can be modulated by cis-elements, trans-factors and other factors.

1) Cis-elements (i.e., DNA sequences):

CircRNA biogenesis can be promoted by RNA pairing between the reversely complementary sequences (RCSs) across flanking introns^87,95 (Fig. 2). Further studies indicated that the flanking introns of circRNA junctions were longer than background introns and harbored more repetitive elements^87,96. Our previous study has demonstrated that RCSs (Alu repeats) across flanking introns can affect the formation of both circRNA and trans-spliced RNA isoforms⁸⁹. Nevertheless, no specific motifs for circRNA formation have been identified in exonic and flanking intron sequences.

2) Trans-factors (i.e., RNA binding proteins, RBPs):

In addition to cis-element, circRNAs can also be facilitated by trans-factors to bridge flanking introns pairing^97,98. Some splicing factors (e.g. Muscleblind (MBL)⁸³, Quaking (QKI)⁹⁷) and RNA-binding proteins (e.g. FUS⁹⁹, ADAR¹⁰⁰) can affect circRNA formation. MBL was shown to bind to its own pre-mRNA and bridging between the two flanking introns to induce backsplicing, which stimulates circMbl production and decrease the expression level of MBL mRNA⁸³. Another regulator of circRNA biogenesis is QKI which binding on intronic QKI binding motifs, then significantly increased circRNA formation⁹⁷. FUS promote backsplicing by binding the flanking introns of circularized exons⁹⁹. Conversely, ADAR1 protein suppresses circRNA formation by disrupting the stem structure. ADARs catalyze A-to-I RNA editing within double stranded RNA pairing structures, resulting in reduced RNA pairing¹⁰⁰.

3) Spliceosome activity and Pol II elongation rate:

A recent study has found that depletion of spliceosome activity increased long and repeat-rich flanking intron¹⁰¹ and non-complementary sequences¹⁰² to pair, facilitating

circRNA formation. Besides, the process of circRNA formation was also influenced by the transcription rate of the corresponding gene. The average Pol II transcription elongation rate of circRNA host genes is higher than that of non-circRNA genes⁸⁴.

1.2.3 Expression of circRNAs

CircRNAs exhibit evolutionary conservation across multiple species, and exists widely in eukaryotes⁸⁷. Several studies reported that circRNAs are enriched in neuronal tissues compared with other tissues^103-105, especially synaptosomes¹⁰⁵. That prompting many researchers to explore the role of circRNAs in neurological diseases. Most of circRNAs are enriched in the cytoplasm^85,106, suggesting that circRNAs regulate gene expression through interfering with miRNAs. In general, circRNAs are expressed in a tissue- or age-dependent manner^107,108, and also show dynamic expression during neuronal differentiation, depolarization and development104,109,110.

Multiple studies have demonstrated that the expression of circular isoform was not correlated with the expression of its cognate linear mRNA¹⁰³. Although most circRNAs are expressed at a much lower level compared with their host gene^81,111,112. However, in some cases, circRNAs even more abundant than their co-linear counterparts^88,89. For example, Morten, et al. found that the expression of three circRNA (circCSPP1, circHDAC2, and circRIMS2) were much higher than those of the linear transcript in porcine brain, and their host gene were associated with synaptic plasticity or brain development¹⁰⁴.

1.2.4 Regulation of circRNAs

Although the function of circRNAs is not entirely clear, the recent studies have shown that circRNAs may have the ability to regulate gene expression through multiple mechanisms¹⁰⁶ (Table 2). The most understood function of circRNAs is the regulatory role of miRNA sponges, suggesting a previously underappreciated regulatory pathway of circRNA-miRNA-mRNA axes. For example, CDR1as is one of the most widely studied circRNA, which has more than 70 binding sites for miR-7 and function as an miRNA sponge to compete with mRNA for miRNA binding¹¹³. In addition, certain circRNAs can regulate transcription. For example, circMbl negatively regulate MBL pre-mRNA splicing by competing the splicing factors⁸³. EIciRNAs are dominantly located in the nucleus and interact with a spliceosomal component U1 snRNPs, which can recruit RNA polymerase II on the promoter of their host genes and thus promote the transcription of the host gene, such as EIciEIF3J, EIciPAIP2 and Ci-ankrd52^93,94. Another study demonstrated that circERBB2 can promotes ribosomal DNA transcription¹¹⁴. In contrast, circSamd4 can represses transcription of the myosin heavy chain protein family by associated with PURA and PURB, two repressors of myogenesis¹¹⁵.

On the other hand, many circRNAs interact with proteins through specific binding sites¹¹⁶. In their function as protein decoys, circPABPN1 serve as a decoy for HuR and suppresses PABPN1 translation¹¹⁷. Besides, circ-Amotl1 bind to c-Myc, promoting their nuclear translocation, and upregulated its targets¹¹⁸. Additionally, circANRIL has similar secondary structure to pre-rRNA, which decoys PES1 to suppress rRNA

processing and maturation¹¹⁹. CircRNAs can also function as scaffolds to facilitate subcellular co-localization of their substrates. Circ-Foxo3 acts as scaffold to interact with CDK2 and p21, and then leading to the inhibition of the CDK function¹²⁰. Circ-Foxo3 also promotes the interaction between MDM2 and p53 to induces apoptosis¹²¹. Specifically, circ-Foxo3 affected ID1, E2F1, HIF1α and FAK subcellular translocation¹²²; circ-Amotl1 interacts with PDK1 and AKT1 to facilitate their nuclear translocation¹²³. Moreover, some protein-coding circRNAs contain internal ribosome entry site (IRES) and open reading frame, such as circ-ZNF609¹²⁴, circ-FBXW7¹²⁵, circ-AKT3¹²⁶ and circPPP1R12A¹²⁷ can be translated to produce peptides (Table 2).

One of the most well-known functions of circRNAs is the role of miRNA sponge to regulate target gene expression¹¹³. Several data indicated that the interactions between circRNAs and miRNAs were important for normal brain function. For example, CDR1as knockdown mice displays impaired sensorimotor gating and abnormal synaptic transmission¹²⁸. Therefore, it is believed that circRNAs can mediate miRNAs and thus regulate the downstream genes at the post-transcriptional level. While some cases of ASD-associated miRNA–mRNA regulatory interactions have been reported^64,129, circRNA–miRNA–mRNA regulatory system may play an important mechanism of epigenetic control over gene expression in ASD and healthy samples.

Table 2. Potential functions of circRNAs.

Function circRNAs References

miRNA sponge CDR1as / Sry / circHIPK3/ circMTO1 /

circITCH / circCCDC66 / circTP63

86,88,113,130-133

Regulation of transcription circMbl / EIciEIF3J / EIciPAIP2 / Ci-ankrd52 / circERBB2 / circSamd4

83,93,94,114,115

Protein decoys circPABPN1 / circ-Amotl1 / circANRIL ^117-119

Protein scaffolds circ-Foxo3 / circ-Amotl1 ^120-123

Translation peptides circ-ZNF609 / circ-FBXW7 / circ-AKT3 / circPPP1R12A

124-127

1.2.5 CircRNAs in neurological diseases

CircRNAs expressed in fly heads or mouse brains are enriched in genes that code for neuronal proteins and synaptic factors, suggesting a potential role for circRNA in the central nervous system^105,107. Moreover, several cases of circRNAs show distinct localization in different parts of neurons¹⁰³, their host genes are related to several synaptic functions, including neurogenesis, neural differentiation, WNT signaling, and synaptic plasticity during neurogenesis^103-105. Therefore, circRNAs have the potential to serve as novel therapeutic targets and diagnosis biomarkers to treat neurological diseases, such as Alzheimer's disease¹³⁴, Parkinson’s disease¹³⁵, major depressive disorder¹³⁶, and many nervous system disorders¹³⁷ (Table 3).

These studies highlight a potential function of circRNAs in the nervous systems and suggest their relevance to pathogenesis of neurodegenerative. However, the biological functions of circRNA regulatory mechanisms in ASD are largely unknown.

Table 3. Representative circRNAs and related regulatory interactions in neurological diseases.

Neurological diseases circRNAs Intersection

molecules* References

Alzheimer’s disease CDR1as →miR-7 →UBE2A

→APP & BACE1

134138139

Parkinson’s disease CDR1as →miR-7 →SNCA ¹¹³

Major depressive disorder

circRNA_103636 ¹³⁶

Neuropathic pain rno_circ_0006298 →miR-184 ¹⁴⁰

Multiple system

atrophy IQCK, MAP4K3,

EFCAB11, DTNA, MCTP1

141

Neurological Tumors circ-FBXW7 →FBXW7-185aa ¹²⁵¹⁴²

Neuroinflammatory circHIPK2 →miR124

* Intersection molecules represent the downstream pathway of circRNAs.