Most circular RNAs (circRNAs) are produced by reverse splicing of exons of precursor mRNAs. In recent years, with the rapid development of transcriptome sequencing technology and computational biology, researchers have discovered tens of thousands of circular RNAs (circRNAs).

Integrating circular RNA sequencing technologies in your research allows for a better understanding of complex biology and enables more discoveries.

History of Circular RNA Research

Unlike most linear messenger RNAs (mRNAs) and long-stranded noncoding RNAs (lncRNAs) that terminate with a 5' N7-methylguanosine (m7G) cap and a 3' polyadenylate tail, circular RNAs are covalently closed single-stranded RNAs (ssRNAs).

The first confirmed circular single-stranded RNAs (ssRNAs) were found in plant viroids, followed by some circular but unknown functional transcripts in eukaryotes by electron microscopy. In the 1980s, circular RNAs were found in some viruses, such as hepatitis δ. In the last decade, RNA sequencing techniques, accompanied by biochemical enrichment of the non-polyadenylated and circular transcriptome, and the development of computational biology applicable to circular RNA annotation, have led to the discovery of widely expressed in postnatal cells and different tissue types of circular RNAs and a fraction of circular RNAs have been found to have important biological functions.

Most circular RNAs are produced by reverse splicing of precursor mRNAs, where the downstream 5' splice donor joins the upstream 3' splice acceptor to produce RNA molecules at the back-splicing junction site (BSJ). RNA molecules are produced at the back-splicing junction site (BSJ). Because reverse splicing is less efficient than classical splicing, the expression of circular RNA is usually not very high in cells and tissues, and most of the identified circular RNAs containing exons are mainly localized in the cytoplasm. It has been shown that in Drosophila cells, the transport of circular RNA is regulated at least in part by the RNA unwinding enzyme Hel25E, while in human cells it is regulated by DDX39B/39A (the two human homologs of Hel25E). Most circular RNAs are relatively stable and have long half-lives: 18.8-23.7 h, while the corresponding linear RNAs have half-lives between 4-7.4 h. The differential expression of circular RNAs in different cells and tissues suggests that they have different functions in different contexts.

Identification of Circular RNA

Since circular RNA has almost the same sequence as its homologous linear RNA, distinguishing circular from its corresponding linear transcript is a great challenge.

RNA-Seq Preparation

enrichment of the circular RNA transcriptome. Because circular RNA lacks a 5' N7-methylguanosine (m7G) cap and a 3' polyadenylate tail, classical poly(A) RNA-seq is unable to capture circular RNA, so researchers have thought of alternative methods to enrich circular RNA. For example, using 3'-5 ' nucleic acid exonuclease, RNase R, can efficiently digest almost all linear RNAs, however, linear RNAs with complex structural regions can block the digestion of RNase R, resulting in the generation of false-positive circular RNAs. subsequently, researchers found that the addition of lithium elements to RNase R can completely digest degraded complex structural linear RNA, which provides a strong and effective strategy for the enrichment of circular RNA.

Annotation

After sequencing, special algorithms are needed to assign reads to the genome, and the core logic here is based on the back-splicing junction site (BSJ), but traditional short-read sequencing has a read length of 150 bases or less, which makes it difficult to distinguish between circular RNA. The recent development of nanopore long-read sequencing, which can reach a read length of 1000 bases, has made it possible to detect circular RNA more efficiently and accurately.

Annotation and functional dissection of circular RNAs. (Liu et al., 2022)

Confirmation of Circular RNA

The candidates obtained by RNA-seq need further experimental validation to finally confirm that they are circular RNAs, and Sanger sequencing is one of the most important detection methods. The presence of the back-splicing junction site (BSJ) is confirmed by targeting the circular RNA candidate of interest by divergent PCR, thus proving whether the candidate is a circular RNA; for candidates with relatively higher expression, northern blotting (NB) is recommended. For candidates with relatively higher expression, Northern Blotting (NB) is recommended, which is also designed to target the back-splicing junction site (BSJ) to prove whether the candidate is a circular RNA.

The Role of Circular RNA in the Cell

Regulation of Transcription

Circular RNA can affect RNA polymerase II (RNA Pol II) transcription through different mechanisms. Ci-ankrd52 is a circular RNA of 444 nucleotides in length, mainly localized in the nucleolus, and Ci-ankrd52 can form an R-loop with the second intron region of the ANKRD52 locus, thus affecting transcriptional elongation at this site, while RNase H1 (a nucleic acid endonuclease) mediates the degradation of ground ci-ankrd52, which can disrupt the R-loop formed at this site, thus enhancing transcriptional elongation at this site. Another example is circKcnt2, which is derived from the fourth to eighth exon of pre-Kcnt2. circKcnt2 can significantly repress Batf gene expression by binding near the promoter region of the Batf gene and by recruiting nucleosome remodeling deacetylase (nucleosome remodeling deacetylase, NuRD) complex, inhibiting its expression and thus promoting the dissipation of colitis.

Regulation of Splicing

SEPALLATA3 (SEP3) is an important transcription factor present in Arabidopsis, whose sixth exon is reverse-spliced to produce circSEP3. Overexpression of circSEP3 and the SEP3.3 mRNA variant can cause a similar phenotype, i.e., defective organ development with few stamens; mechanistically, circSEP3 interacts with the parental DNA motif, forming an R-loop, leading to a transcriptional pause and an exon skipping event in the sixth exon of this motif, which results in the mRNA variant, SEP3.3, which in turn leads to the above phenotype.

Cellular roles of circular RNAs. (Liu et al., 2022)

Protein Sponges

The first circular RNA identified as a protein sponge is circMbl, which is regulated in a feedback loop with MBL (multifunctional protein muscleblind), which promotes the biosynthesis of circMbl by binding to the lateral end of the circMbl intron, and accordingly, increasing MBL decreases Mbl mRNA splicing, which leads to the above phenotype. Conversely, high expression of circMbl binds to MBL, which isolates the protein and thus affects its function in other neurons.

Formation of Functional CircRNP Complexes

There are many examples of circular RNAs and proteins forming circRNP complexes to regulate signaling pathways, one of the classic cases is SCAR (Steatohepatitis-associated circRNA ATP5B regulator), which is a highly expressed circular RNA in mitochondria. RNA-pull down experiments demonstrated that SCAR binds directly to ATP5B (the regulator of mitochondrial permeability transition pore (mPTP), and this binding can prevent mPTP This binding can prevent the opening of mPTP, thus reducing the output of mitochondrial reactive oxygen species (mROS) and maintaining the dynamic balance of mROS in vivo.

It is worth mentioning that, unlike linear RNA, circular RNA has different conformation, stability and immunogenicity, which makes circular RNA has very broad clinical translation prospects in the medical field, such as RNA ligands to interfere with intracellular processes, modulate natural immune responses, act as sponges to isolate disease-related miRNAs and proteins, antisense RNAs, protein translation vectors and disease-related molecules. The potential of RNAs in the medical field is very promising. It is foreseen that with the development of biotechnology, more and more circular RNAs will be discovered and the biomedical applications based on circular RNAs will also shine.

Reference:

1. Liu, Chu-Xiao, and Ling-Ling Chen. "Circular RNAs: Characterization, cellular roles, and applications." Cell (2022).