Circular RNA - A Candidate for Next Generation RNA Therapy

What is Circular RNA?

Circular RNA (circRNA) is a class of non-coding RNA molecules without a 5' terminal cap and a 3' terminal poly(A) tail, and form a ring structure by covalent bonding, which are objectively present in organisms, and are significantly different from linear RNAs. Discovered in 1976, the circRNA molecule was once ignored as 'junk' after RNA splicing. However, after more than 30 years of inactivity, circRNAs burst onto the scene in 2013 and have become important candidates for the development of next-generation RNA therapeutics.

Why circRNA is Always Overlooked?

• When using the linear genome as a template for primer design, conventional reverse transcriptase-rtPCR is unable to distinguish between circRNA and linear RNA.
• The circRNA does not map to a linear reference genome, so sequence reads are discarded in the sequencing data.
• circRNA lacks a polyA tail, and RNA sequencing library preparation usually removes the polyA of rRNA.

Figure.1 Three types of circular RNA. (L, P, Qiu.; et al, 2018)

Features of circRNA

• circRNA is produced by specific variable shearing and is present in large quantities in the cytoplasm of eukaryotic cells, mainly from exons, with a small proportion of intron-derived circRNAs present in the nucleus.
• The expression level of circRNA is species-, tissue-, and time-specific.
• The circRNA has a closed loop structure, is not easily degraded by nucleic acid exonucleases, and is more stable than linear RNA.
• circRNA has a certain sequence conservatism.
• circRNA plays a regulatory role at the transcriptional or post-transcriptional level.
• The vast majority of circRNAs are non-coding, but a few can be translated into polypeptides.

How circRNA is Formed

circRNAs can be categorized into exonic circRNAs, retained-intron circRNAs, and circular intronic RNAs according to their origin. circRNAs are formed differently from the standard shearing model of linear RNAs, which are sheared by backsplicing. There are five main existing models of circRNA formation as follows:

• Exon skipping
• Direct backsplicing
• circular intronic RNAs (ciRNAs) formation models
• RBPs-dependent circularization model
• Variable shear-like variable circularization model

Mechanism of action of circRNA

circRNAs were once thought to be errors in the normal splicing process. In recent years, there has been an explosion of circRNA-related research. circRNAs have been found to be important players in normal cellular differentiation and tissue homeostasis as well as in disease development, and the expression of circRNAs is usually not correlated with host gene expression. This suggests that circRNAs are not merely homeostatic byproducts of mRNA splicing, but rather products of novel regulated variable splicing. Sequence conservation analysis also demonstrated that circRNAs have important noncoding functions.

Figure 2. Metabolism of circRNA. (W, Y, Zhou .; et al, 2020)

• Acting as miRNA Sponges

circRNAs contain a large number of miRNA binding sites and act as miRNA sponges, which in turn indirectly regulate the expression of miRNA downstream target genes. For example, ciRS7, the first circRNA revealed to have a regulatory function, acts as a sponge for miR7 and contains 470 conserved binding sites for miR7. ciRS7 is stably expressed in many tissues of the human body, and increases the expression level of miR7 target genes by inhibiting miR7 activity. And miR7 directly targets several oncogenes involved in many different human cancers.

• Acting as Protein Molecule Sponges

Some circular RNAs have one or more binding sites for RNA-binding proteins and can act as sponges for protein molecules.

• Inducing Immune Responses

Some endogenous circular RNAs play a role in the antiviral response, while others are associated with immune response. Exogenous cyclic RNAs can stimulate immune signaling in mammalian cells by activating the pattern recognition receptor RIG-I.

• Coding Function

Although circRNAs are non-coding RNAs, there are a few circRNAs that can encode polypeptides, through which they exercise regulatory functions.

New Research Advances in circRNA

• Functional Circular RNA Screening Tool

A CRISPR-Cas13d-based screening tool, CRISPR-RfxCas13d, has been researched and developed to enable rapid screening and discovery of functional circRNAs.This screening tool targets the reverse splicing site of circRNAs through gRNAs Based on this technology, the research team constructed a lentiviral library targeting the reverse splice site sequences of highly expressed human circRNAs, and using this screening library, the team identified a set of functional circRNAs that are important for cell growth and embryonic development.

• Mitochondrial circRNA as a New Target for Nonalcoholic Steatohepatitis

The study identifies a mitochondria-specific circRNA, SCAR, whose expression is down-regulated in non-alcoholic steatohepatitis (NASH). Delivering SCAR specifically to mitochondria reduces mitochondrial reactive oxygen species (mROS) output and reduces inflammation, and could be a therapeutic target for non-alcoholic steatohepatitis (NASH). therapeutic target.

• Specific Knock-down of circRNA in vivo

The study describes an in vivo specific knockdown of circRNAs at tissue and cellular resolution, utilizing shRNAs targeted to circRNA-specific reverse splice sites to specifically knock down circRNAs with no off-target effects.

References

1. L, P, Qiu.; et al. The Emerging Role of Circular RNAs in Hepatocellular Carcinoma. Journal of Cancer. 2018, 9(9):1548-1559.
2. W, Y, Zhou.; et al. Circular RNA: Metabolism, Functions And Interactions With Proteins. Molecular Cancer. 2020, 19:172.