“Endless RNAs” open up new therapeutic potentials

    RNAs are known to be unstable and easily degradable, as they are single-stranded and contain ribose, which is more easily hydrolyzed than the DNA sugar base deoxyribose. However, this commonly known idea is now challenged by one subtype called circular RNA (circRNA). 

    Although circRNAs were considered “useless” because they are part of the non-coding regions, researchers discovered the connection between untranslated regions (UTR) and gene expression regulations during the past decade and opened up therapeutic potentials of circRNA in human diseases, such as Alzheimer’s disease, diabetes, and digestive tract tumors1. 

    Start-up biopharmaceutical companies, such as Laronde2 and oRNA3, are marketing circRNAs as an “endless RNA” sequence or highlighting its ring structure, to emphasize on the revolutionary therapeutic potential that comes from its stability and many other characteristics. 

Figure 1. Screenshots taken from the website of Laronde

Structure

circRNA has a special closed-ring structure, consisting of 30-50 phosphodiester bonds4. The ring structure provides more stability because the end of the strand can no longer be accessible for nucleases to “chew up.” Therefore, circRNA is a more ideal biomarker compared to the commonly seen linear counterparts: circRNA can last for more than 48 hours, while the half-life for linear RNAs lasts no more than 10 hours6. The 5’ and 3’ caps are joined together through a process called reverse splicing. 

There are three types of reverse splicing: 1) exon-skipping, 2) intron-pairing, and 3) RNA-binding protein (RBP) pairing (Figure 1). As shown in Figure 1, during exon-skipping, the two ends of exon 2 and exon 3 on the pre-mRNA strand covalently bond together, leaving exon 1 and exon 4 out and forming an intermediate product called a lariat RNA. As to intron-pairing and RBP pairing, the introns are removed by either binding the complementary bases or connecting the introns using RBP before splicing3.

The different types of circRNAs are all named based on how they are formed during reverse splicing: exon-derived circular RNA (ecircRNA), intron-derived circular RNA (ciRNA), and circRNA containing both exons and introns (EIcircRNA). circRNA can be found everywhere in the human body. ecircRNA is the most common type, usually found in the cytoplasm. EIcircRNA and ciRNA, meanwhile, are mostly found in the nucleus6. 

Figure 2. The reverse splicing process of circRNA3 (Adopted from Li et al. 2021.) EIciRNA or ecircRNA are formed either through exon-skipping, intron-pairing, or RBP pairing.

Function

As previously mentioned, circRNA was underappreciated until a decade ago. Researchers are still trying to understand and verify its functions and applications. Some examples of circRNA potential functions are briefly described below.

circRNAs can inhibit microRNA (miRNA), which is another type of RNA that regulates post-transcriptional gene expression of mRNAs7. miRNA can target mRNA strands to promote mRNA degradation or repress translation6. Overexpression of miRNA can be suppressed by circRNA, also referred to as “miRNA sponges.” These sponges “cover” the miRNAs and decrease the availability of miRNA that targets mRNA. circRNAs can also work as RBP “sponges”, with a similar mechanism7.

Figure 2. Circular RNA acts like sponges to inhibit miRNA7 (adopted from Barrett et al., 2016). Argonaute (AGO) proteins or RBPs bind to the surfaces of circRNA.

           Another possible function is to compete with the regular splicing sequences of mRNA strands. Since the reverse splicing process has an extra step of stitching itself back to form a ring, and it can possibly slow down the splicing process. More repetitions of this experiment are still being conducted to make sure whether this finding is valid7.

Possible Therapeutic Application

circRNAs are widely used in various types of cancers. For example, the expression of circTADA2A-E6 in triple-negative breast cancer (TNBC) tissues acts like miR-203-3p sponges, and downregulates the gene expression. This suppresses the proliferation and migration of the cancer cells6. circRNAs also contribute therapeutically to other cancer types such as lung cancer, liver cancer, and gastric cancer6.

circRNA also has a significant role in cardiovascular disease. Huang et al. discovered that downregulation of a particular circRNA, circNfix, in the adult hearts in humans, mice, or rats leads to the proliferation of cardiac cells and inhibits their apoptosis after myocardial infarction (MI), also commonly known as a heart attack. The research implies that circNfix can be a novel therapeutic target in preventing the development of MI8.

Other potential therapeutic applications of circRNA include treating central nervous system diseases, diabetes, and musculoskeletal diseases.

Conclusion & Future Steps

As you might have noticed, most articles cited in this article were published very recently. Researchers are still in the progress of understanding the functions of circRNAs, and no clinical trials on humans have been conducted as of 2022. Nevertheless, the special characteristics of circRNAs make them promising biomarkers and worth more investigation. Future research will likely expand our knowledge of various aspects of circRNA, including the function, mechanism, and side effects of circRNAs if they are applied clinically. 

Definitions

• Untranslated Region (UTR)

Can be found at both ends of mRNA or DNA strands. As the term implies, the UTRs don’t carry genetic information. However, they still have many other essential functions, for example, protecting the strands from degradation.

• Reverse Splicing

RNA splicing is the process of a pre-mRNA strand getting transcribed into an mRNA strand. Introns are removed and exons are stitched together. In reverse splicing, the intron self-splicing process is reversed.

• Lariat

The intermediate product of pre-mRNA splicing.

• Downregulation

Decreasing formation of a certain product through direct inactivation, or activation of its inhibitor. The opposite is upregulation. 

• Overexpression

Too many copies of certain gene information. For example, cancer might be a result of too many, or the overexpression, or certain cancer cells.

• Myocardial infarction (MI)

Commonly known as a heart attack.

References

1. Research Outreach. Synthesising circular RNA is as easy as PIE. 2021. https://researchoutreach.org/articles/synthesising-circular-rna-easy-pie/ 

2. Laronde. “Translating medicines for life.” https://www.laronde.bio/erna-science (Accessed Apr. 9, 2022.)

3. Orna Therapeutics. “The power of oRNA.” https://www.ornatx.com/the-power-of-rna/ (Accessed Apr. 21, 2022.)

4. Li, J.; Xu, Q.; Huang, Z. CircRNAs: a new target for the diagnosis and treatment of digestive system neoplasms. Cell Death Dis. 2021, (12)205. https://doi.org/10.1038/s41419-021-03495-0

5. Guria, A.; Sharma, P.; Natesan, S.; Pandi, G. Circular RNAs – The Road Less Traveled. Front. Mol. Biosci. 2020. https://doi.org/10.3389/fmolb.2019.00146

6. He, A.T.; Liu, J.; Li, F.; Yang, B.B. Targeting circular RNAs as a therapeutic approach: current strategies and challenges. Sig Transduct Target Ther. 2021, (6)185. https://doi.org/10.1038/s41392-021-00569-5

7. Barrett, S.P.; Salzman. J. Circular RNAs: analysis, expression and potential functions. Development. 2016, 143 (11), 1838–1847. doi: https://doi.org/10.1242/dev.128074

8. Huang, S.; Li, X.; Zheng, H.; Si, X.; Li, B.; Wei, G.; Li, C.; Chen, Y.; Chen, Y.; Liao, W.; Liao, Y.; Bin, J. Loss of Super-Enhancer-Regulated circRNA Nfix Induces Cardiac Regeneration After Myocardial Infarction in Adult Mice. Circulation. 2019, 139(25), 2857-2876. doi: 10.1161/CIRCULATIONAHA.118.038361