circRNA – Introduction
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circRNA Characteristics and Biogenesis
circular RNA (circRNA) is a novel type of single-stranded, non-coding RNA molecule widely expressed in eukaryotes. Unlike typical RNA molecules, these circRNAs possess no 5’ cap or 3’ end polyA tail and instead form a ring structure joined by a covalent bond.
circRNAs are mainly formed by a mechanism known as back-splicing which is carried out by the spliceosome machinery. This process involves a downstream splice donor site joining with an upstream splice acceptor site in reverse order across one or multiple exons to form a circRNA (1). There are two main models for the biogenesis of circRNAs including direct back-splicing and indirect or lariat intermediate model (2). In the direct back splicing model, a branch point upstream of the circularized exon attacks the downstream splice donor site which results in a pre-mRNA intermediate containing a 2’,5’-phosphodiester link. In a subsequent step, the upstream splice acceptor site is attacked by the free 3’ hydroxyl group which then forms the circRNA. In the indirect back splicing (lariat intermediate) model, circRNA is formed from an exon-containing lariat intermediate following a forward splicing event that subsequently undergoes an internal splicing reaction resulting in the excision of a skipped exon to form the final circRNA.
Figure 1 – Models for the biogenesis of linear and circular RNA including forward splicing (A), direct back splicing (B) and indirect or lariat intermediate back splicing (C).
circRNA Functions
Historically circRNAs were thought to be rare byproducts of aberrant splicing events. However, recent findings in the past two decades have revealed that they are widely expressed and have functionally significant roles in eukaryotes (3). circRNA is a fairly new area of research and thus the extent of their roles in biology is not fully elucidated. Currently, a growing number of publications have implicated aberrant expression of circRNAs to be associated with diseases and this will likely remain a key focus point for researchers as circRNAs have been specifically linked to diabetes mellitus, cardiovascular disease and cancer.
Table 1: Summary of circRNA functions and their potential applications
| Function |
Description |
Potential Application |
Reference |
| miRNA sponge |
circRNAs can contain multiple miRNA binding sites, thus work to decrease miRNA availability and in turn upregulate gene expression |
Cancer therapy
Tumor suppressor
Cardiovascular disease therapy
Neurological disorder therapy
Disease biomarkers
|
(4) |
| Regulating transcription and translation |
circRNAs can interact with RNA Polymerase (RNAP) complexes and in turn enhance transcription. circRNAs can bind directly to mRNA thereby suppressing translation |
Transcription and translation regulators
|
(5, 6) |
| Sequestering and translocating proteins |
circRNAs can function as protein sponges and can facilitate protein trafficking within the cell |
Cancer biomarker
|
(7, 8) |
| Facilitating interactions between proteins |
circRNAs can act as scaffolds and facilitate complex protein interactions resulting in various physiological functions |
Transcription regulators
Cell cycle regulators
|
(9) |
| Translating into proteins |
circRNAs can be translated into proteins in a 5’ cap independent and IRES dependent manner |
Neurological disorder therapy
Cancer therapeutics
Disease biomarkers
|
(10, 11) |
References
- Chen, L. (2020). The expanding regulatory mechanisms and cellular functions of circular RNAs. Nature Reviews Molecular Cell Biology, 21(8), 475–490. https://doi.org/10.1038/s41580-020-0243-y
- Barrett, S. P., Wang, P. L., & Salzman, J. (2015). Circular RNA biogenesis can proceed through an exon-containing lariat precursor. eLife, 4. https://doi.org/10.7554/elife.07540
- Liang, Y., Liu, N., Yang, L., Tang, J., Wang, Y., & Meng, M. (2021). A brief review of CirCRNA biogenesis, detection, and function. Current Genomics, 22(7), 485–495. https://doi.org/10.2174/1389202922666210331130722
- Hansen, T. B., Jensen, T. I., Clausen, B. H., Bramsen, J. B., Finsen, B., Damgaard, C. K., & Kjems, J. (2013). Natural RNA circles function as efficient microRNA sponges. Nature, 495(7441), 384–388. https://doi.org/10.1038/nature11993
- Zhang, Y., Zhang, X., Tian, C., Xiang, J., Yin, Q., Xing, Y., Zhu, S., Yang, L., & Chen, L. (2013). Circular intronic Long Noncoding RNAs. Molecular Cell, 51(6), 792–806. https://doi.org/10.1016/j.molcel.2013.08.017
- Wu, N., Yuan, Z., Du, K. Y., Fang, L., Lyu, J., Zhang, C., He, A. T., Eshaghi, E., Zeng, K., Ma, J., Du, W. W., & Yang, B. B. (2019). Translation of yes-associated protein (YAP) was antagonized by its circular RNA via suppressing the assembly of the translation initiation machinery. Cell Death & Differentiation, 26(12), 2758–2773. https://doi.org/10.1038/s41418-019-0337-2
- Du, W. W., Yang, W., Chen, Y., Wu, Z., Foster, F. S., Yang, Z., Li, X., & Yang, B. B. (2016). Foxo3 circular RNA promotes cardiac senescence by modulating multiple factors associated with stress and senescence responses. European Heart Journal, ehw001. https://doi.org/10.1093/eurheartj/ehw001
- Yang, Q., Du, W. W., Wu, N., Yang, W., Awan, F. M., Fang, L., Ma, J., Li, X., Zeng, Y., Yang, Z., Dong, J., Khorshidi, A., & Yang, B. B. (2017). A circular RNA promotes tumorigenesis by inducing c-myc nuclear translocation. Cell Death & Differentiation, 24(9), 1609–1620. https://doi.org/10.1038/cdd.2017.86
- Du, W. W., Yang, W., Liu, E., Yang, Z., Dhaliwal, P., & Yang, B. B. (2016). Foxo3 circular RNA retards cell cycle progression via forming ternary complexes with p21 and CDK2. Nucleic Acids Research, 44(6), 2846–2858. https://doi.org/10.1093/nar/gkw027
- Legnini, I., Di Timoteo, G., Rossi, F. M., Morlando, M., Briganti, F., Sthandier, O., Fatica, A., Santini, T., Andronache, A., Wade, M., Laneve, P., Rajewsky, N., & Bozzoni, I. (2017). Circ-ZNF609 Is a Circular RNA that Can Be Translated and Functions in Myogenesis. Molecular Cell, 66(1), 22-37.e9. https://doi.org/10.1016/j.molcel.2017.02.017
- Mo, D., Li, X., Raabe, C. A., Rozhdestvensky, T. S., Skryabin, B. V., & Brosius, J. (2020). Circular RNA encoded amyloid beta peptides—A novel putative player in Alzheimer’s disease. Cells, 9(10), 2196. https://doi.org/10.3390/cells9102196
- Obi, P., & Chen, Y. G. (2021b). The design and synthesis of circular RNAs. Methods, 196, 85–103. https://doi.org/10.1016/j.ymeth.2021.02.020
- Ashwal-Fluss, R., Meyer, M., Pamudurti, N. R., Ivanov, A., Bartok, O., Hanan, M., Evantal, N., Memczak, S., Rajewsky, N., & Kadener, S. (2014). circRNA Biogenesis Competes with Pre-mRNA Splicing. Molecular Cell, 56(1), 55–66. https://doi.org/10.1016/j.molcel.2014.08.019
- Zhao, J., Lee, E. E., Kim, J., Yang, R., Chamseddin, B., Ni, C., Gusho, E., Xie, Y., Chiang, C. M., Buszczak, M., Zhan, X., Laimins, L. A., & Wang, R. C. (2019). Transforming activity of an oncoprotein-encoding circular RNA from human papillomavirus. Nature Communications, 10(1). https://doi.org/10.1038/s41467-019-10246-5
- CRostain, W., Shen, S., Cordero, T., Rodrigo, G., & Jaramillo, A. (2020). Engineering a Circular Riboregulator in Escherichia coli. Biodesign Research, 2020. https://doi.org/10.34133/2020/1916789
- Litke, J. L., & Jaffrey, S. R. (2019). Highly efficient expression of circular RNA aptamers in cells using autocatalytic transcripts. Nature Biotechnology, 37(6), 667–675. https://doi.org/10.1038/s41587-019-0090-6
- Gholamalipour, Y., Mudiyanselage, A. K., & Martin, C. T. (2018). 3′ end additions by T7 RNA polymerase are RNA self-templated, distributive and diverse in character—RNA-Seq analyses. Nucleic Acids Research, 46(18), 9253–9263. https://doi.org/10.1093/nar/gky796
- Dolinnaya, N. G., Sokolova, N. I., Ashirbekova, D., & Shabarova, Z. A. (1991). The use of BrCN for assembling modified DNA duplexes and DNA-RNA hybrids; comparison with water-soluble carbodiimide. Nucleic Acids Research, 19(11), 3067–3072. https://doi.org/10.1093/nar/19.11.3067
- Wang, L., & Ruffner, D. E. (1998). Oligoribonucleotide circularization by “template-mediated” ligation with T4 RNA ligase: Synthesis of circular hammerhead ribozymes. Nucleic Acids Research, 26(10), 2502–2504. https://doi.org/10.1093/nar/26.10.2502
- Petkovic, S., & Müller, S. (2013). RNA self‐processing: Formation of cyclic species and concatemers from a small engineered RNA. FEBS Letters, 587(15), 2435–2440. https://doi.org/10.1016/j.febslet.2013.06.013
- AShi, H., Zhou, Y., Jia, E., Liu, Z., Pan, M., Bai, Y. M., Zhao, X., & Ge, Q. (2021). Comparative analysis of circular RNA enrichment methods. RNA Biology, 19(1), 55–67. https://doi.org/10.1080/15476286.2021.2012632
- Panda, A. C., & Gorospe, M. (2018). Detection and analysis of circular RNAs by RT-PCR. Bio-protocol, 8(6). https://doi.org/10.21769/bioprotoc.2775
- Chen, L., Wang, C., Sun, H., Wang, J., Liang, Y., Wang, Y., & Wong, G. (2020). The bioinformatics toolbox for circRNA discovery and analysis. Briefings in Bioinformatics, 22(2), 1706–1728. https://doi.org/10.1093/bib/bbaa001
