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The biogenesis, biology and characterization of circular RNAs

Abstract

Circular RNAs (circRNAs) are covalently closed, endogenous biomolecules in eukaryotes with tissue-specific and cell-specific expression patterns, whose biogenesis is regulated by specific cis-acting elements and trans-acting factors. Some circRNAs are abundant and evolutionarily conserved, and many circRNAs exert important biological functions by acting as microRNA or protein inhibitors (‘sponges’), by regulating protein function or by being translated themselves. Furthermore, circRNAs have been implicated in diseases such as diabetes mellitus, neurological disorders, cardiovascular diseases and cancer. Although the circular nature of these transcripts makes their detection, quantification and functional characterization challenging, recent advances in high-throughput RNA sequencing and circRNA-specific computational tools have driven the development of state-of-the-art approaches for their identification, and novel approaches to functional characterization are emerging.

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Fig. 1: The biogenesis of circRNAs.
Fig. 2: Methodologies used to detect and quantify the expression of circRNAs.
Fig. 3: General mechanisms of circRNA functions.
Fig. 4: circRNAs acting as miRNA or protein sponges.
Fig. 5: Methodologies for the functional characterization of circRNAs.

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References

  1. Sanger, H. L., Klotz, G., Riesner, D., Gross, H. J. & Kleinschmidt, A. K. Viroids are single-stranded covalently closed circular RNA molecules existing as highly base-paired rod-like structures. Proc. Natl Acad. Sci. USA 73, 3852–3856 (1976).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Hsu, M. T. & Coca-Prados, M. Electron microscopic evidence for the circular form of RNA in the cytoplasm of eukaryotic cells. Nature 280, 339–340 (1979).

    Article  CAS  PubMed  Google Scholar 

  3. Cocquerelle, C., Mascrez, B., Hetuin, D. & Bailleul, B. Mis-splicing yields circular RNA molecules. FASEB J 7, 155–160 (1993).

    Article  CAS  PubMed  Google Scholar 

  4. Capel, B. et al. Circular transcripts of the testis-determining gene Sry in adult mouse testis. Cell 73, 1019–1030 (1993).

    Article  CAS  PubMed  Google Scholar 

  5. Wang, P. L. et al. Circular RNA is expressed across the eukaryotic tree of life. PLOS ONE 9, e90859 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Ivanov, A. et al. Analysis of intron sequences reveals hallmarks of circular RNA biogenesis in animals. Cell Rep. 10, 170–177 (2015).

    Article  CAS  PubMed  Google Scholar 

  7. Jeck, W. R. et al. Circular RNAs are abundant, conserved, and associated with ALU repeats. RNA 19, 141–157 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Salzman, J., Chen, R. E., Olsen, M. N., Wang, P. L. & Brown, P. O. Cell-type specific features of circular RNA expression. PLOS Genet. 9, e1003777 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Westholm, J. O. et al. Genome-wide analysis of drosophila circular RNAs reveals their structural and sequence properties and age-dependent neural accumulation. Cell Rep. 9, 1966–1980 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Maass, P. G. et al. A map of human circular RNAs in clinically relevant tissues. J. Mol. Med. 95, 1179–1189 (2017).

    Article  CAS  Google Scholar 

  11. Xia, S. et al. Comprehensive characterization of tissue-specific circular RNAs in the human and mouse genomes. Brief. Bioinform. 18, 984–992 (2017).

    CAS  PubMed  Google Scholar 

  12. Guo, J. U., Agarwal, V., Guo, H. & Bartel, D. P. Expanded identification and characterization of mammalian circular RNAs. Genome Biol. 15, 409 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Zhang, X. O. et al. Diverse alternative back-splicing and alternative splicing landscape of circular RNAs. Genome Res. 26, 1277–1287 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Memczak, S. et al. Circular RNAs are a large class of animal RNAs with regulatory potency. Nature 495, 333–338 (2013). This article identifies circRNAs as a large class of post-transcriptional regulators that compete with other RNAs for binding by miRNAs and RBPs.

    Article  CAS  PubMed  Google Scholar 

  15. Salzman, J., Gawad, C., Wang, P. L., Lacayo, N. & Brown, P. O. Circular RNAs are the predominant transcript isoform from hundreds of human genes in diverse cell types. PLOS ONE 7, e30733 (2012). This article reveals that the production of circRNA is a general feature of gene expression in human cells.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Li, Z. et al. Exon–intron circular RNAs regulate transcription in the nucleus. Nat. Struct. Mol. Biol. 22, 256–264 (2015).

    Article  CAS  PubMed  Google Scholar 

  17. Zhang, Y. et al. Circular intronic long noncoding RNAs. Mol. Cell 51, 792–806 (2013).

    Article  CAS  PubMed  Google Scholar 

  18. Veno, M. T. et al. Spatio-temporal regulation of circular RNA expression during porcine embryonic brain development. Genome Biol. 16, 245 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Rybak-Wolf, A. et al. Circular RNAs in the mammalian brain are highly abundant, conserved, and dynamically expressed. Mol. Cell 58, 870–885 (2015). This article provides a circRNA brain expression atlas and shows that circRNA expression correlates negatively with the expression of ADAR1.

    Article  CAS  PubMed  Google Scholar 

  20. Enuka, Y. et al. Circular RNAs are long-lived and display only minimal early alterations in response to a growth factor. Nucleic Acids Res. 44, 1370–1383 (2016).

    Article  CAS  PubMed  Google Scholar 

  21. Hansen, T. B. et al. Natural RNA circles function as efficient microRNA sponges. Nature 495, 384–388 (2013). This study is the first to functionally characterize naturally expressed circRNAs.

    Article  CAS  PubMed  Google Scholar 

  22. Izuogu, O. G. et al. Analysis of human ES cell differentiation establishes that the dominant isoforms of the lncRNAs RMST and FIRRE are circular. BMC Genomics 19, 276 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Szabo, L. et al. Statistically based splicing detection reveals neural enrichment and tissue-specific induction of circular RNA during human fetal development. Genome Biol. 16, 126 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. You, X. et al. Neural circular RNAs are derived from synaptic genes and regulated by development and plasticity. Nat. Neurosci. 18, 603–610 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. van Rossum, D., Verheijen, B. M. & Pasterkamp, R. J. Circular RNAs: novel regulators of neuronal development. Front. Mol. Neurosci. 9, 74 (2016).

    PubMed  PubMed Central  Google Scholar 

  26. Bachmayr-Heyda, A. et al. Correlation of circular RNA abundance with proliferation — exemplified with colorectal and ovarian cancer, idiopathic lung fibrosis, and normal human tissues. Sci. Rep. 5, 8057 (2015). This study is the first to report a global reduction in circRNA abundance in cancer relative to normal tissues and presents a model for how circRNAs may accumulate in non-proliferating cells.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Moldovan, L.-I. et al. High-throughput RNA sequencing from paired lesional- and non-lesional skin reveals major alterations in the psoriasis circRNAome. Preprint at bioRxiv https://doi.org/10.1101/581066 (2019).

  28. Fang, Y. et al. Screening of circular RNAs and validation of circANKRD36 associated with inflammation in patients with type 2 diabetes mellitus. Int. J. Mol. Med. 42, 1865–1874 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Hanan, M., Soreq, H. & Kadener, S. CircRNAs in the brain. RNA Biol. 14, 1028–1034 (2017).

    Article  PubMed  Google Scholar 

  30. Holdt, L. M. et al. Circular non-coding RNA ANRIL modulates ribosomal RNA maturation and atherosclerosis in humans. Nat. Commun. 7, 12429 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Kristensen, L. S., Hansen, T. B., Veno, M. T. & Kjems, J. Circular RNAs in cancer: opportunities and challenges in the field. Oncogene 37, 555–565 (2018).

    Article  CAS  PubMed  Google Scholar 

  32. Li, H. et al. Comprehensive circular RNA profiles in plasma reveals that circular RNAs can be used as novel biomarkers for systemic lupus erythematosus. Clin. Chim. Acta 480, 17–25 (2018).

    Article  CAS  PubMed  Google Scholar 

  33. Aufiero, S., Reckman, Y.J., Pinto, Y.M. & Creemers, E.E. Circular RNAs open a new chapter in cardiovascular biology. Nat. Rev. Cardiol. 16, 503–514 (2019).

  34. Vo, J. N. et al. The landscape of circular RNA in. Cancer. Cell 176, 869–881.e813 (2019).

    CAS  Google Scholar 

  35. Cortes-Lopez, M. et al. Global accumulation of circRNAs during aging in Caenorhabditis elegans. BMC Genomics 19, 8 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Gruner, H., Cortes-Lopez, M., Cooper, D. A., Bauer, M. & Miura, P. CircRNA accumulation in the aging mouse brain. Sci. Rep. 6, 38907 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Fu, X. D. & Ares, M. Jr. Context-dependent control of alternative splicing by RNA-binding proteins. Nat. Rev. Genet. 15, 689–701 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Ashwal-Fluss, R. et al. circRNA biogenesis competes with pre-mRNA splicing. Mol. Cell 56, 55–66 (2014).

    Article  CAS  PubMed  Google Scholar 

  39. Starke, S. et al. Exon circularization requires canonical splice signals. Cell Rep. 10, 103–111 (2015).

    Article  CAS  PubMed  Google Scholar 

  40. Liang, D. et al. The output of protein-coding genes shifts to circular RNAs when the pre-mRNA processing machinery is limiting. Mol. Cell 68, 940–954.e943 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Kramer, M. C. et al. Combinatorial control of Drosophila circular RNA expression by intronic repeats, hnRNPs, and SR proteins. Genes Dev. 29, 2168–2182 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Kelly, S., Greenman, C., Cook, P. R. & Papantonis, A. Exon skipping is correlated with exon circularization. J. Mol. Biol. 427, 2414–2417 (2015).

    Article  CAS  PubMed  Google Scholar 

  43. Zhang, X. O. et al. Complementary sequence-mediated exon circularization. Cell 159, 134–147 (2014). This article shows that exon circularization is often dependent on flanking intronic complementary sequences.

    Article  CAS  PubMed  Google Scholar 

  44. Conn, S. J. et al. The RNA binding protein quaking regulates formation of circRNAs. Cell 160, 1125–1134 (2015).

    Article  CAS  PubMed  Google Scholar 

  45. Errichelli, L. et al. FUS affects circular RNA expression in murine embryonic stem cell-derived motor neurons. Nat. Commun. 8, 14741 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Eisenberg, E. & Levanon, E. Y. A-to-I RNA editing — immune protector and transcriptome diversifier. Nat. Rev. Genet. 19, 473–490 (2018).

    Article  CAS  PubMed  Google Scholar 

  47. Koh, H. R., Xing, L., Kleiman, L. & Myong, S. Repetitive RNA unwinding by RNA helicase A facilitates RNA annealing. Nucleic Acids Res. 42, 8556–8564 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Aktas, T. et al. DHX9 suppresses RNA processing defects originating from the Alu invasion of the human genome. Nature 544, 115–119 (2017). This study shows that DHX9 suppresses RNA processing defects, including exon circularization, originating from the Alu invasion of the human genome.

    Article  CAS  PubMed  Google Scholar 

  49. Wen, X. et al. NF90 exerts antiviral activity through regulation of PKR phosphorylation and stress granules in infected cells. J. Immunol. 192, 3753–3764 (2014).

    Article  CAS  PubMed  Google Scholar 

  50. Li, X. et al. Coordinated circRNA biogenesis and function with NF90/NF110 in viral infection. Mol. Cell 67, 214–227 (2017).

    Article  CAS  PubMed  Google Scholar 

  51. Eger, N., Schoppe, L., Schuster, S., Laufs, U. & Boeckel, J. N. Circular RNA splicing. Adv. Exp. Med. Biol. 1087, 41–52 (2018).

    Article  CAS  PubMed  Google Scholar 

  52. Ferreira, H. J. et al. Circular RNA CpG island hypermethylation-associated silencing in human cancer. Oncotarget 9, 29208–29219 (2018).

    PubMed  PubMed Central  Google Scholar 

  53. Kristensen, L. S., Okholm, T. L. H., Veno, M. T. & Kjems, J. Circular RNAs are abundantly expressed and upregulated during human epidermal stem cell differentiation. RNA Biol. 15, 280–291 (2018).

    Article  PubMed  Google Scholar 

  54. Shukla, S. et al. CTCF-promoted RNA polymerase II pausing links DNA methylation to splicing. Nature 479, 74–79 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Bentley, D. L. Coupling mRNA processing with transcription in time and space. Nat. Rev. Genet. 15, 163–175 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Rinaldi, L. et al. Dnmt3a and Dnmt3b associate with enhancers to regulate human epidermal stem cell homeostasis. Cell Stem Cell 19, 491–501 (2016).

    Article  CAS  PubMed  Google Scholar 

  57. Chen, N. et al. A novel FLI1 exonic circular RNA promotes metastasis in breast cancer by coordinately regulating TET1 and DNMT1. Genome Biol. 19, 218 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. He, Y. F. et al. Tet-mediated formation of 5-carboxylcytosine and its excision by TDG in mammalian DNA. Science 333, 1303–1307 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Zhang, Y. et al. The biogenesis of nascent circular RNAs. Cell Rep. 15, 611–624 (2016).

    Article  CAS  PubMed  Google Scholar 

  60. Huang, C., Liang, D., Tatomer, D. C. & Wilusz, J. E. A length-dependent evolutionarily conserved pathway controls nuclear export of circular RNAs. Genes Dev. 32, 639–644 (2018). This study provides the first data on how circRNAs are exported from the nucleus to the cytoplasm.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Park, O. H. et al. Endoribonucleolytic cleavage of m(6)A-containing RNAs by RNase P/MRP complex. Mol. Cell 74, 494–507 (2019).

    Article  CAS  PubMed  Google Scholar 

  62. Liu, C. X. et al. Structure and degradation of circular RNAs regulate PKR activation in innate immunity. Cell 177, 865–880 (2019). This study indicates that circRNAs are important players in innate immunity as they form duplexes of 16–26 bp, which bind and regulate PKR activity.

    Article  CAS  PubMed  Google Scholar 

  63. Hansen, T. B. et al. miRNA-dependent gene silencing involving Ago2-mediated cleavage of a circular antisense RNA. EMBO J. 30, 4414–4422 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Kleaveland, B., Shi, C. Y., Stefano, J. & Bartel, D. P. A network of noncoding regulatory RNAs acts in the mammalian brain. Cell 174, 350–362 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Hammond, S. M., Boettcher, S., Caudy, A. A., Kobayashi, R. & Hannon, G. J. Argonaute2, a link between genetic and biochemical analyses of RNAi. Science 293, 1146–1150 (2001).

    Article  CAS  PubMed  Google Scholar 

  66. Lasda, E. & Parker, R. Circular RNAs co-precipitate with extracellular vesicles: a possible mechanism for circRNA clearance. PLOS ONE 11, e0148407 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Dou, Y. et al. Circular RNAs are down-regulated in KRAS mutant colon cancer cells and can be transferred to exosomes. Sci. Rep. 6, 37982 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. Li, Y. et al. Circular RNA is enriched and stable in exosomes: a promising biomarker for cancer diagnosis. Cell Res. 25, 981–984 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Preusser, C. et al. Selective release of circRNAs in platelet-derived extracellular vesicles. J. Extracell. Vesicles 7, 1424473 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  70. Glazar, P., Papavasileiou, P. & Rajewsky, N. circBase: a database for circular RNAs. RNA 20, 1666–1670 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  71. Szabo, L. & Salzman, J. Detecting circular RNAs: bioinformatic and experimental challenges. Nat. Rev. Genet. 17, 679–692 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Rigatti, R., Jia, J. H., Samani, N. J. & Eperon, I. C. Exon repetition: a major pathway for processing mRNA of some genes is allele-specific. Nucleic Acids Res. 32, 441–446 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  73. Chuang, T.J. et al. Integrative transcriptome sequencing reveals extensive alternative trans-splicing and cis-backsplicing in human cells. Nucleic Acids Res. 46, 3671-3691 (2018).

  74. Dahl, M. et al. Enzyme-free digital counting of endogenous circular RNA molecules in B-cell malignancies. Lab. Invest. 98, 1657–1669 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  75. Jeck, W. R. & Sharpless, N. E. Detecting and characterizing circular RNAs. Nat. Biotechnol. 32, 453–461 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  76. Li, X., Yang, L. & Chen, L. L. The biogenesis, functions, and challenges of circular RNAs. Mol. Cell 71, 428–442 (2018).

    Article  CAS  PubMed  Google Scholar 

  77. Hansen, T. B. Improved circRNA identification by combining prediction algorithms. Front. Cell Dev. Biol. 6, 20 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  78. Gao, Y., Zhang, J. & Zhao, F. Circular RNA identification based on multiple seed matching. Brief. Bioinform. 19, 803-810 (2017).

  79. Hansen, T. B., Veno, M. T., Damgaard, C. K. & Kjems, J. Comparison of circular RNA prediction tools. Nucleic Acids Res. 44, e58 (2016).

    Article  PubMed  Google Scholar 

  80. Chen, X. et al. PRMT5 circular RNA promotes metastasis of urothelial carcinoma of the bladder through sponging miR-30c to induce epithelial–mesenchymal transition. Clin. Cancer Res. 24, 6319–6330 (2018).

  81. Bustin, S. A. et al. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin. Chem. 55, 611–622 (2009).

    Article  CAS  PubMed  Google Scholar 

  82. Chen, D.-F., Zhang, L.-J., Tan, K. & Jing, Q. Application of droplet digital PCR in quantitative detection of the cell-free circulating circRNAs. Biotechnol. Biotechnol. Equip. 32, 116-123 (2018).

  83. Li, T. et al. Plasma circular RNA profiling of patients with gastric cancer and their droplet digital RT-PCR detection. J. Mol. Med. 96, 85–96 (2018).

    Article  CAS  Google Scholar 

  84. Geiss, G. K. et al. Direct multiplexed measurement of gene expression with color-coded probe pairs. Nat. Biotechnol. 26, 317–325 (2008).

    Article  CAS  PubMed  Google Scholar 

  85. Baker, A. M. et al. Robust RNA-based in situ mutation detection delineates colorectal cancer subclonal evolution. Nat. Commun. 8, 1998 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  86. Erben, L., He, M. X., Laeremans, A., Park, E. & Buonanno, A. A novel ultrasensitive in situ hybridization approach to detect short sequences and splice variants with cellular resolution. Mol. Neurobiol. 55, 6169–6181 (2018).

    Article  CAS  PubMed  Google Scholar 

  87. Granados-Riveron, J. T. & Aquino-Jarquin, G. CRISPR–Cas13 precision transcriptome engineering in cancer. Cancer Res. 78, 4107–4113 (2018).

    Article  CAS  PubMed  Google Scholar 

  88. Abudayyeh, O. O. et al. RNA targeting with CRISPR-Cas13. Nature 550, 280–284 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  89. Hsu, P. D., Lander, E. S. & Zhang, F. Development and applications of CRISPR–Cas9 for genome engineering. Cell 157, 1262–1278 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  90. Piwecka, M. et al. Loss of a mammalian circular RNA locus causes miRNA deregulation and affects brain function. Science 357 (2017). This study presents the first circRNA knockout mouse model, which indicates that interactions between miRNA and circRNA are important for normal brain function.

  91. Zheng, Q. et al. Circular RNA profiling reveals an abundant circHIPK3 that regulates cell growth by sponging multiple miRNAs. Nat. Commun. 7, 11215 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  92. Dudekula, D. B. et al. CircInteractome: a web tool for exploring circular RNAs and their interacting proteins and microRNAs. RNA Biol. 13, 34–42 (2016).

    Article  PubMed  Google Scholar 

  93. Abdelmohsen, K. et al. Identification of HuR target circular RNAs uncovers suppression of PABPN1 translation by circPABPN1. RNA Biol. 14, 361–369 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  94. Zeng, Y. et al. A circular RNA binds to and activates AKT phosphorylation and nuclear localization reducing apoptosis and enhancing cardiac repair. Theranostics 7, 3842–3855 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  95. Du, W. W. et al. Foxo3 circular RNA retards cell cycle progression via forming ternary complexes with p21 and CDK2. Nucleic Acids Res. 44, 2846–2858 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  96. Du, W. W. et al. Induction of tumor apoptosis through a circular RNA enhancing Foxo3 activity. Cell Death Differ. 24, 357–370 (2017).

    Article  CAS  PubMed  Google Scholar 

  97. Legnini, I. et al. circ-ZNF609 is a circular RNA that can be translated and functions in myogenesis. Mol. Cell 66, 22–37 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  98. Pamudurti, N. R. et al. Translation of circRNAs. Mol. Cell 66, 9–21.e27 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  99. Yang, Y. et al. Extensive translation of circular RNAs driven by N6-methyladenosine. Cell Res 27, 626–641 (2017).

  100. Yang, Y. et al. Novel role of FBXW7 circular RNA in repressing glioma tumorigenesis. J. Natl Cancer Inst. 110, 304–315 (2018).

  101. Zhang, M. et al. A novel protein encoded by the circular form of the SHPRH gene suppresses glioma tumorigenesis. Oncogene 37, 1805–1814 (2018).

    Article  CAS  PubMed  Google Scholar 

  102. Zhang, M. et al. A peptide encoded by circular form of LINC-PINT suppresses oncogenic transcriptional elongation in glioblastoma. Nat. Commun. 9, 4475 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  103. Stagsted, L.V., Nielsen, K.M., Daugaard, I. & Hansen, T. B. Noncoding AUG circRNAs constitute an abundant and conserved subclass of circles. Life Sci. Alliance 2, e201900398 (2019).

  104. Denzler, R., Agarwal, V., Stefano, J., Bartel, D. P. & Stoffel, M. Assessing the ceRNA hypothesis with quantitative measurements of miRNA and target abundance. Mol. Cell 54, 766–776 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  105. Thomson, D. W. & Dinger, M. E. Endogenous microRNA sponges: evidence and controversy. Nat. Rev. Genet. 17, 272–283 (2016).

    Article  CAS  PubMed  Google Scholar 

  106. Weng, W. et al. Circular RNA ciRS-7 — a promising prognostic biomarker and a potential therapeutic target in colorectal cancer. Clin. Cancer Res. 23, 3918-3928 (2017).

  107. Yu, L. et al. The circular RNA Cdr1as act as an oncogene in hepatocellular carcinoma through targeting miR-7 expression. PLOS ONE 11, e0158347 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  108. Yu, C. Y. et al. The circular RNA circBIRC6 participates in the molecular circuitry controlling human pluripotency. Nat. Commun. 8, 1149 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  109. Barbollat-Boutrand, L. et al. MicroRNA-23b-3p regulates human keratinocyte differentiation through repression of TGIF1 and activation of the TGF-ss-SMAD2 signalling pathway. Exp. Dermatol. 26, 51–57 (2017).

    Article  CAS  PubMed  Google Scholar 

  110. Hsiao, K.Y. et al. Non-coding effects of circular RNA CCDC66 promote colon cancer growth and metastasis. Cancer Res. 77, 2339-2350 (2017).

  111. Verduci, L. et al. The oncogenic role of circPVT1 in head and neck squamous cell carcinoma is mediated through the mutant p53/YAP/TEAD transcription-competent complex. Genome Biol. 18, 237 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  112. Xia, P. et al. A circular RNA protects dormant hematopoietic stem cells from DNA sensor cGAS-mediated exhaustion. Immunity 48, 688–701.e687 (2018).

    Article  CAS  PubMed  Google Scholar 

  113. Chen, Q., Sun, L. & Chen, Z. J. Regulation and function of the cGAS-STING pathway of cytosolic DNA sensing. Nat. Immunol. 17, 1142–1149 (2016).

    Article  CAS  PubMed  Google Scholar 

  114. Sato, T. et al. Interferon regulatory factor-2 protects quiescent hematopoietic stem cells from type I interferon-dependent exhaustion. Nat. Med. 15, 696–700 (2009).

    Article  CAS  PubMed  Google Scholar 

  115. Essers, M. A. et al. IFNα activates dormant haematopoietic stem cells in vivo. Nature 458, 904–908 (2009).

    Article  CAS  PubMed  Google Scholar 

  116. Schlee, M. & Hartmann, G. Discriminating self from non-self in nucleic acid sensing. Nat. Rev. Immunol. 16, 566–580 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  117. Chen, Y. G. et al. Sensing self and foreign circular RNAs by intron identity. Mol. Cell 67, 228–238 (2017). This study indicates that circRNAs play important roles in immunobiology and that self–non-self discrimination depends on the introns that flank circRNAs.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  118. Wesselhoeft, R. A. et al. RNA circularization diminishes immunogenicity and can extend translation duration in vivo. Mol. Cell 74, 508–520 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  119. Abe, N. et al. Rolling circle translation of circular RNA in living human cells. Sci. Rep. 5, 16435 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  120. Meyer, K. D. et al. 5′ UTR m(6)A promotes cap-independent translation. Cell 163, 999–1010 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  121. Zhou, J. et al. Dynamic m(6)A mRNA methylation directs translational control of heat shock response. Nature 526, 591–594 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  122. Chen, C. Y. & Sarnow, P. Initiation of protein synthesis by the eukaryotic translational apparatus on circular RNAs. Science 268, 415–417 (1995).

    Article  CAS  PubMed  Google Scholar 

  123. Perriman, R. & Ares, M. Jr. Circular mRNA can direct translation of extremely long repeating-sequence proteins in vivo. RNA 4, 1047–1054 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  124. Wang, Y. & Wang, Z. Efficient backsplicing produces translatable circular mRNAs. RNA 21, 172–179 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  125. Chen, X. et al. circRNADb: a comprehensive database for human circular RNAs with protein-coding annotations. Sci. Rep. 6, 34985 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  126. Ng, S. Y., Bogu, G. K., Soh, B. S. & Stanton, L. W. The long noncoding RNA RMST interacts with SOX2 to regulate neurogenesis. Mol. Cell 51, 349–359 (2013).

    Article  CAS  PubMed  Google Scholar 

  127. Ng, S. Y., Johnson, R. & Stanton, L. W. Human long non-coding RNAs promote pluripotency and neuronal differentiation by association with chromatin modifiers and transcription factors. EMBO J. 31, 522–533 (2012).

    Article  CAS  PubMed  Google Scholar 

  128. Barra, J. & Leucci, E. Probing long non-coding RNA–protein interactions. Front. Mol. Biosci. 4, 45 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  129. Du, W. W. et al. Identifying and characterizing circRNA–protein interaction. Theranostics 7, 4183–4191 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  130. Schneider, T. et al. circRNA–protein complexes: IMP3 protein component defines subfamily of circRNPs. Sci. Rep. 6, 31313 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  131. Bramsen, J. B. et al. A large-scale chemical modification screen identifies design rules to generate siRNAs with high activity, high stability and low toxicity. Nucleic Acids Res. 37, 2867–2881 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  132. Bramsen, J. B. et al. Improved silencing properties using small internally segmented interfering RNAs. Nucleic Acids Res. 35, 5886–5897 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  133. de Bruyns, A., Geiling, B. & Dankort, D. Construction of modular lentiviral vectors for effective gene expression and knockdown. Methods Mol. Biol. 1448, 3–21 (2016).

    Article  CAS  PubMed  Google Scholar 

  134. Herrera-Carrillo, E., Harwig, A. & Berkhout, B. Influence of the loop size and nucleotide composition on AgoshRNA biogenesis and activity. RNA Biol. 14, 1559–1569 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  135. Liu, Y. P., Schopman, N. C. & Berkhout, B. Dicer-independent processing of short hairpin RNAs. Nucleic Acids Res. 41, 3723–3733 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  136. Barrett, S. P., Parker, K. R., Horn, C., Mata, M. & Salzman, J. ciRS-7 exonic sequence is embedded in a long non-coding RNA locus. PLOS Genet. 13, e1007114 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  137. Barrett, S. P. & Salzman, J. Circular RNAs: analysis, expression and potential functions. Development 143, 1838–1847 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  138. Liang, D. & Wilusz, J. E. Short intronic repeat sequences facilitate circular RNA production. Genes Dev. 28, 2233–2247 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  139. Schmidt, C. A., Noto, J. J., Filonov, G. S. & Matera, A. G. A method for expressing and imaging abundant, stable, circular RNAs in vivo using tRNA splicing. Methods Enzymol. 572, 215–236 (2016).

    Article  CAS  PubMed  Google Scholar 

  140. Deng, Q., Ramskold, D., Reinius, B. & Sandberg, R. Single-cell RNA-seq reveals dynamic, random monoallelic gene expression in mammalian cells. Science 343, 193–196 (2014).

    Article  CAS  PubMed  Google Scholar 

  141. Zhu, Q., Shah, S., Dries, R., Cai, L. & Yuan, G.C. Identification of spatially associated subpopulations by combining scRNAseq and sequential fluorescence in situ hybridization data. Nat. Biotechnol. 36, 1183–1190 (2018).

  142. Fan, X. et al. Single-cell RNA-seq transcriptome analysis of linear and circular RNAs in mouse preimplantation embryos. Genome Biol. 16, 148 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  143. Verboom, K. et al. SMARTer single cell total RNA sequencing. Nucleic Acids Res. https://doi.org/10.1093/nar/gkz535 (2019).

  144. Amaria, R. N. et al. Neoadjuvant immune checkpoint blockade in high-risk resectable melanoma. Nat. Med. 24, 1649–1654 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  145. Blank, C.U. et al. Neoadjuvant versus adjuvant ipilimumab plus nivolumab in macroscopic stage III melanoma. Nat. Med. 24, 1655–1661 (2018).

  146. Jain, M. et al. Nanopore sequencing and assembly of a human genome with ultra-long reads. Nat. Biotechnol. 36, 338–345 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  147. Li, R. C. et al. CiRS-7 promotes growth and metastasis of esophageal squamous cell carcinoma via regulation of miR-7/HOXB13. Cell Death Dis. 9, 838 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  148. Barbagallo, D. et al. circSMARCA5 inhibits migration of glioblastoma multiforme cells by regulating a molecular axis involving splicing factors SRSF1/SRSF3/PTB. Int. J. Mol. Sci. 19, E480 (2018).

  149. Begum, S., Yiu, A., Stebbing, J. & Castellano, L. Novel tumour suppressive protein encoded by circular RNA, circ-SHPRH, in glioblastomas. Oncogene 37, 4055–4057 (2018).

    Article  CAS  PubMed  Google Scholar 

  150. Zeng, K. et al. CircHIPK3 promotes colorectal cancer growth and metastasis by sponging miR-7. Cell Death Dis. 9, 417 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  151. Okholm, T. L. H. et al. Circular RNA expression is abundant and correlated to aggressiveness in early-stage bladder cancer. NPJ Genom. Med. 2, 36 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  152. Li, Y. et al. CircHIPK3 sponges miR-558 to suppress heparanase expression in bladder cancer cells. EMBO Rep. 18, 1646–1659 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  153. Smid, M. et al. The circular RNome of primary breast cancer. Genome Res. 29, 356–366 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  154. Bahn, J. H. et al. The landscape of microRNA, Piwi-interacting RNA, and circular RNA in human saliva. Clin. Chem. 61, 221–230 (2015).

    Article  CAS  PubMed  Google Scholar 

  155. Memczak, S., Papavasileiou, P., Peters, O. & Rajewsky, N. Identification and characterization of circular RNAs as a new class of putative biomarkers in human blood. PLOS ONE 10, e0141214 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  156. Stoll, L. et al. Circular RNAs as novel regulators of beta-cell functions in normal and disease conditions. Mol. Metab. 9, 69–83 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  157. Xu, H., Guo, S., Li, W. & Yu, P. The circular RNA Cdr1as, via miR-7 and its targets, regulates insulin transcription and secretion in islet cells. Sci. Rep. 5, 12453 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  158. Wang, K. et al. A circular RNA protects the heart from pathological hypertrophy and heart failure by targeting miR-223. Eur. Heart J. 37, 2602–2611 (2016).

    Article  CAS  PubMed  Google Scholar 

  159. Miao, Q. et al. RNA-seq of circular RNAs identified circPTPN22 as a potential new activity indicator in systemic lupus erythematosus. Lupus 28, 520–528 (2019).

    Article  CAS  PubMed  Google Scholar 

  160. Lukiw, W. J. Circular RNA (circRNA) in Alzheimer’s disease (AD). Front. Genet. 4, 307 (2013).

    PubMed  PubMed Central  Google Scholar 

  161. Shi, Z. et al. The circular RNA ciRS-7 promotes APP and BACE1 degradation in an NF-kappaB-dependent manner. FEBS J. 284, 1096–1109 (2017).

    Article  CAS  PubMed  Google Scholar 

  162. Kumar, L. et al. Functional characterization of novel circular RNA molecule, circzip-2 and its synthesizing gene zip-2 in C. elegans model of Parkinson’s disease. Mol. Neurobiol. 55, 6914–6926 (2018).

    Article  CAS  PubMed  Google Scholar 

  163. Tagawa, T. et al. Discovery of Kaposi’s sarcoma herpesvirus-encoded circular RNAs and a human antiviral circular RNA. Proc. Natl Acad. Sci. USA (2018).

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Acknowledgements

This work was supported by a grant to L.S.K. from the Carlsberg Foundation (CF16-0087).

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L.S.K. researched data for the article. All of the authors wrote and edited the manuscript before submission.

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Correspondence to Lasse S. Kristensen.

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Glossary

Viroids

Small, circular, single-stranded RNA molecules (246–401 nucleotides) that are uncoated and do not encode proteins. They are pathogenic to higher plants.

Alternative splicing

A mechanism by which different forms of a mature RNA can be generated from the same primary RNA by the use of different splice sites.

Alu elements

The most abundant primate-specific DNA transposable elements. These are highly repetitive and composed of ~300 bases.

Innate immune system

The first-line host defence to confine and combat infection.

Lariat formation

Splicing intermediates formed when the 5′ end of the intron being removed is joined to the branch-point adenosine with a 2′,5′-phosphodiester linkage, creating a lasso-shaped molecule.

Debranching

The hydrolysis of 2′,5′-phosphodiester bonds in intron lariats by the lariat debranching enzyme, encoded by DBR1. This hydrolysis converts the intron lariat into a linearized intron, which is subsequently degraded.

Backsplice junction (BSJ) region

The only region of a circular RNA (circRNA) that is distinct from the corresponding linear RNA at the primary sequence level. It is generated through the backsplicing event that generates the circRNA and is composed of a canonical 5′ splice site sequence joined to an upstream 3′ splice site sequence.

Droplet digital PCR

A quantitative PCR method that uses microfluidics (oil–water separation) to amplify individual nucleic acids within individual droplets in the same tube. By measuring the fluorescence signal in each droplet, the copy number of the target molecule can be determined.

Long-term haematopoietic stem cells

Haematopoietic stem cells that are defined by specific surface markers and can self-renew infinitely and differentiate to all cell types within the blood and immune system.

Pattern recognition receptor

Host receptors that recognize molecules typical for pathogens. Upon recognition of pathogen-associated patterns, the innate immune system is activated.

Internal ribosome entry sites

Structural RNA elements that enable the initiation of a cap-independent translation.

Argonaute-crosslinking and immunoprecipitation

A method to identify and map microRNAs bound to AGO proteins and the target transcripts associated with them.

Polysome profiling

A technique to study the translatome based on a sucrose-gradient separation of untranslated and translated RNA transcripts; translated RNA transcripts are associated with polysomes.

Ribosome footprinting

A technique to measure translation by the high-throughput sequencing of ribosome-protected RNA fragments, which determines the position of ribosomes at codon resolution.

Locked nucleic acids

A modified RNA nucleotide in which the ribose moiety is modified with a methylene bridge connecting the 2′ oxygen and 4′ carbon. It has an increased affinity for its complementary nucleotide relative to traditional DNA or RNA oligonucleotides.

Unlocked nucleic acids

An acyclic RNA nucleotide that lacks the C2′–C3′ bond of the ribose moiety found in traditional RNA. It has a decreased affinity for its complementary nucleotide relative to traditional DNA or RNA oligonucleotides.

Passenger disabled siRNA

A small interfering RNA (siRNA) in which an intact antisense strand is complemented with a fragmented sense strand. These siRNAs, which are known as small internally segmented interfering RNAs, eliminate off-target effects by only allowing the functional incorporation of the antisense strand into the RNA-induced silencing complex (RISC).

AgoshRNAs

Short hairpin RNAs that are characterized by a relatively short base-paired stem, which allows them to avoid cleavage by Dicer. Instead, they are processed by the slicer activity of Ago2, which creates a single guide RNA strand that targets a specific RNA for degradation and has less off-target effects as no passenger strand is created.

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Kristensen, L.S., Andersen, M.S., Stagsted, L.V.W. et al. The biogenesis, biology and characterization of circular RNAs. Nat Rev Genet 20, 675–691 (2019). https://doi.org/10.1038/s41576-019-0158-7

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