Characterization of Circular RNAs

  • Yang Zhang
  • Li YangEmail author
  • Ling-Ling ChenEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1402)


Accumulated lines of evidence reveal that a large number of circular RNAs are produced in transcriptomes from fruit fly to mouse and human. Unlike linear RNAs shaped with 5′ cap and 3′ tail, circular RNAs are characterized by covalently closed loop structures without open terminals, thus requiring specific treatments for their identification and validation. Here, we describe a detailed pipeline for the characterization of circular RNAs. It has been successfully applied to the study of circular intronic RNAs derived from intron lariats (ciRNAs) and circular RNAs produced from back spliced exons (circRNAs) in human.

Key words

Circular RNAs ciRNAs circRNAs RNA fractionation RNase R 



This work was supported by 31271390, 31322018, 91440202 from NSFC to L.Y. and L.L.C.


  1. 1.
    Sanger HL et al (1976) Viroids are single-stranded covalently closed circular RNA molecules existing as highly base-paired rod-like structures. Proc Natl Acad Sci U S A 73:3852–3856CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Arnberg AC et al (1980) Some yeast mitochondrial RNAs are circular. Cell 19:313–319CrossRefPubMedGoogle Scholar
  3. 3.
    Kos A et al (1986) The hepatitis delta (delta) virus possesses a circular RNA. Nature 323:558–560CrossRefPubMedGoogle Scholar
  4. 4.
    Nigro JM et al (1991) Scrambled exons. Cell 64:607–613CrossRefPubMedGoogle Scholar
  5. 5.
    Cocquerelle C et al (1992) Splicing with inverted order of exons occurs proximal to large introns. EMBO J 11:1095–1098PubMedPubMedCentralGoogle Scholar
  6. 6.
    Capel B et al (1993) Circular transcripts of the testis-determining gene Sry in adult mouse testis. Cell 73:1019–1030CrossRefPubMedGoogle Scholar
  7. 7.
    Burd CE et al (2010) Expression of linear and novel circular forms of an INK4/ARF-associated non-coding RNA correlates with atherosclerosis risk. PLoS Genet 6:e1001233CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Hansen TB et al (2011) miRNA-dependent gene silencing involving Ago2-mediated cleavage of a circular antisense RNA. EMBO J 30:4414–4422CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Hansen TB et al (2013) Natural RNA circles function as efficient microRNA sponges. Nature 495:384–388CrossRefPubMedGoogle Scholar
  10. 10.
    Memczak S et al (2013) Circular RNAs are a large class of animal RNAs with regulatory potency. Nature 495:333–338CrossRefPubMedGoogle Scholar
  11. 11.
    Graveley BR et al (2008) Molecular biology: power sequencing. Nature 453:1197–1198CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Yang L et al (2011) Genomewide characterization of non-polyadenylated RNAs. Genome Biol 12:R16CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Zhang Y et al (2013) Circular intronic long noncoding RNAs. Mol Cell 51:792–806CrossRefPubMedGoogle Scholar
  14. 14.
    Salzman J et al (2012) Circular RNAs are the predominant transcript isoform from hundreds of human genes in diverse cell types. PLoS One 7:e30733CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Zhang XO et al (2014) Complementary sequence-mediated exon circularization. Cell 159:134–147CrossRefPubMedGoogle Scholar
  16. 16.
    Zhang Y, Yang L, Chen LL (2014) Life without A tail: new formats of long noncoding RNAs. Int J Biochem Cell Biol 54:338–349CrossRefPubMedGoogle Scholar
  17. 17.
    Jeck WR et al (2013) Circular RNAs are abundant, conserved, and associated with ALU repeats. RNA 19:141–157CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Salzman J et al (2013) Cell-type specific features of circular RNA expression. PLoS Genet 9:e1003777CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Wang PL et al (2014) Circular RNA is expressed across the eukaryotic tree of life. PLoS One 9:e90859CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Westholm JO et al (2014) Genome-wide analysis of drosophila circular RNAs reveals their structural and sequence properties and age-dependent neural accumulation. Cell Rep 9:1966–1980CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Yin QF, Chen LL, Yang L (2015) Fractionation of non-polyadenylated and ribosomal-free RNAs from mammalian cells. Methods Mol Biol 1206:69–80CrossRefPubMedGoogle Scholar
  22. 22.
    Suzuki H et al (2006) Characterization of RNase R-digested cellular RNA source that consists of lariat and circular RNAs from pre-mRNA splicing. Nucleic Acids Res 34:e63CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.State Key Laboratory of Molecular Biology, Shanghai Key Laboratory of Molecular Andrology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological SciencesChinese Academy of SciencesShanghaiChina
  2. 2.Key Laboratory of Computational Biology, CAS-MPG Partner Institute for Computational Biology, CAS Center for Excellence in Brain Science, Shanghai Institutes for Biological SciencesChinese Academy of SciencesShanghaiChina
  3. 3.School of Life Science and TechnologyShanghaiTech UniversityShanghaiChina

Personalised recommendations