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Circular RNAs are a large class of animal RNAs with regulatory potency

Abstract

Circular RNAs (circRNAs) in animals are an enigmatic class of RNA with unknown function. To explore circRNAs systematically, we sequenced and computationally analysed human, mouse and nematode RNA. We detected thousands of well-expressed, stable circRNAs, often showing tissue/developmental-stage-specific expression. Sequence analysis indicated important regulatory functions for circRNAs. We found that a human circRNA, antisense to the cerebellar degeneration-related protein 1 transcript (CDR1as), is densely bound by microRNA (miRNA) effector complexes and harbours 63 conserved binding sites for the ancient miRNA miR-7. Further analyses indicated that CDR1as functions to bind miR-7 in neuronal tissues. Human CDR1as expression in zebrafish impaired midbrain development, similar to knocking down miR-7, suggesting that CDR1as is a miRNA antagonist with a miRNA-binding capacity ten times higher than any other known transcript. Together, our data provide evidence that circRNAs form a large class of post-transcriptional regulators. Numerous circRNAs form by head-to-tail splicing of exons, suggesting previously unrecognized regulatory potential of coding sequences.

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Figure 1: Detection, classification and evolutionary conservation of circRNAs.
Figure 2: CircRNAs are stable transcripts with robust expression.
Figure 3: The circRNA CDR1as is bound by the miRNA effector protein AGO, and is cytoplasmic.
Figure 4: CDR1as and miR-7 have overlapping and specific expression in neuronal tissues.
Figure 5: In zebrafish, knockdown of miR-7 or expression of CDR1as causes midbrain defects.

Accession codes

Primary accessions

Gene Expression Omnibus

Data deposits

Sequencing data have been deposited at GEO under accession number GSE43574.

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)

    CAS  Article  ADS  PubMed  PubMed Central  Google Scholar 

  2. Grabowski, P. J., Zaug, A. J. & Cech, T. R. The intervening sequence of the ribosomal RNA precursor is converted to a circular RNA in isolated nuclei of Tetrahymena. Cell 23, 467–476 (1981)

    CAS  Article  PubMed  Google Scholar 

  3. Danan, M., Schwartz, S., Edelheit, S. & Sorek, R. Transcriptome-wide discovery of circular RNAs in Archaea. Nucleic Acids Res. 40, 3131–3142 (2012)

    CAS  Article  PubMed  Google Scholar 

  4. Nigro, J. M. et al. Scrambled exons. Cell 64, 607–613 (1991)

    CAS  Article  PubMed  Google Scholar 

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

    CAS  Article  PubMed  Google Scholar 

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

    CAS  Article  PubMed  Google Scholar 

  7. Chao, C. W., Chan, D. C., Kuo, A. & Leder, P. The mouse formin (Fmn) gene: abundant circular RNA transcripts and gene-targeted deletion analysis. Mol. Med. 4, 614–628 (1998)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  8. Burd, C. E. et al. Expression of linear and novel circular forms of an INK4/ARF-associated non-coding RNA correlates with atherosclerosis risk. PLoS Genet. 6, e1001233 (2010)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  10. 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)

    CAS  Article  ADS  PubMed  PubMed Central  Google Scholar 

  11. Ambros, V. The functions of animal microRNAs. Nature 431, 350–355 (2004)

    CAS  Article  ADS  PubMed  Google Scholar 

  12. Baek, D. et al. The impact of microRNAs on protein output. Nature 455, 64–71 (2008)

    CAS  Article  ADS  PubMed  PubMed Central  Google Scholar 

  13. Selbach, M. et al. Widespread changes in protein synthesis induced by microRNAs. Nature 455, 58–63 (2008)

    CAS  Article  ADS  PubMed  Google Scholar 

  14. Bartel, D. P. MicroRNAs: target recognition and regulatory functions. Cell 136, 215–233 (2009)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  15. Krek, A. et al. Combinatorial microRNA target predictions. Nature Genet. 37, 495–500 (2005)

    CAS  Article  PubMed  Google Scholar 

  16. Lewis, B. P., Burge, C. B. & Bartel, D. P. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 120, 15–20 (2005)

    CAS  Article  PubMed  Google Scholar 

  17. Xie, X. et al. Systematic discovery of regulatory motifs in human promoters and 3′ UTRs by comparison of several mammals. Nature 434, 338–345 (2005)

    CAS  Article  ADS  PubMed  PubMed Central  Google Scholar 

  18. Friedman, R. C., Farh, K. K., Burge, C. B. & Bartel, D. P. Most mammalian mRNAs are conserved targets of microRNAs. Genome Res. 19, 92–105 (2009)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  19. Ebert, M. S., Neilson, J. R. & Sharp, P. A. MicroRNA sponges: competitive inhibitors of small RNAs in mammalian cells. Nature Methods 4, 721–726 (2007)

    CAS  Article  PubMed  Google Scholar 

  20. Franco-Zorrilla, J. M. et al. Target mimicry provides a new mechanism for regulation of microRNA activity. Nature Genet. 39, 1033–1037 (2007)

    CAS  Article  PubMed  Google Scholar 

  21. Poliseno, L. et al. A coding-independent function of gene and pseudogene mRNAs regulates tumour biology. Nature 465, 1033–1038 (2010)

    CAS  Article  ADS  PubMed  PubMed Central  Google Scholar 

  22. Tay, Y. et al. Coding-independent regulation of the tumor suppressor PTEN by competing endogenous mRNAs. Cell 147, 344–357 (2011)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  23. Cesana, M. et al. A long noncoding RNA controls muscle differentiation by functioning as a competing endogenous RNA. Cell 147, 358–369 (2011)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  24. Ebert, M. S. & Sharp, P. A. Emerging roles for natural microRNA sponges. Curr. Biol. 20, R858–R861 (2010)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  25. Vivancos, A. P., Guell, M., Dohm, J. C., Serrano, L. & Himmelbauer, H. Strand-specific deep sequencing of the transcriptome. Genome Res. 20, 989–999 (2010)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  26. Huang, R. et al. An RNA-Seq strategy to detect the complete coding and non-coding transcriptome including full-length imprinted macro ncRNAs. PLoS ONE 6, e27288 (2011)

    CAS  Article  ADS  PubMed  PubMed Central  Google Scholar 

  27. Kent, W. J. et al. The human genome browser at UCSC. Genome Res. 12, 996–1006 (2002)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  28. Pruitt, K. D., Tatusova, T. & Maglott, D. R. NCBI Reference Sequence (RefSeq): a curated non-redundant sequence database of genomes, transcripts and proteins. Nucleic Acids Res. 33, D501–D504 (2005)

    CAS  Article  PubMed  Google Scholar 

  29. Cabili, M. N. et al. Integrative annotation of human large intergenic noncoding RNAs reveals global properties and specific subclasses. Genes Dev. 25, 1915–1927 (2011)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  30. Suzuki, H. et al. Characterization of RNase R-digested cellular RNA source that consists of lariat and circular RNAs from pre-mRNA splicing. Nucleic Acids Res. 34, e63 (2006)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Iwai, Y., Akahane, K., Pluznik, D. H. & Cohen, R. B. Ca2+ ionophore A23187-dependent stabilization of granulocyte-macrophage colony-stimulating factor messenger RNA in murine thymoma EL-4 cells is mediated through two distinct regions in the 3′-untranslated region. J. Immunol. 150, 4386–4394 (1993)

    CAS  PubMed  Google Scholar 

  32. Hafner, M. et al. Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP. Cell 141, 129–141 (2010)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  33. Lebedeva, S. et al. Transcriptome-wide analysis of regulatory interactions of the RNA-binding protein HuR. Mol. Cell 43, 340–352 (2011)

    CAS  Article  PubMed  Google Scholar 

  34. Baltz, A. G. et al. The mRNA-bound proteome and its global occupancy profile on protein-coding transcripts. Mol. Cell 46, 674–690 (2012)

    CAS  Article  PubMed  Google Scholar 

  35. Dropcho, E. J., Chen, Y. T., Posner, J. B. & Old, L. J. Cloning of a brain protein identified by autoantibodies from a patient with paraneoplastic cerebellar degeneration. Proc. Natl Acad. Sci. USA 84, 4552–4556 (1987)

    CAS  Article  ADS  PubMed  PubMed Central  Google Scholar 

  36. Wee, L. M., Flores-Jasso, C. F., Salomon, W. E. & Zamore, P. D. Argonaute divides its RNA guide into domains with distinct functions and RNA-binding properties. Cell 151, 1055–1067 (2012)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

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

    CAS  Article  Google Scholar 

  38. Landgraf, P. et al. A mammalian microRNA expression atlas based on small RNA library sequencing. Cell 129, 1401–1414 (2007)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  39. Shaw, G., Morse, S., Ararat, M. & Graham, F. L. Preferential transformation of human neuronal cells by human adenoviruses and the origin of HEK 293 cells. FASEB J. 16, 869–871 (2002)

    CAS  Article  PubMed  Google Scholar 

  40. Kaufman, M. H. & Bard, J. B. L. The Anatomical Basis of Mouse Development (Academic, 1999)

    Google Scholar 

  41. Schambra, U. Prenatal Mouse Brain Atlas (Springer, 2008)

    Book  Google Scholar 

  42. Kapsimali, M. et al. MicroRNAs show a wide diversity of expression profiles in the developing and mature central nervous system. Genome Biol. 8, R173 (2007)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Jacobs, T. et al. Localized activation of p21-activated kinase controls neuronal polarity and morphology. J. Neurosci. 27, 8604–8615 (2007)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  44. Chacon, M. R. et al. Focal adhesion kinase regulates actin nucleation and neuronal filopodia formation during axonal growth. Development 139, 3200–3210 (2012)

    CAS  Article  PubMed  Google Scholar 

  45. Kato, M. et al. Cell-free formation of RNA granules: low complexity sequence domains form dynamic fibers within hydrogels. Cell 149, 753–767 (2012)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  46. Romeo, T. Global regulation by the small RNA-binding protein CsrA and the non-coding RNA molecule CsrB. Mol. Microbiol. 29, 1321–1330 (1998)

    CAS  Article  PubMed  Google Scholar 

  47. Gottesman, S. The small RNA regulators of Escherichia coli: roles and mechanisms. Annu. Rev. Microbiol. 58, 303–328 (2004)

    CAS  Article  PubMed  Google Scholar 

  48. Huelsken, J., Vogel, R., Erdmann, B., Cotsarelis, G. & Birchmeier, W. β-Catenin controls hair follicle morphogenesis and stem cell differentiation in the skin. Cell 105, 533–545 (2001)

    CAS  Article  PubMed  Google Scholar 

  49. Park, H. C. et al. Analysis of upstream elements in the HuC promoter leads to the establishment of transgenic zebrafish with fluorescent neurons. Dev. Biol. 227, 279–293 (2000)

    CAS  Article  PubMed  Google Scholar 

  50. Peri, F. & Nusslein-Volhard, C. Live imaging of neuronal degradation by microglia reveals a role for v0-ATPase a1 in phagosomal fusion in vivo. Cell 133, 916–927 (2008)

    CAS  Article  PubMed  Google Scholar 

  51. Jeck, W. R. et al. Circular RNAs are abundant, conserved, and associated with ALU repeats. RNA 19, 1–17 (2013)

    Article  CAS  Google Scholar 

  52. Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nature Methods 9, 357–359 (2012)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  53. Aroian, R. V., Field, C., Pruliere, G., Kenyon, C. & Alberts, B. M. Isolation of actin-associated proteins from Caenorhabditis elegans oocytes and their localization in the early embryo. EMBO J. 16, 1541–1549 (1997)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  54. L’Hernault, S. W. & Roberts, T. M. Cell biology of nematode sperm. Methods Cell Biol. 48, 273–301 (1995)

    Article  PubMed  Google Scholar 

  55. Stoeckius, M. et al. Large-scale sorting of C. elegans embryos reveals the dynamics of small RNA expression. Nature Methods 6, 745–751 (2009)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  56. Trapnell, C. et al. Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nature Biotechnol. 28, 511–515 (2010)

    CAS  Article  Google Scholar 

  57. Hinrichs, A. S. et al. The UCSC Genome Browser Database: update 2006. Nucleic Acids Res. 34, D590–D598 (2006)

    CAS  Article  PubMed  Google Scholar 

  58. Pollard, K. S. et al. Detection of nonneutral substitution rates on mammalian phylogenies. Genome Res. 20, 110–121 (2010)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  59. Blankenberg, D. et al. Galaxy: a web-based genome analysis tool for experimentalists. Curr. Protoc. Mol. Biol. Ch. 19, Unit 19 10 11-21. (2010)

  60. Giardine, B. et al. Galaxy: a platform for interactive large-scale genome analysis. Genome Res. 15, 1451–1455 (2005)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  61. Goecks, J., Nekrutenko, A., Taylor, J. & Galaxy, T. Galaxy: a comprehensive approach for supporting accessible, reproducible, and transparent computational research in the life sciences. Genome Biol. 11, R86 (2010)

    Article  PubMed  PubMed Central  Google Scholar 

  62. Griffiths-Jones, S. The microRNA Registry. Nucleic Acids Res. 32, D109–D111 (2004)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  63. Landthaler, M. et al. Molecular characterization of human Argonaute-containing ribonucleoprotein complexes and their bound target mRNAs. RNA 14, 2580–2596 (2008)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  64. Rudel, S., Flatley, A., Weinmann, L., Kremmer, E. & Meister, G. A multifunctional human Argonaute2-specific monoclonal antibody. RNA 14, 1244–1253 (2008)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25, 1754–1760 (2009)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  66. Zisoulis, D. G. et al. Comprehensive discovery of endogenous Argonaute binding sites in Caenorhabditis elegans. Nature Struct. Mol. Biol. 17, 173–179 (2010)

    CAS  Article  Google Scholar 

  67. Langmead, B., Trapnell, C., Pop, M. & Salzberg, S. L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. Kent, W. J. BLAT–the BLAST-like alignment tool. Genome Res. 12, 656–664 (2002)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  69. Goujon, M. et al. A new bioinformatics analysis tools framework at EMBL-EBI. Nucleic Acids Res. 38, W695–W699 (2010)

    CAS  Article  ADS  PubMed  PubMed Central  Google Scholar 

  70. Bernhart, S. H. et al. Partition function and base pairing probabilities of RNA heterodimers. Algorithms Mol. Biol. 1, 3 (2006)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  71. Ventura, A. et al. Cre-lox-regulated conditional RNA interference from transgenes. Proc. Natl Acad. Sci. USA 101, 10380–10385 (2004)

    CAS  Article  ADS  PubMed  PubMed Central  Google Scholar 

  72. Westerfield, M. The Zebrafish Book: A Guide for the Laboratory Use of Zebrafish (Brachydanio rerio) 2nd edn (Univ. Oregon Press, 1993)

    Google Scholar 

  73. Krueger, J. et al. Flt1 acts as a negative regulator of tip cell formation and branching morphogenesis in the zebrafish embryo. Development 138, 2111–2120 (2011)

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  74. Friedländer, M. R., Mackowiak, S. D., Li, N., Chen, W. & Rajewsky, N. miRDeep2 accurately indentifies known and hundreds of novel microRNA genes in seven animal clades. Nucleic Acids Res. 40, 37–52 (2012)

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We thank M. Feldkamp and C. Langnick (laboratory of W. Chen) for Illumina sequencing runs. We thank J. Kjems for sending us a plasmid encoding circular human CDR1as for our zebrafish experiments. We thank K. Meier for technical assistance with zebrafish experiments and A. Sporbert from the confocal imaging facility. We thank A. Ivanov for assisting in bioinformatic analysis. N.R. thanks E. Westhof for useful discussions. We acknowledge the following funding sources: PhD program of the Max-Delbrück-Center (MDC) (S.M., F.T., L.H.G.); the MDC-NYU exchange program (M.M.); BMBF project 1210182, ‘MiRNAs as therapeutic targets’ (A.E.); DFG for KFO218 (U.Z.); Helmholtz Association for the ‘MDC Systems Biology Network’, MSBN (S.D.M.); BMBF support for the DZHK (F.l.N. and N.R.); Center for Stroke Research Berlin (J.K., F.l.N.). Funding for the group of M.L. is supported by BMBF-funding for the Berlin Institute for Medical Systems Biology (0315362C).

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Contributions

S.M., M.J., A.E. and F.T. contributed equally. S.M. performed many experiments, assisted by L.M. M.J. and A.E. carried out most of the computation, with contributions from N.R. and S.D.M. F.T. performed the circRNA validation experiments. A.R. performed all northern experiments. L.H.G. and M.M. contributed AGO PAR-CLIP experiments and HEK293 ribominus data, supervised by M.L. C.K. designed and carried out the single molecule experiments, in part together with A.L. U.Z. performed the mouse experiments. J.K. contributed the zebrafish experiments, supervised by F.l.N. N.R. designed and supervised the project. N.R. and M.J. wrote the paper.

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Correspondence to Nikolaus Rajewsky.

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Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-10 and Supplementary Tables 4, 5 and 7. (PDF 13652 kb)

Supplementary Table 1

This file contains a description of Ribominus DeepSequencing libraries. (XLS 11 kb)

Supplementary Table 2

This file contains predicted circRNA candidates. (XLS 5421 kb)

Supplementary Table 3

This file contains a summary of circRNA validation experiments. (XLS 21 kb)

Supplementary Table 6

This file contains primer standard curves. (XLS 107 kb)

Supplementary Table 8

This file contains DNA oligos. (XLS 41 kb)

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Memczak, S., Jens, M., Elefsinioti, A. et al. Circular RNAs are a large class of animal RNAs with regulatory potency. Nature 495, 333–338 (2013). https://doi.org/10.1038/nature11928

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