Circular RNAs (circRNAs) are a diverse and abundant class of hyper-stable, non-canonical RNAs that arise through a form of alternative splicing (AS) called back-splicing. These single-stranded, covalently-closed circRNA molecules have been identified in all eukaryotic kingdoms of life1, yet their functions have remained elusive. Here, we report that circRNAs can be used as bona fide biomarkers of functional, exon-skipped AS variants in Arabidopsis, including in the homeotic MADS-box transcription factor family. Furthermore, we demonstrate that circRNAs derived from exon 6 of the SEPALLATA3 (SEP3) gene increase abundance of the cognate exon-skipped AS variant (SEP3.3 which lacks exon 6), in turn driving floral homeotic phenotypes. Toward demonstrating the underlying mechanism, we show that the SEP3 exon 6 circRNA can bind strongly to its cognate DNA locus, forming an RNA:DNA hybrid, or R-loop, whereas the linear RNA equivalent bound significantly more weakly to DNA. R-loop formation results in transcriptional pausing, which has been shown to coincide with splicing factor recruitment and AS2,​3,​4. This report presents a novel mechanistic insight for how at least a subset of circRNAs probably contribute to increased splicing efficiency of their cognate exon-skipped messenger RNA and provides the first evidence of an organismal-level phenotype mediated by circRNA manipulation.

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  1. 1.

    et al. Circular RNA is expressed across the eukaryotic tree of life. PLoS ONE 9, e90859 (2014).

  2. 2.

    , , & Quantitative model of R-loop forming structures reveals a novel level of RNA–DNA interactome complexity. Nucleic Acids Res. 40, e16 (2012).

  3. 3.

    , , & Genome-wide distribution of RNA-DNA hybrids identifies RNase H targets in tRNA genes, retrotransposons and mitochondria. PLoS Genet. 10, e1004716 (2014).

  4. 4.

    , , & Splicing-dependent RNA polymerase pausing in yeast. Mol. Cell 40, 582–593 (2010).

  5. 5.

    & Expansion of the eukaryotic proteome by alternative splicing. Nature 463, 457–463 (2010).

  6. 6.

    Shaping the Arabidopsis transcriptome through alternative splicing. Adv. Bot. 2015, 419428 (2015).

  7. 7.

    , , , & Widespread noncoding circular RNAs in plants. New Phytol. 208, 88–95 (2015).

  8. 8.

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

  9. 9.

    , , & Exon skipping is correlated with exon circularization. J. Mol. Biol. 427, 2414–2417 (2015).

  10. 10.

    The biogenesis and emerging roles of circular RNAs. Nat. Rev. Mol. Cell Biol. 17, 205–211 (2016).

  11. 11.

    et al. Integrative analysis of Arabidopsis thaliana transcriptomics reveals intuitive splicing mechanism for circular RNA. FEBS Lett. 590, 3510–3516 (2016).

  12. 12.

    et al. Design of RNA splicing analysis null models for post hoc filtering of Drosophila head RNA-Seq data with the splicing analysis kit (Spanki). BMC Bioinform. 14, 320 (2013).

  13. 13.

    & Plant biology. Floral quartets. Nature 409, 469–471 (2001).

  14. 14.

    et al. Predicting the impact of alternative splicing on plant MADS domain protein function. PLoS ONE 7, e30524 (2012).

  15. 15.

    et al. Temperature-dependent regulation of flowering by antagonistic FLM variants. Nature 503, 414–417 (2013).

  16. 16.

    et al. Regulation of temperature-responsive flowering by MADS-box transcription factor repressors. Science 342, 628–632 (2013).

  17. 17.

    , , , & Nonsense-mediated mRNA decay modulates FLM-dependent thermosensory flowering response in Arabidopsis. Nat. Plants 2, 16055 (2016).

  18. 18.

    et al. Structural basis for the oligomerization of the MADS domain transcription factor SEPALLATA3 in Arabidopsis. Plant Cell 26, 3603–3615 (2014).

  19. 19.

    et al. Target genes of the MADS transcription factor SEPALLATA3: integration of developmental and hormonal pathways in the Arabidopsis flower. PLoS Biol. 7, e1000090 (2009).

  20. 20.

    et al. Evolution of the plant reproduction master regulators LFY and the MADS transcription factors: the role of protein structure in the evolutionary development of the flower. Front. Plant Sci. 6, 1193 (2015).

  21. 21.

    & Detecting and characterizing circular RNAs. Nat. Biotechnol. 32, 453–461 (2014).

  22. 22.

    , , , & The Vienna RNA websuite. Nucleic Acids Res. 36, W70–W74 (2008).

  23. 23.

    et al. How slow RNA polymerase II elongation favors alternative exon skipping. Mol. Cell 54, 683–690 (2014).

  24. 24.

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

  25. 25.

    et al. Circular RNAs are a large class of animal RNAs with regulatory potency. Nature 495, 333–338 (2013).

  26. 26.

    et al. Natural RNA circles function as efficient microRNA sponges. Nature 495, 384–388 (2013).

  27. 27.

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

  28. 28.

    et al. Analysis of an activated ABI5 allele using a new selection method for transgenic Arabidopsis seeds. FEBS Lett. 561, 127–131 (2004).

  29. 29.

    , , & Protocol: a simple phenol-based method for 96-well extraction of high quality RNA from Arabidopsis. Plant Methods 7, 7 (2011).

  30. 30.

    et al. Cell-specific vacuolar calcium storage mediated by CAX1 regulates apoplastic calcium concentration, gas exchange, and plant productivity in Arabidopsis. Plant Cell 23, 240–257 (2011).

  31. 31.

    , , , & R loops regulate promoter-proximal chromatin architecture and cellular differentiation. Nat. Struct. Mol. Biol. 22, 999–1007 (2015).

  32. 32.

    , & The homologous recombination machinery modulates the formation of RNA-DNA hybrids and associated chromosome instability. eLife 2, e00505 (2013).

  33. 33.

    & Splint ligation of RNA with T4 DNA ligase. Methods Mol. Biol. Clifton NJ 941, 257–269 (2012).

  34. 34.

    & Initiation of protein synthesis by the eukaryotic translational apparatus on circular RNAs. Science 268, 415–417 (1995).

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Research reported in this publication was supported by the Agence Nationale de la Recherche (Projet FLOPINET), Centre National de la Recherche Scientifique funding to S.J.C., NHMRC project grant funding (GNT1089167) to S.J.C., Australian Research Council Future Fellowship (FT160100318) to S.J.C., ATIP-Avenir and LabEx GRAL (ANR-10-LABX-49-01) program funding to C.Z., Premier's Research and Industry Fund grant provided by the South Australian Government Department of State Development to V.T. and CEA Irtelis fellowship to A.N. Mouse monoclonal S9.6 antibody was kindly donated by S. Leppla (National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA).

Author information


  1. Laboratoire de Physiologie Cellulaire and Végétale, CNRS, CEA, INRA, Université Grenoble-Alpes, BIG, UMR 5168, Grenoble 38000, France

    • Vanessa M. Conn
    • , Véronique Hugouvieux
    • , Aditya Nayak
    • , Agnès Jourdain
    • , Chloe Zubieta
    •  & Simon J. Conn
  2. Centre for Cancer Biology, SA Pathology and the University of South Australia, Adelaide, SA 5000, Australia

    • Vanessa M. Conn
    • , Stephanie A. Conos
    • , Gökhan Cildir
    • , Vinay Tergaonkar
    •  & Simon J. Conn
  3. Department of Molecular Biology, Max Planck Institute for Developmental Biology, Tübingen 72076, Germany

    • Giovanna Capovilla
    •  & Markus Schmid
  4. Laboratory of NF-κB Signaling, Institute of Molecular and Cell Biology (IMCB), 61 Biopolis Drive, Proteos, Singapore 138673, Singapore

    • Vinay Tergaonkar
  5. Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore (NUS), Singapore 117597, Singapore

    • Vinay Tergaonkar
  6. Umeå Plant Science Centre, Department of Plant Physiology, Umeå University, SE-901 87 Umeå, Sweden

    • Markus Schmid


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S.J.C. and C.Z. conceived the project. V.M.C., S.J.C., V.H., A.N., S.A.C., Gi.C., Go.C., A.J., V.T. and M.S. performed all experiments, provided material and analysed the results. S.J.C. wrote the manuscript. All authors discussed the results and commented on the manuscript.

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The authors declare no competing financial interests.

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Correspondence to Simon J. Conn.

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