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A circRNA from SEPALLATA3 regulates splicing of its cognate mRNA through R-loop formation

Nature Plants volume 3, Article number: 17053 (2017) Cite this article

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Abstract

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 AS2–4. 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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Figure 1: CircRNAs are bona fide biomarkers of AS in Arabidopsis MADS TFs.
Figure 2: Circular RNA skews AS of its cognate mRNA driving homeotic phenotypes.
Figure 3: CircRNAs can form stable R-loops (RNA:DNA hybrids), which are stronger than with linear RNA.

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Acknowledgements

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).

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Authors and Affiliations

  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, 5000, SA, 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, 138673, Singapore, Singapore

    Vinay Tergaonkar

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

    Vinay Tergaonkar

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

    Markus Schmid

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  1. Vanessa M. Conn
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Contributions

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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Correspondence to Simon J. Conn.

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Supplementary Figures 1–8, Supplementary Table 1, References. (PDF 1203 kb)

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Conn, V., Hugouvieux, V., Nayak, A. et al. A circRNA from SEPALLATA3 regulates splicing of its cognate mRNA through R-loop formation. Nature Plants 3, 17053 (2017). https://doi.org/10.1038/nplants.2017.53

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