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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.

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Primary accessions

Gene Expression Omnibus

Data deposits

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

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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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Authors

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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The authors declare no competing financial interests.

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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