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Site-specific DICER and DROSHA RNA products control the DNA-damage response

Nature volume 488, pages 231–235 (2012)Cite this article

Abstract

Non-coding RNAs (ncRNAs) are involved in an increasingly recognized number of cellular events1. Some ncRNAs are processed by DICER and DROSHA RNases to give rise to small double-stranded RNAs involved in RNA interference (RNAi)2. The DNA-damage response (DDR) is a signalling pathway that originates from a DNA lesion and arrests cell proliferation3. So far, DICER and DROSHA RNA products have not been reported to control DDR activation. Here we show, in human, mouse and zebrafish, that DICER and DROSHA, but not downstream elements of the RNAi pathway, are necessary to activate the DDR upon exogenous DNA damage and oncogene-induced genotoxic stress, as studied by DDR foci formation and by checkpoint assays. DDR foci are sensitive to RNase A treatment, and DICER- and DROSHA-dependent RNA products are required to restore DDR foci in RNase-A-treated cells. Through RNA deep sequencing and the study of DDR activation at a single inducible DNA double-strand break, we demonstrate that DDR foci formation requires site-specific DICER- and DROSHA-dependent small RNAs, named DDRNAs, which act in a MRE11–RAD50–NBS1-complex-dependent manner (MRE11 also known as MRE11A; NBS1 also known as NBN). DDRNAs, either chemically synthesized or in vitro generated by DICER cleavage, are sufficient to restore the DDR in RNase-A-treated cells, also in the absence of other cellular RNAs. Our results describe an unanticipated direct role of a novel class of ncRNAs in the control of DDR activation at sites of DNA damage.

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Figure 1: DICER or DROSHA inactivation impairs DDR foci formation in irradiated cells.
Figure 2: Irradiation-induced DDR foci are sensitive to RNase A treatment and are restored by small and DICER-dependent RNAs.
Figure 3: Site-specific DDR focus formation is RNase A sensitive and can be restored by site-specific RNA in a MRN-dependent manner.
Figure 4: Chemically synthesized small RNAs and in vitro -generated DICER RNA products are sufficient to restore DDR focus formation in RNase-A-treated cells in a sequence-specific manner.

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Data deposits

Sequence data have been deposited in the DNA Data Bank of Japan under accession code DRA000540.

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Acknowledgements

We thank E. Soutoglou, W. C. Hahn, M. Kastan, V. Orlando, R. Shiekhattar, J. Amatruda, T. Halazonetis, E. Dejana, P. Ng and F. Nicassio for sharing reagents, M. Fumagalli and F. Rossiello for reading the manuscript, M. Dobreva, V. Matti and F. Pezzimenti for technical support, G. D’Ario for help with statistical analyses, B. Amati, M. Foiani, V. Costanzo and F.d.A.d.F. group members for help and discussions. The F.d.A.d.F. laboratory was supported by Fondazione Italiana Ricerca Sul Cancro (FIRC), Associazione Italiana Ricerca sul Cancro (AIRC) European Community’s 7th Framework Programme (FP7/2007-2013) under grant agreement no. 202230, acronym “GENINCA”, HFSP, AICR, the EMBO Young Investigator Program. The initial part of this project was supported by Telethon grant no. GGP08183. P.C. was supported by 7th Framework of the European Union commission to the Dopaminet consortium, a Grant-in-Aids for Scientific Research (A) no. 20241047, Funding Program for the Next Generation World-Leading Researchers (NEXT Program) to P.C. and a Research Grant to RIKEN Omics Science Center from MEXT. S.F. is supported by Center for Genomic Science of IIT@SEMM (Scuola Europea di Medicina Molecolare) and AIRC. M.M. was supported by Cariplo (grant no. 2007-5500) and AIRC. A.S. is supported by a JSPS fellowship P09745 and grant in aid by JSPS, and D.T. is supported by the European Union 7th Framework Programme under grant agreement FP7-People-ITN-2008-238055 (“BrainTrain” project) to P.C.

Author information

Author notes

  1. Viviana Anelli & Marina Mione

    Present address: Present addresses: Departments of Surgery and Medicine, Weill Cornell Medical College and New York Presbyterian Hospital, 1300 York Avenue, New York, New York 10065, USA (V.A.); Institute of Toxicology and Genetics, Karlsruhe Institute of Technology, 76344 Karlsruhe, Germany (M.M.).,

Authors and Affiliations

  1. IFOM Foundation - FIRC Institute of Molecular Oncology Foundation, Via Adamello 16, 20139 Milan, Italy ,

    Sofia Francia, Flavia Michelini, Viviana Anelli, Marina Mione & Fabrizio d’Adda di Fagagna

  2. Center for Genomic Science of IIT@SEMM, Istituto Italiano di Tecnologia, at the IFOM-IEO Campus, Via Adamello 16, 20139 Milan, Italy ,

    Sofia Francia

  3. Omics Science Center, RIKEN Yokohama Institute, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa 230-0045, Japan ,

    Alka Saxena, Dave Tang, Michiel de Hoon & Piero Carninci

  4. Istituto di Genetica Molecolare, Consiglio Nazionale delle Ricerche, Pavia, 27100, Italy

    Fabrizio d’Adda di Fagagna

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Contributions

A.S., D.T. and P.C. planned, generated and analysed the genomics data presented in Supplementary Figs 20a–e, 21, 22b and 23. M.d.H. performed statistical analysis of the genomics data. A.S. and P.C. also edited the manuscript. M.M. and V.A. generated the data presented in Supplementary Figs 14 and 15. F.M. generated the data shown in Figs 2b, 3d, e, 4b and Supplementary Figs 2b, e, 3e, 4b, 5f, g, 6b–d, 7d, 9, 13d–f, 14d, f, 17f, g, 18a, b, 19, 20g, h and 22a and generated RNA for deep sequencing; contributed to: Supplementary Figs 16a, 5d, e, 11c, d and edited the manuscript. S.F. generated the data shown in remaining figures, contributed to experimental design and edited the manuscript. F.d.A.d.F. conceived the study, designed the experiments and wrote the manuscript.

Corresponding author

Correspondence to Fabrizio d’Adda di Fagagna.

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The authors declare no competing financial interests.

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This file contains Supplementary Introduction, Supplementary Figures 1-23, a Supplementary Discussion and additional references. (PDF 9693 kb)

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Francia, S., Michelini, F., Saxena, A. et al. Site-specific DICER and DROSHA RNA products control the DNA-damage response. Nature 488, 231–235 (2012). https://doi.org/10.1038/nature11179

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