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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Sequence data have been deposited in the DNA Data Bank of Japan under accession code DRA000540.
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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.
The authors declare no competing financial interests.
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