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

Non-coding RNAs (ncRNAs) are involved in an increasing number of cellular events(1). Some ncRNAs are processed by DICER and DROSHA ribonucleases to give rise to small double-stranded RNAs involved in RNA interference (RNAi)(2). The DNA-damage response (DDR) is a signaling pathway that originates from the DNA lesion and arrests cell proliferation(3). So far, DICER or DROSHA RNA products have not been reported to control DDR activation. Here we show that DICER and DROSHA, but not downstream elements of the RNAi pathway, are necessary to activate DDR upon oncogene-induced genotoxic stress and exogenous DNA damage, as studied also by DDR foci formation in mammalian cells and zebrafish 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 treated cells. Through RNA deep sequencing and studies of DDR activation at an inducible unique DNA double-strand break (DSB), we demonstrate that DDR foci formation requires site-specific DICER- and DROSHA-dependent small RNAs, named DDRNAs, which act in a MRE11-RAD50-NBS1 (MRN) complex-dependent manner. Chemically synthesized or in vitro-generated by DICER cleavage, DDRNAs are sufficient to restore 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.