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

DICER and DROSHA are crucial ribonucleases involved in RNA interference (RNAi). RNA products generated by DICER and DROSHA are involved in chromatin assembly, gene silencing and cancer. The DNA damage response (DDR) is a signaling pathway that arrests the proliferation of cells undergoing genotoxic events. So far, RNAi-RNA products have never been demonstrated to directly stimulate DDR signaling. Here we show that DICER and DROSHA are necessary to activate DDR upon oncogene-induced genotoxic stress and exogenous DNA damage. In an in vitro cellular system, DDR foci are sensitive to Rnase A, and DICER- and DROSHA-RNA products are required to restore DDR foci in Rnase A-treated cells. Through RNA sequencing and studies of DDR activation at a unique and traceable DNA double-strand break, we demonstrate that DDR focus formation requires DICER dependent, locus-specific short RNA molecules, which we call DDRNAs. Importantly, we show that DDRNAs act in a MRN-complex dependent manner. When chemically synthesized or generated in vitro by DICER cleavage, DDRNAs are sufficient to restore DDR in Rnase A-treated cells. All together, our results describe an unanticipated, direct role of short RNAs in the control of DDR activation at sites of DNA damage revealing a novel layer of modulation of the DDR pathway.