We have recently discovered a novel class of short nuclear non-coding RNAs that are generated at sites of DNA damage in vertebrates and that are necessary for focal accumulation and local activation of DNA damage response (DDR) proteins, including ATM. Although these RNAs (named DDRNAs) are products of DICER and DROSHA, their effects on DDR are fully independent from canonical RNAi mechanisms, as demonstrated by their independence from GW182 factors (that are necessary for mRNA translational control) and by their activity in in vitro permeabilized and RNAseA-treated cells, thus in the absence of any potential target mRNA.

The DDRNAs that we have identified and validated so far originated from an exogenous construct integrated in the host genome of mammalian cells. We now plan to comprehensively study the biogenesis and mechanism of action of endogenous human DDRNAs in a cell system in which we can induce DNA double-strand breaks (DSB) in a sequence-specific manner. Towards this end, we will combine the expertise in molecular biology of Fabrizio d’Adda di Fagagna, who will perform the experiments in this system, with that in genomics technologies of Piero Carninci, who will develop methods to capture and profile the expression of the short RNAs under various conditions, and with that in computational biology of Mihaela Zavolan, who will analyze the genome-wide data and will contribute experimental analyses of RNA-RNA-binding protein interactions.  We aim to unravel the genome-wide distribution of DDRNAs by standard Next Generation Sequencing (NGS) and by a novel approach being developed by Piero Carninci and to determine the mechanisms behind their synthesis, stability and processing, including the involvement of DICER and DROSHA.

Then, by focusing on few site-specific DSB, we will validate the functions of the identified site-specific DDRNAs through reconstitution experiments, in which we introduce synthetic DDRNAs with the sequence as identified by the approaches described above. We will test how their level at different genomic locations impacts on DDRs signalling. We then plan to study the DDRNAs-dependent stepwise assembly of DDR protein complexes at damaged loci by chromatin immunoprecipitations (ChIP) and qPCR and to uncover the network of RNA-protein interactions at DDR foci through CrossLinking and ImmunoPrecipitation (CLIP).

Finally we will investigate whether DDRNAs play a role not only in DDR signalling but also in DNA repair and whether DDRNAs may act as template for DNA repair reactions.