Università degli Studi di Milano, 2014-03-25

DNA is the most precious molecule in our cells, thus it has to be protected from damage and alterations and, if damaged, it has to be repaired efficiently. The DNA damage response (DDR) is a signaling cascade that follows the generation of a lesion in the DNA double helix and promptly arrests cell proliferation in order to attempt DNA repair. It has been proposed that mammalian genomes are pervasively transcribed, also in non-coding regions. Non-coding RNAs (ncRNAs) have been involved in an increasing number of cellular events and some of them are processed by members of the RNA interference (RNAi) pathway. So far, RNAi and DDR pathways have not been demonstrated to directly interact. During my PhD, I contributed to uncover an unexpected layer of DDR regulation by a new class of DICER- and DROSHA-dependent small non-coding RNA, named DDRNA. DDR foci stability is sensitive to RNA polymerase II inhibition and to RNase A treatment. Incubation of RNase A-treated cells with DICER- and DROSHA-dependent RNA products restores focal accumulation of DDR factors. DICER and DROSHA are indeed necessary to trigger DDR upon exogenous DNA damage in human cells, and DICER processing activity is necessary to activate DDR. Moreover, DICER and DROSHA knockdown impacts on checkpoint activation and allows senescent cells to re-enter S-phase. Differently, inactivation of GW182, a component of the RNAi machinery involved downstream of DICER and DROSHA in mRNA translational control, does not impact on DDR foci formation and detection. In a mammalian cell system in which a single DNA double-strand break can be generated at a defined exogenous integrated locus, DDR focus formation requires site-specific RNA molecules. RNA deep sequencing confirmed the presence of 22-23-nucleotide sequence-specific transcripts arising from the damaged locus, which are DICER-dependent. These DDR-regulating RNAs (DDRNAs) act at the first steps of the DDR cascade, in an MRN-dependent manner and have an impact also on DNA damage repair. Importantly, DDRNAs, both chemically synthesized or generated in vitro by DICER cleavage, are biologically active and antisense LNA oligonucleotides reduce DDR activation in living cells. Finally, by the use of fluorescently labeled molecules, DDRNAs have been demonstrated to localize at the site-specific damaged locus. Collectively these results suggest an unanticipated direct role of DICER and DROSHA in the production of small ncRNAs that control DDR activation at sites of DNA damage. Given the known tumor suppressive functions of DDR and the implication of its activation in a number of biological relevant processes, such as senescence, this discovery may have a significant impact on our understanding of ageing and cancer.

diritti: info:eu-repo/semantics/openAccess
Supervisor: F. D'Adda Di Fagagna ; Internal Co-supervisor: G. Natoli ; External Supervisor: J. Martinez ; Internal Examiner: M. Foiani ; External Examiner: W. Filipowicz
Settore BIO/11 - - Biologia Molecolare

Tesi di dottorato. | Lingua: Inglese. | Paese: | BID: TD16001645