First name
Flavia
Last name
Michelini
Year of Study
Thesis Title
A new class of non-coding RNA Controls the DNA damage Response and DNA repair
Thesis Abstract
The DNA damage response (DDR) is a signaling cascade that follows the generation of a lesion inthe DNA double helix and promptly arrests cell proliferation in order to attempt DNA repair. Noncoding RNAs have been involved in an increasing number of cellular events and some of them are processed by members of the RNA interference (RNAi) pathway. During my PhD, I contributed to uncover an unexpected layer of DDR regulation by a new class of DICER- and DROSHAdependent small non-coding RNA, named DDRNA.
I demonstrated that 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, in a miRNA-independent manner. In a mammalian cell system in which a single DNA double-strand break can be generated at a defined locus, DDR focus formation requires site-specific RNA molecules. RNA deep sequencing confirmed the presence of DICER-dependent 22-23-nucleotide transcripts arising from the damaged locus. These DDR-regulating RNAs (DDRNAs) act at the first steps of the DDR cascade, in an MRN-dependent manner. Importantly, DDRNAs, both chemically synthesized or generated in vitro by DICER cleavage, are biologically active. Finally, fluorescently labeled DDRNAs have been demonstrated to localize site-specifically at the damaged locus. Collectively these results suggest an unanticipated direct role of DICER and DROSHA in the production of small non-coding RNAs that control DDR activation at sites of DNA damage.
I demonstrated that 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, in a miRNA-independent manner. In a mammalian cell system in which a single DNA double-strand break can be generated at a defined locus, DDR focus formation requires site-specific RNA molecules. RNA deep sequencing confirmed the presence of DICER-dependent 22-23-nucleotide transcripts arising from the damaged locus. These DDR-regulating RNAs (DDRNAs) act at the first steps of the DDR cascade, in an MRN-dependent manner. Importantly, DDRNAs, both chemically synthesized or generated in vitro by DICER cleavage, are biologically active. Finally, fluorescently labeled DDRNAs have been demonstrated to localize site-specifically at the damaged locus. Collectively these results suggest an unanticipated direct role of DICER and DROSHA in the production of small non-coding RNAs that control DDR activation at sites of DNA damage.
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