Recent studies suggest that transcription takes place at DNA double-strand breaks (DSBs), that transcripts at DSBs are processed by Drosha and Dicer into damage-induced small RNAs (diRNAs), and that diRNAs are required for DNA repair. However, diRNAs have been mostly detected in reporter constructs or repetitive sequences, and their existence at endogenous loci has been questioned by recent reports. Using the homing endonuclease I-PpoI, we have investigated diRNA production in genetically unperturbed human and mouse cells. I-PpoI is an ideal tool to clarify the requirements for diRNA production because it induces DSBs in different types of loci: the repetitive 28S locus, unique genes and intergenic loci. We show by extensive sequencing that the rDNA locus produces substantial levels of diRNAs, whereas unique genic and intergenic loci do not. Further characterization of diRNAs emerging from the 28S locus reveals the existence of two diRNA subtypes. Surprisingly, Drosha and its partner DGCR8 are dispensable for diRNA production and only one diRNAs subtype depends on Dicer processing. Furthermore, we provide evidence that diRNAs are incorporated into Argonaute. Our findings provide direct evidence for diRNA production at endogenous loci in mammalian cells and give insights into RNA processing at DSBs.

The RNA exosome acts on different RNA substrates and plays important roles in RNA metabolism. The fact that short non-coding RNAs are involved in the DNA damage response led us to investigate whether the exosome plays a role in DNA repair. We have shown that the exosome catalytic subunit RRP6/EXOSC10 is recruited to DNA double-strand breaks (DSBs) in Drosophila S2 cells and human HeLa cells exposed to either ionizing radiation or I-PpoI endonuclease cleavage. DIS3, the other catalytic subunit of the nuclear exosome, is also recruited to DSBs, whereas the exosome core subunit EXOSC7 is not. Depletion of different exosome subunits does not interfere with the phosphorylation of the histone variants H2Av (Drosophila) or H2AX (humans), but depletion of RRP6/EXOSC10 impairs the recruitment of the homologous recombination factor RAD51 to the damaged sites, without affecting RAD51 levels. The recruitment of RAD51 to DSBs in S2 cells is also inhibited by overexpression of RRP6-Y361A–V5, a catalytically inactive RRP6 mutant. Furthermore, cells depleted of RRP6 or EXOSC10 are more sensitive to radiation, which is consistent with RRP6/EXOSC10 playing a role in DNA repair. RRP6/EXOSC10 can be co-immunoprecipitated with RAD51, which links RRP6/EXOSC10 to the homologous recombination pathway in animal cells. Taken together, our results suggest that a 3’-5’ ribonucleolytic activity is required for efficient DNA repair. 

The RNA exosome is a ribonucleolytic complex that acts on different RNA substrates and plays important roles in RNA metabolism. In recent years, the synthesis and the processing of RNA have been directly linked to the integrity of the genome. RNAs can either be the responsible for genomic instability or, on the contrary, can participate in the DNA damage response. Damage-induced RNAs (diRNAs) are short non-coding RNAs that have been implicated in the repair of DNA double-strand breaks (DSBs) by homologous recombination. The implication of specialized RNAs in DNA damage and repair led us to investigate whether the exosome was involved in DNA repair.

In Paper I, we have shown by fluorescence microscopy and chromatin immunoprecipitation that the exosome catalytic subunit RRP6/EXOSC10 is recruited to DSBs in Drosophila and human cells. Depletion of this subunit or overexpression of a catalytically inactive mutant makes the cells more sensitive to radiation and unable to recruit the homologous recombination factor RAD51 to DSBs, which is consistent with RRP6/EXOSC10 playing a role in homologous recombination, both in insect and mammalian cells. The results obtained with the RRP6 inactive mutant also suggest that the ribonucleolytic activity of RRP6 is required for DNA repair. However, the mechanisms by which RNAs and the exosome are implicated in DNA repair need to be further investigated.

In Paper II, we describe how transcription of DSB-flanking sequences by RNA polymerase II gives rise to damage-induced long non-coding RNAs that are processed into diRNAs. The direct detection of diRNAs had been elusive and their existence had been questioned, but our results show that damage-induced transcription and diRNA production occur at DSBs in endogenous, repetitive genomic sequences in mammalian cells. However, our exhaustive next-generation sequencing failed to detect diRNAs derived from DSBs in unique sequences. The diRNAs produced at repetitive loci bind to Argonaute and belong to two different subpopulations. One of them is Dicer-dependent and has a length of 21-22 nucleotides. The other one is not yet well characterized and is probably composed of degradation products from other ribonucleases.

Finally, in Paper III, we have demonstrated that EXOSC10 is one of the ribonucleases involved in RNA degradation at DSBs. By strand-specific quantitative PCR and RNA-seq, we show that the levels of diRNA precursors and diRNAs are increased in the absence of EXOSC10. Moreover, EXOSC10-depleted cells fail to recruit RPA to DSBs, and this defect is restored by RNase A digestion. Depletion of EXOSC10 also results in extended DNA resected tracks, as shown by both single-molecule analysis of resected tracks and quantitative amplification of single-stranded DNA. These results suggest that EXOSC10 is involved in RNA degradation at DSBs to allow RPA recruitment and regulated resection.

The work presented in this thesis supports the conclusion that damage-induced RNAs are synthesized de novo by RNA polymerase II at DSBs in mammalian cells. In repetitive genomic loci, these RNAs are processed into diRNAs that bind Argonaute. Regardless of whether diRNAs are functional or not, their precursors have to be degraded. The main function of the exosome, and more specifically EXOSC10, in the maintenance of the integrity of the genome is to degrade these transcripts in order to allow faithful repair of DNA double-strand breaks by homologous recombination.

The exosome is a ribonucleolytic complex that plays important roles in RNA metabolism. Here we show that the exosome is necessary for the repair of DNA double-strand breaks (DSBs) in human cells and that RNA clearance is an essential step in homologous recombination. Transcription of DSB-flanking sequences results in the production of damage-induced long non-coding RNAs (dilncRNAs) that engage in DNA-RNA hybrid formation. Depletion of EXOSC10, an exosome catalytic subunit, leads to increased dilncRNA and DNA-RNA hybrid levels. Moreover, the targeting of the ssDNA-binding protein RPA to sites of DNA damage is impaired whereas DNA end resection is hyper-stimulated in EXOSC10-depleted cells. The DNA end resection deregulation is abolished by transcription inhibitors, and RNase H1 overexpression restores the RPA recruitment defect caused by EXOSC10 depletion, which suggests that RNA clearance of newly synthesized dilncRNAs is required for RPA recruitment, controlled DNA end resection and assembly of the homologous recombination machinery.

The exosome acts on different RNA substrates and plays important roles in RNA metabolism. The fact that short non-coding RNAs are involved in the DNA damage response led us to investigate whether the exosome factor RRP6 of Drosophila melanogaster and its human ortholog EXOSC10 play a role in DNA repair. Here, we show that RRP6 and EXOSC10 are recruited to DNA double-strand breaks (DSBs) in S2 cells and HeLa cells, respectively. Depletion of RRP6/ EXOSC10 does not interfere with the phosphorylation of the histone variant H2Av (Drosophila) or H2AX (humans), but impairs the recruitment of the homologous recombination factor RAD51 to the damaged sites, without affecting RAD51 levels. The recruitment of RAD51 to DSBs in S2 cells is also inhibited by overexpression of RRP6-Y361A-V5, a catalytically inactive RRP6 mutant. Furthermore, cells depleted of RRP6 or EXOSC10 are more sensitive to radiation, which is consistent with RRP6/EXOSC10 playing a role in DNA repair. RRP6/EXOSC10 can be co-immunoprecipitated with RAD51, which links RRP6/EXOSC10 to the homologous recombination pathway. Taken together, our results suggest that the ribonucleolytic activity of RRP6/EXOSC10 is required for the recruitment of RAD51 to DSBs.

Long intergenic non-coding RNA, linc RNA, are often produced from intergenic sequences and have been ascribed diverse functions, such as regulating mRNA levels and being involved in the formation of heterochromatin. We show here that the intergenic spacer region (IGS) of the ribosomal DNA gene repeat in human cells is transcribed. Three ncRNAs, the IGS_19asRNA, the IGS_32asRNA and the IGS₃₈RNA, of 500, 800 and 1300 bases, respectively, were isolated and investigated. Two of them, the IGS_19asRNA and the IGS_32asRNA_, were transcribed in the antisense direction with respect to the rRNA and in the sense direction for the IGS₃₈RNA. We also showed that the ncRNAs were transcribed by different RNA polymerases; the IGS_19asRNA and the IGS₃₈RNA were transcribed by RNA polymerase II and the IGS_32asRNA were transcribed by RNA polymerase I. The three ncRNAs were also differentially regulated; IGS_19asRNA induced upon heat shock and the level of the IGS_32asRNA increased upon glucose feeding, similar to the 45S rRNA. In addition, the ncRNAs IGS_19asRNA and IGS_32asRNA were found at different locations in the nucleus, with IGS_19asRNA located in a speckled pattern in the nucleus and IGS_32asRNA associated with chromatin bound to heterochromatin protein 1. This suggests that the IGS_32asRNA has a role in heterochromatin formation.