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The RNA exosome and the maintenance of genome integrity
Stockholm University, Faculty of Science, Department of Molecular Biosciences, The Wenner-Gren Institute. (Neus Visa)
2018 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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.

Place, publisher, year, edition, pages
Stockholm: Department of Molecular Biosciences, The Wenner-Gren Institute , 2018. , p. 25
Keywords [en]
EXOSC10, RRP6, DNA damage, diRNA, DDRNA, DNA end resection, homologous recombination
National Category
Cell and Molecular Biology
Research subject
Molecular Bioscience
Identifiers
URN: urn:nbn:se:su:diva-155691ISBN: 978-91-7797-298-3 (print)ISBN: 978-91-7797-299-0 (electronic)OAI: oai:DiVA.org:su-155691DiVA, id: diva2:1201560
Public defence
2018-06-15, E306, Arrheniuslaboratorierna, Svante Arrhenius väg 20C, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Submitted. Paper 3: Manuscript.

Available from: 2018-05-23 Created: 2018-04-26 Last updated: 2018-05-23Bibliographically approved
List of papers
1. RRP6/EXOSC10 is required for the repair of DNA double-strand breaks by homologous recombination
Open this publication in new window or tab >>RRP6/EXOSC10 is required for the repair of DNA double-strand breaks by homologous recombination
2015 (English)In: Journal of Cell Science, ISSN 0021-9533, E-ISSN 1477-9137, Vol. 128, no 6, p. 1097-1107Article in journal (Refereed) Published
Abstract [en]

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.

Keywords
RRP6, EXOSC10, DNA repair, Exosome, Non-coding RNA, RAD51
National Category
Cell Biology
Research subject
Molecular Bioscience
Identifiers
urn:nbn:se:su:diva-116613 (URN)10.1242/jcs.158733 (DOI)000350999500005 ()25632158 (PubMedID)
Note

AuthorCount:4;

Available from: 2015-04-28 Created: 2015-04-22 Last updated: 2018-05-08Bibliographically approved
2. Next-generation sequencing reveals two populations of damage- induced small RNAs at endogenous DNA double-strand breaks
Open this publication in new window or tab >>Next-generation sequencing reveals two populations of damage- induced small RNAs at endogenous DNA double-strand breaks
Show others...
(English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962Article in journal (Refereed) Submitted
National Category
Cell and Molecular Biology
Research subject
Molecular Bioscience
Identifiers
urn:nbn:se:su:diva-155685 (URN)
Available from: 2018-04-26 Created: 2018-04-26 Last updated: 2018-05-08Bibliographically approved
3. EXOSC10 is required for RPA assembly and controlled DNA resection at DNA dobule-strand breaks
Open this publication in new window or tab >>EXOSC10 is required for RPA assembly and controlled DNA resection at DNA dobule-strand breaks
Show others...
(English)Manuscript (preprint) (Other academic)
National Category
Cell and Molecular Biology
Research subject
Molecular Bioscience
Identifiers
urn:nbn:se:su:diva-155687 (URN)
Available from: 2018-04-26 Created: 2018-04-26 Last updated: 2018-05-08Bibliographically approved

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