Academic dissertation for the Degree of Doctor of Philosophy in Molecular Bioscience at Stockholm University to be publicly defended on Friday 15 June 2018 at 10.00 in E306, Svante Arrehnius väg 20 C

By: Judit Domingo Prim

Title: The RNA exosome and the maintenance of genome integrity

Examination board

Andrés Aguilera, Department of Dept. Genetics, University of Sevilla, Spain (opponent)
Lena Ström, Department of Cell and Molecular Biology, Karolinska Institutet
Claudia Kutter Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet
Stefan Åström, Department of Molecular Biosciences, Wenner-Gren institute, Stockholm University
Marie Öhman, Deptartment of Molecular Biosciences, Wenner-Gren institute, Stockholm University (Chairman)

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 damageinduced 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.

Keywords: EXOSC10, RRP6, DNA damage, diRNA, DDRNA, DNA end resection, homologous recombination.