EXOSC10 is required for RPA assembly and controlled DNA end resection at DNA double-strand breaks

Abstract

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.

RNA at DNA Double‐Strand Breaks: The Challenge of Dealing with DNA:RNA Hybrids

“…A variety of experimental strategies have been applied in recent years to study the occurrence of DNA:RNA hybrids at sites of DNA damage. [ 28,29,52,56,57 ] These studies support the conclusion that DNA lesions in active transcription units cause RNAPII arrest and promote R‐loop formation, and DNA:RNA hybrids have been proposed to play a key role in promoting accurate DSB repair by contributing to the recruitment of DNA repair factors such as RAD52. [ 58 ] Interestingly, not only promoter‐dependent transcripts but also dilncRNAs can engage in DNA:RNA hybrid formation.…”

“…They have been identified by ssRT‐qPCR in genic and intergenic regions as well as in CTD Y1P RIP experiments following DSB induction in genic regions, [ 22,25 ] but could not be confirmed in another study of sequence specific DSBs. [ 29 ] It is possible that transcription toward the break occurs, but that the resulting transcripts are short lived and their abundance is near the detection limit of standard RT‐qPCR methods. On the other hand, dilncRNAs synthesized by RNAPIIs that initiate transcription at the DSB and elongate away from the break could anneal with an already existing transcript (Figure 2b).…”

“…Single‐gene studies and a genome wide transcriptional analysis of DSBs induced at AsiSI sites could not identify dilncRNAs originating from transcriptionally inactive regions. [ 28,29 ] However, case studies of specific, unique intergenic DSBs as well as an in vitro model of linearized transcriptionally inactive plasmids that mimics DSBs, could detect dilncRNAs even from silent loci. [ 22 ]…”

“…[ 48,49 ] Cells depleted of Exosome Component 10 (EXOSC10), one of the catalytic subunits of the exosome, show increased levels of dilncRNAs produced at sequence‐specific DSBs, which suggests that EXOSC10 is involved in the degradation of damage‐induced transcripts. [ 29 ]…”

EXOSC10 sculpts the transcriptome during the growth-to-maturation transition in mouse oocytes

“…The association of the core complex with DIS3, DIS3L or EXOSC10 RNases provides the required enzymatic activity. EXOSC10 is a nuclear RNase, the absence of which causes RNA processing defects in yeast (Carneiro et al, 2007) and increased sensitivity to DNA damage in fly and human cells (Domingo-Prim et al, 2019;Rolfsmeier et al, 2011). EXOSC10 has been documented to promote mRNA turnover (van Dijk et al, 2007), 3' pre-rRNA processing (Knight et al, 2016) and long noncoding/enhancer RNA degradation (Pefanis et al, 2015).…”