Abstract

The DNA damage response (DDR) preserves genomic integrity. Small non-coding RNAs termed DDRNAs are generated at DNA double-strand breaks (DSBs) and are critical for DDR activation. Here we show that active DDRNAs specifically localize to their damaged homologous genomic sites in a transcription-dependent manner. Following DNA damage, RNA polymerase II (RNAPII) binds to the MRE11–RAD50–NBS1 complex, is recruited to DSBs and synthesizes damage-induced long non-coding RNAs (dilncRNAs) from and towards DNA ends. DilncRNAs act both as DDRNA precursors and by recruiting DDRNAs through RNA–RNA pairing. Together, dilncRNAs and DDRNAs fuel DDR focus formation and associate with 53BP1. Accordingly, inhibition of RNAPII prevents DDRNA recruitment, DDR activation and DNA repair. Antisense oligonucleotides matching dilncRNAs and DDRNAs impair site-specific DDR focus formation and DNA repair. We propose that DDR signalling sites, in addition to sharing a common pool of proteins, individually host a unique set of site-specific RNAs necessary for DDR activation.

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References

  1. 1.

    & Dynamics of DNA damage response proteins at DNA breaks: a focus on protein modifications. Genes Dev. 25, 409–433 (2011).

  2. 2.

    & Non-coding RNAs in DNA damage and repair. FEBS Lett. 587, 1832–1839 (2013).

  3. 3.

    & A role for reverse transcripts in gene conversion. Nature 361, 170–173 (1993).

  4. 4.

    , , , & RNA-templated DNA repair. Nature 447, 338–341 (2007).

  5. 5.

    et al. Transcript-RNA-templated DNA recombination and repair. Nature 515, 436–439 (2014).

  6. 6.

    et al. A role for small RNAs in DNA double-strand break repair. Cell 149, 101–112 (2012).

  7. 7.

    & Small RNAs recruit chromatin-modifying enzymes MMSET and Tip60 to reconfigure damaged DNA upon double-strand break and facilitate repair. Cancer Res. 76, 1904–1915 (2016).

  8. 8.

    et al. Ago2 facilitates Rad51 recruitment and DNA double-strand break repair by homologous recombination. Cell Res. 24, 532–541 (2014).

  9. 9.

    , , & Histone H2AX and the small RNA pathway modulate both non-homologous end-joining and homologous recombination in plants. Mutat. Res. 783, 9–14 (2016).

  10. 10.

    & RNA-directed repair of DNA double-strand breaks. DNA Repair 32, 82–85 (2015).

  11. 11.

    et al. Transient RNA-DNA hybrids are required for efficient double-strand break repair. Cell 167, 1001–1013 e1007 (2016).

  12. 12.

    et al. Classical non-homologous end-joining pathway utilizes nascent RNA for error-free double-strand break repair of transcribed genes. Nat. Commun. 7, 13049 (2016).

  13. 13.

    , , & Analysis of a piwi-related gene implicates small RNAs in genome rearrangement in tetrahymena. Cell 110, 689–699 (2002).

  14. 14.

    , , & Homology directed repair is unaffected by the absence of siRNAs in Drosophila melanogaster. Nucleic Acids Res. 44, 8261–8271 (2016).

  15. 15.

    et al. Efficient generation of diRNAs requires components in the posttranscriptional gene silencing pathway. Sci. Rep. 7, 301 (2017).

  16. 16.

    et al. Site-specific DICER and DROSHA RNA products control the DNA-damage response. Nature 488, 231–235 (2012).

  17. 17.

    , , , & DICER, DROSHA and DNA damage response RNAs are necessary for the secondary recruitment of DNA damage response factors. J. Cell Sci. 129, 1468–1476 (2016).

  18. 18.

    et al. DNA damage response inhibition at dysfunctional telomeres by modulation of telomeric DNA damage response RNAs. Nat. Commun. 8, 13980 (2017).

  19. 19.

    , & A small RNA response at DNA ends in Drosophila. Nucleic Acids Res. 40, 9596–9603 (2012).

  20. 20.

    et al. Positional stability of single double-strand breaks in mammalian cells. Nat. Cell Biol. 9, 675–682 (2007).

  21. 21.

    , & Intracellular single molecule microscopy reveals two kinetically distinct pathways for microRNA assembly. EMBO Rep. 13, 709–715 (2012).

  22. 22.

    , , & Dissecting non-coding RNA mechanisms in cellulo by single-molecule high-resolution localization and counting. Methods 63, 188–199 (2013).

  23. 23.

    , , , & Resolving subcellular miRNA trafficking and turnover at single-molecule resolution. Cell Rep. 19, 630–642 (2017).

  24. 24.

    Inhibiting eukaryotic transcription: which compound to choose? How to evaluate its activity? Transcription 2, 103–108 (2011).

  25. 25.

    , , , & Imaging individual mRNA molecules using multiple singly labeled probes. Nat. Methods 5, 877–879 (2008).

  26. 26.

    et al. The nucleoporin 153, a novel factor in double-strand break repair and DNA damage response. Oncogene 31, 4803–4809 (2012).

  27. 27.

    et al. Nuclear position dictates DNA repair pathway choice. Genes Dev. 28, 2450–2463 (2014).

  28. 28.

    et al. Spatial dynamics of chromosome translocations in living cells. Science 341, 660–664 (2013).

  29. 29.

    , , & DNAPKcs-dependent arrest of RNA polymerase II transcription in the presence of DNA breaks. Nat. Struct. Mol. Biol. 19, 276–282 (2012).

  30. 30.

    et al. High-resolution profiling of γH2AX around DNA double strand breaks in the mammalian genome. EMBO J. 29, 1446–1457 (2010).

  31. 31.

    et al. Rescuing dicer defects via inhibition of an anti-dicing nuclease. Cell Rep. 9, 1471–1481 (2014).

  32. 32.

    , , , & TERRA biogenesis, turnover and implications for function. FEBS Lett. 584, 3812–3818 (2010).

  33. 33.

    , , & Super-resolution microscopy reveals decondensed chromatin structure at transcription sites. Sci. Rep. 4, 4477 (2014).

  34. 34.

    et al. A forward chemical genetic screen reveals an inhibitor of the Mre11–Rad50–Nbs1 complex. Nat. Chem. Biol. 4, 119–125 (2008).

  35. 35.

    & RIF1: a novel regulatory factor for DNA replication and DNA damage response signaling. DNA Repair (Amst) 15, 54–59 (2014).

  36. 36.

    & The heterochromatic barrier to DNA double strand break repair: how to get the entry visa. Int. J. Mol. Sci. 13, 11844–11860 (2012).

  37. 37.

    & Writers, readers, and erasers of histone ubiquitylation in DNA double-strand break repair. Front. Genet. 7, 122 (2016).

  38. 38.

    et al. 53BP1 exchanges slowly at the sites of DNA damage and appears to require RNA for its association with chromatin. J. Cell Sci. 118, 2043–2055 (2005).

  39. 39.

    et al. 53BP1 is a reader of the DNA-damage-induced H2A Lys 15 ubiquitin mark. Nature 499, 50–54 (2013).

  40. 40.

    et al. Nuclear phosphorylated Dicer processes double-stranded RNA in response to DNA damage. J. Cell Biol. 216, 2373–2389 (2017).

  41. 41.

    & RNA-mediated epigenetic regulation of gene expression. Nat. Rev. Genet. 16, 71–84 (2015).

  42. 42.

    & RNA-RNA interactions in gene regulation: the coding and noncoding players. Trends Biochem. Sci. 40, 248–256 (2015).

  43. 43.

    et al. Recent advancements in DNA damage-transcription crosstalk and high-resolution mapping of DNA breaks. Annu. Rev. Genomics Hum. Genet. 18, 87–113 (2017).

  44. 44.

    Transcriptional termination in mammals: stopping the RNA polymerase II juggernaut. Science 352, aad9926 (2016).

  45. 45.

    , & R-loops induce repressive chromatin marks over mammalian gene terminators. Nature 516, 436–439 (2014).

  46. 46.

    , , & Express or repress? The transcriptional dilemma of damaged chromatin. FEBS J. 284, 2133–2147 (2017).

  47. 47.

    et al. A damaged genome’s transcriptional landscape through multilayered expression profiling around in situ-mapped DNA double-strand breaks. Nat. Commun. 8, 15656 (2017).

  48. 48.

    et al. DNA damage triggers SAF-A and RNA biogenesis factors exclusion from chromatin coupled to R-loops removal. Nucleic Acids Res. 42, 9047–9062 (2014).

  49. 49.

    et al. Multiomic analysis of the UV-induced DNA damage response. Cell Rep. 15, 1597–1610 (2016).

  50. 50.

    et al. Requirement for PBAF in transcriptional repression and repair at DNA breaks in actively transcribed regions of chromatin. Mol. Cell 55, 723–732 (2014).

  51. 51.

    , , & Mammalian NET-seq analysis defines nascent RNA profiles and associated RNA processing genome-wide. Nat. Protoc. 11, 413–428 (2016).

  52. 52.

    Telomere measurement by quantitative PCR. Nucleic Acids Res. 30, e47 (2002).

  53. 53.

    , & Roles of ATM and NBS1 in chromatin structure modulation and DNA double-strand break repair. Nat. Cell Biol. 9, 683–690 (2007).

  54. 54.

    , & Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Res. 11, 1475–1489 (1983).

  55. 55.

    & Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359 (2012).

  56. 56.

    et al. The sequence alignment/map format and SAMtools. Bioinformatics 25, 2078–2079 (2009).

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Acknowledgements

We thank E. Soutoglou (Institut de Génétique et de Biologie Moléculaire et Cellulaire, Strasbourg, France), T. Misteli (National Cancer Institute, Bethesda, USA), G. Legube (Centre de Biologie Intégrative, Toulouse, France), M. Kastan (Duke Cancer Institute, Durham, USA), A. Aguilera, (Centro Andaluz de Biología Molecular y Medicina Regenerativa, Sevilla, Spain), Y. Xu (University of California, San Diego, USA), S. P. Jackson (Gurdon Institute, Cambridge, UK), D. Durocher (The Lunenfeld-Tanenbaum Research Institute, Toronto, Canada), B. Amati (European Institute of Oncology, Milan, Italy), A. Verrecchia (European Institute of Oncology, Milan, Italy), P. Pellanda (European Institute of Oncology, Milan, Italy) for reagents; Single Molecule Analysis in Real-Time Center (University of Michigan, USA) for instruments; M. Bedford (MD Anderson Cancer Center, Texas, USA), C. A. Sagum (MD Anderson Cancer Center, Texas, USA) and M. Roncador (Italian National Research Council, Pavia, Italy) for their help during the revision of the manuscript and all F.d’A.d.F. group members for reading the manuscript, support and constant discussions. F.M. was supported by Fondazione Italiana Ricerca Sul Cancro (FIRC, 12491). S.F. was supported by Collegio Ghislieri and Fondazione Cariplo (Grant rif. 2014-1215). G.V.S. is supported by Mechanobiology Institute (MBI) and Singapore Ministry of Education Academic Research Fund Tier3 (MOE2012-T3-1-001). N.G.W. is supported by NIH grants 2R01 GM062357, 1R01 GM098023 and 1R21 AI109791. F.d’A.d.F. was supported by the Associazione Italiana per la Ricerca sul Cancro, AIRC (application 12971), Human Frontier Science Program (contract RGP 0014/2012), Cariplo Foundation (grant 2010.0818 and 2014-0812), Marie Curie Initial Training Networks (FP7 PEOPLE 2012 ITN (CodAge)), Fondazione Telethon (GGP12059), Association for International Cancer Research (AICR-Worldwide Cancer Research Rif. N. 14-1331), Progetti di Ricerca di Interesse Nazionale (PRIN) 2010–2011, the Italian Ministry of Education Universities and Research EPIGEN Project, a European Research Council advanced grant (322726) and AriSLA (project ‘DDRNA and ALS’).

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Author notes

    • Sethuramasundaram Pitchiaya

    Present address: Michigan Center for Translational Pathology, University of Michigan Cancer Center, Ann Arbor, Michigan 48109-0940, USA.

Affiliations

  1. IFOM—The FIRC Institute of Molecular Oncology, Milan 20139, Italy

    • Flavia Michelini
    • , Valerio Vitelli
    • , Sheetal Sharma
    • , Ubaldo Gioia
    • , Fabio Pessina
    • , Fabio Iannelli
    • , Valentina Matti
    • , Sofia Francia
    • , G. V. Shivashankar
    •  & Fabrizio d’Adda di Fagagna
  2. Single Molecule Analysis Group and Center for RNA Biomedicine, Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109-1055, USA

    • Sethuramasundaram Pitchiaya
    •  & Nils G. Walter
  3. Istituto di Genetica Molecolare, CNR - Consiglio Nazionale delle Ricerche, Pavia 27100, Italy

    • Matteo Cabrini
    • , Ilaria Capozzo
    • , Sofia Francia
    •  & Fabrizio d’Adda di Fagagna
  4. Mechanobiology Institute, National University Singapore, 117411 Singapore, Singapore

    • Yejun Wang
    •  & G. V. Shivashankar

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Contributions

S.P. conceived and performed all microinjection assays, intracellular single-molecule imaging of DDRNA, FISH and kinetics experiments. V.V. generated the lentiviral I-SceI-GR construct, produced strand-specific RT–qPCR data for dilncRNA detection (NIH2/4 cells, NIH3T3duo cells, U2OS19ptight cells, HeLa111 cells, HeLa I-PpoI cells cut in the DAB1 gene) and qPCR analyses of ChIP experiments in NIH2/4 and determined dilncRNA polyadenylation status. S.S. performed all in vitro DSB-induced transcription assays and 5′ RACE and produced samples for sequencing. U.G. performed the RNA pulldown experiments without ASOs and RIP experiments, performed RT–qPCR analyses of DDRNA and generated the GFP-53BP1ΔTUD construct. M.C. performed ChIP experiments of RNAPII and the phosphorylated forms in NIH2/4 cells and the ChIP in AsiSI-ER BJ-5Ta cells treated with DRB. F.P. performed the in vitro binding assay of RNAPII to DNA ends, the co-immunoprecipitations of RNAPII with MRN and the ChIP of RNAPII in HeLa I-PpoI cells. Y.W. performed confocal and super-resolution analyses of RNAPII localization on damaged chromatin. F.I. conducted bioinformatics analyses of next-generation sequencing data. I.C. detected dilncRNA in the AsiSI system. V.M. contributed with technical support. S.F. supervised M.C. and I.C. G.V.S. supervised Y.W. N.G.W. initiated the single-molecule experiments, advised S.P. in their execution, provided critical input in experimental design and result interpretation, and edited the manuscript. F.M. designed and performed all of the remaining experiments and wrote the manuscript. F.d’A.d.F. conceived the study and, together with F.M., assembled and revised the manuscript. All authors commented on the manuscript.

Competing interests

F.d’A.d.F., F.M. and S.F. are inventors on the following patent application: PCT/EP2013/059753.

Corresponding author

Correspondence to Fabrizio d’Adda di Fagagna.

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