RNase A treatment and reconstitution with DNA damage response RNA in living cells as a tool to study the role of non-coding RNA in the formation of DNA damage response foci

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Non-coding RNA (ncRNA) molecules have been shown to play a variety of cellular roles; however, the contributions of different types of RNA to specific phenomena are often hard to dissect. To study the role of RNA in the assembly of DNA damage response (DDR) foci, we developed the RNase A treatment and reconstitution (RATaR) method, in which cells are mildly permeabilized, incubated with recombinant RNase A and subsequently reconstituted with different RNA species, under conditions of RNase A inactivation and inhibition of endogenous transcription. The block of transcription right after RNase A removal represents a key innovation of RATaR, preventing potential contributions of endogenously neo-synthesized transcripts to the phenotypes studied. A critical aspect of this technique is the balance between sufficient permeabilization of membranes to allow enzyme/RNA access into the cell nucleus and cell viability. Here, we present our protocol for RNA-dependent DDR foci disassembly and reassembly using fluorescent DDR RNAs (DDRNAs) in NIH2/4 cells, an engineered NIH3T3-derived cell line. The use of sequence-specific, fluorescent RNA molecules permits the concomitant determination of their subcellular localization and biological functions. We also outline adaptations of RATaR when implemented in different cell lines exposed to various genotoxic treatments, such as γ-radiation, restriction enzymes and telomere deprotection. In all these cases, the entire procedure can be completed within 2 h without the need for special equipment or uncommon skills. We believe this technique will prove useful for investigating the contribution of RNA to a variety of relevant cellular processes.

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Fig. 1: Outline of RATaR with synthetic fluorescent DDRNAs in NIH2/4 cells.
Fig. 2: RATaR shows site-specific DDRNA localization to the damage locus and 53BP1 focus reformation in NIH2/4 cells.


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The laboratory of F.d’A.d.F. was supported by the Associazione Italiana per la Ricerca sul Cancro, AIRC (application 12971), the Human Frontier Science Program (contract RGP 0014/2012), the Cariplo Foundation (grant nos. 2010-0818 and 2014-0812), Fondazione Telethon (GGP12059), the Association for International Cancer Research (AICR-Worldwide Cancer Research Rif. N. 14-1331), Progetti di Ricerca di Interesse Nazionale (PRIN) 2010/2011/2015, the Italian Ministry of Education Universities and Research EPIGEN and INTEROMICs project, the AMANDA project Accordo Quadro Regione Lombardia–CNR, AIRC Special Program ‘5 per mille’ metastases project no. 21091 and a European Research Council advanced grant (322726). S.F. was supported by Collegio Ghislieri, Fondazione Cariplo (2014-1215) and AriSLA (project ‘DDRNA&ALS’).

Author information

S.F. and F.d’A.d.F. developed the initial version of the protocol. F.d’A.d.F. conceived the use of the procedure for discovering DDRNA and its functions. F.M. performed experiments with fluorescent RNA in NIH2/4 cells. F.R. adapted RATaR for Rosa26-CreERT2 Trf2F/F MEFs. S.F. adapted RATaR for U2OS-TRE19 cells. F.M. assembled the figures. F.M. and S.F. wrote the manuscript. F.R. integrated the parts relative to Rosa26-CreERT2 Trf2F/F MEFs and F.d’A.d.F. edited the manuscript.

Correspondence to Sofia Francia.

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The autors declare no competing interests.

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Journal peer review information: Nature Protocols thanks Bernd Kaina, Kum Khanna and other anonymous reviewer(s) for their contribution to the peer review of this work.

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Related links

Key reference(s) using this protocol

Francia, S. et al. Nature 488, 231–235 (2012): https://doi.org/10.1038/nature11179

Francia, S. et al. J. Cell Sci. 129, 1468–1476 (2016): https://doi.org/10.1242/jcs.182188

Rossiello, F. et al. Nat. Commun. 8, 13980 (2017): https://doi.org/10.1038/ncomms13980

Michelini, F. et al. Nat. Cell Biol. 19, 1400–1411 (2017): https://doi.org/10.1038/ncb3643

Integrated supplementary information

Supplementary Figure 1 Examples of permeabilized NIH2/4 cells.

Images of non-permeabilized and permeabilized NIH2/4 cells, captured with a transmitted light microscope, show a clear difference in cell transparency and thickness. Margins of nuclei and nucleoli are more evident in permeabilized cells. As an example, the outlines of a non-permeabilized and a permeabilized cell membrane (green), nucleus (red) and nucleoli (blue) are depicted. Scale bar, 50 μM.

Supplementary Figure 2 RATaR shows site-specific DDRNA localization to the damage locus and 53BP1 focus reformation in U2OS TRE/I-SceI-19 cells.

U2OS TRE/I-SceI-19 cells were co-transfected with YFP-TetR- and I-SceI-expressing vectors. 24 hours later, doxycycline (1 μg/mL) was added to the medium to induce YFP-TetR binding to the TetO array, enabling visualization of the locus, and after 3 hours cells were permeabilized and treated with RNase A. Cells were then reconstituted by incubation with synthetic DDRNA-Cy5 matching TetO sequences (Tet DDRNA, 100 μM) or DDRNA-Cy5 matching LacO sequences (Lac DDRNA, 100 μM) as control, mixed with tRNA (200 ng). Images show that only DDRNAs matching TetO sequences (magenta) localize to the YFP-TetR-marked locus (green) and reconstitute 53BP1 (blue) focus post RNase A treatment, while DDRNAs matching LacO sequences do not. Images were captured by a confocal microscope. The dotted outline depicts the nucleus. Arrows indicate YFP-TetR-marked locus. Scale bar, 5 μM.

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Supplementary Figures 1 and 2

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Michelini, F., Rossiello, F., d’Adda di Fagagna, F. et al. RNase A treatment and reconstitution with DNA damage response RNA in living cells as a tool to study the role of non-coding RNA in the formation of DNA damage response foci. Nat Protoc 14, 1489–1508 (2019) doi:10.1038/s41596-019-0147-5

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