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BRCA2 prevents R-loop accumulation and associates with TREX-2 mRNA export factor PCID2

Nature volume 511, pages 362–365 (2014)Cite this article

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Abstract

Genome instability is central to ageing, cancer and other diseases. It is not only proteins involved in DNA replication or the DNA damage response (DDR) that are important for maintaining genome integrity: from yeast to higher eukaryotes, mutations in genes involved in pre-mRNA splicing and in the biogenesis and export of messenger ribonucleoprotein (mRNP) also induce DNA damage and genome instability. This instability is frequently mediated by R-loops formed by DNA–RNA hybrids and a displaced single-stranded DNA1. Here we show that the human TREX-2 complex, which is involved in mRNP biogenesis and export, prevents genome instability as determined by the accumulation of γ-H2AX (Ser-139 phosphorylated histone H2AX) and 53BP1 foci and single-cell electrophoresis in cells depleted of the TREX-2 subunits PCID2, GANP and DSS1. We show that the BRCA2 repair factor, which binds to DSS1, also associates with PCID2 in the cell. The use of an enhanced green fluorescent protein-tagged hybrid-binding domain of RNase H1 and the S9.6 antibody did not detect R-loops in TREX-2-depleted cells, but did detect the accumulation of R-loops in BRCA2-depleted cells. The results indicate that R-loops are frequently formed in cells and that BRCA2 is required for their processing. This link between BRCA2 and RNA-mediated genome instability indicates that R-loops may be a chief source of replication stress and cancer-associated instability.

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Figure 1: TREX-2 associates with BRCA2 and affects genome integrity.
Figure 2: Detection of DNA–RNA hybrids in mammalian cells in vivo.
Figure 3: DNA–RNA hybrids accumulate in actively transcribed genes in BRCA2 and THOC1-depleted cells.
Figure 4: R-loops trigger genome instability in BRCA2-deficient cells.

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Acknowledgements

We thank J. C. Reyes and A. G. Rondón for comments on the manuscript, and D. Haun for style supervision. Research was funded by grants from the Spanish Ministry of Economy and Competitiveness (Consolider CSD2007-00015 and BFU2010-16372), the Junta de Andalucía (CVI4567) and the European Union (FEDER).

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Authors and Affiliations

  1. Centro Andaluz de Biología Molecular y Medicina Regenerativa CABIMER, Universidad de Sevilla, Avenida Américo Vespucio s/n, 41092 Seville, Spain ,

    Vaibhav Bhatia, Sonia I. Barroso, María L. García-Rubio, Emanuela Tumini, Emilia Herrera-Moyano & Andrés Aguilera

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  1. Vaibhav Bhatia
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  2. Sonia I. Barroso
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  3. María L. García-Rubio
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  4. Emanuela Tumini
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  6. Andrés Aguilera
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Contributions

V.B., S.B., M.G.R., E.T. and E.H.M. performed the experiments. V.B. and A.A. designed the experiments and wrote the paper.

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Correspondence to Andrés Aguilera.

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Extended data figures and tables

Extended Data Figure 1 Subcellular localization of PCID2.

Immunofluorescence of endogenous PCID2 in HeLa cells. a, Without permeabilization. b, With pre- permeabilization (see Methods).

Extended Data Figure 2 Validation of siRNAs.

a, Relative mRNA quantification. Means and s.e.m. are plotted. b, Western blot analysis of siRNA-treated HeLa cells.

Extended Data Figure 3 Effect of GANP depletion in genomic instability.

a, γ-H2AX and 53BP1 foci. b, Single-cell electrophoresis. c, DNA-combing analysis in GANP-depleted cells. Details as in Fig. 1.

Extended Data Figure 4 Proximity ligation assay in PCID2-depleted cells.

a, Staining pattern observed with both anti-BRCA2 and anti-PCID2 antibodies in conditions used for proximity ligation assay in Fig. 1d. b, Anti-PCID2 immunofluorescence analysis. c, Proximity ligation assay in control and PCID2 siRNA-treated cells (see Fig. 1d).

Extended Data Figure 5 HB–GFP interacts with chromatin and chromatin-associated proteins by means of DNA–RNA hybrids.

a, HB–GFP-expressing HEK293 lysate fractionated on Superose 6 size-exclusion columns (17-5172-01; Gelifesciences) and analysed by western blotting. b, HB–GFP co-immunoprecipitated proteins by using the ReCLIP method33. c, HB–GFP ChIP in the ribosomal DNA region in TOP1-depleted HeLa cells (n = 3). Means and s.e.m. are plotted. d, RNH1-dependent release of HB–GFP from chromatin of HeLa cells. e, Scheme and representative plot of FACS assays used to quantify DNA–RNA hybrids. PI, propidium iodide (see Fig. 2f). f, FACS assay to quantify DNA–RNA hybrids in TOP1-depleted cells. Means and s.e.m. are plotted (n = 3).

Extended Data Figure 6 HB–GFP ChIP of the UTRN and ACTB gene regions.

a, b, Normalized siRNA control-relative values for the immunoprecipitated DNA–RNA hybrids plotted relative to the siRNA control in the UTRN (a) and ACTB (b) genes. Means and s.e.m. are shown (n = 3). Amplicon positions used are indicated in the gene diagram above each graph.

Extended Data Figure 7 Accumulation of DNA–RNA hybrids in cells depleted of both PCID2 and BRCA2.

HeLa cells were treated with siBRCA2 in combination with siRNA control or siPCID2, and processed. Means and s.e.m. are plotted (n = 3). Details as in Fig. 3b.

Extended Data Figure 8 Chromosomal aberrations in cells expressing HB–GFP.

a, Metaphase spreads of HeLa cells expressing GFP (control) or HB–GFP. Fragmentation and sister chromatid exchange events are indicated by arrowheads and arrows, respectively. b, Quantification of chromosome breaks in RPE cells expressing HB–GFP. c, Adhesion-independent proliferation assay. Cell proliferation relative to control siRNA-treated RPE cells is shown. Means and s.e.m. are plotted (n = 3). *P ≤ 0.05 (two-tailed Student’s t-test).

Extended Data Figure 9 Model to explain the role of BRCA2 preventing R-loops as a source of genome instability.

a, RNA–DNA hybrids may form both in the interior and at the periphery of the nucleus. mRNP biogenesis factors such as the TREX-2 complex may help recruit or stabilize BRCA2 near transcribed regions, whether or not these are in proximity to the nuclear pore complex. BRCA2 and other related proteins could bind to the branched structure generated by the ssDNA displaced in the R-loop, facilitating the action of enzymes that remove R-loops, such as specific RNases or DNA–RNA helicases. This could occur in non-replicating chromatin. b, In replicating chromatin, BRCA2 and, presumably, other Fanconi anaemia proteins may act directly at putatively stalled RFs in front of an R-loop to impede the collapse or reversal of the replication fork, probably impeding R-loop extension. Subsequently, R-loop removal could be promoted by the passage of the replication fork.

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Bhatia, V., Barroso, S., García-Rubio, M. et al. BRCA2 prevents R-loop accumulation and associates with TREX-2 mRNA export factor PCID2. Nature 511, 362–365 (2014). https://doi.org/10.1038/nature13374

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