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A genome-wide homologous recombination screen identifies the RNA-binding protein RBMX as a component of the DNA-damage response

Abstract

Repair of DNA double-strand breaks is critical to genomic stability and the prevention of developmental disorders and cancer. A central pathway for this repair is homologous recombination (HR). Most knowledge of HR is derived from work in prokaryotic and eukaryotic model organisms. We carried out a genome-wide siRNA-based screen in human cells. Among positive regulators of HR we identified networks of DNA-damage-response and pre-mRNA-processing proteins, and among negative regulators we identified a phosphatase network. Three candidate proteins localized to DNA lesions, including RBMX, a heterogeneous nuclear ribonucleoprotein that has a role in alternative splicing. RBMX accumulated at DNA lesions through multiple domains in a poly(ADP-ribose) polymerase 1-dependent manner and promoted HR by facilitating proper BRCA2 expression. Our screen also revealed that off-target depletion of RAD51 is a common source of RNAi false positives, raising a cautionary note for siRNA screens and RNAi-based studies of HR.

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Figure 1: A genome-wide siRNA-based screen for HR genes.
Figure 2: Rescreen and validation of candidate HR genes.
Figure 3: Off-target RAD51-depletion was a major source of false positives among Dharmacon siRNAs identified by the primary screen.
Figure 4: RBMX accumulates transiently at sites of DNA damage in a PARP-dependent manner.
Figure 5: RBMX promotes HR and resistance to DNA-damaging agents.
Figure 6: RBMX promotes formation of ionizing-radiation-induced RAD51 foci by facilitating proper expression of BRCA2.

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Acknowledgements

We thank M. Jasin (Memorial Sloan-Kettering Cancer Center, USA) for DR-U2OS cells, P. Ng and F. Graham (McMaster University, Canada) for the AdNGUS24i, P. D. Adams (Institute of Cancer Sciences, CR-UK Beatson Labs, UK) for antibodies, S. J. Boulton (DNA Damage Response Laboratory, London Research Institute, UK) for the PARP inhibitor and C. Cotta-Ramusino and members of the Elledge laboratory for advice and discussion. We thank the Institute of Chemistry and Cell Biology (ICCB)-Longwood screening facility, including C. Shamu, S. Rudnicki, S. M. Johnston and T. Xie. This work was supported by a grant from the National Institutes of Health to S.J.E. A.S. was supported by T32CA09216 to the Pathology Department at the Massachusetts General Hospital and by Burroughs Wellcome Fund Career Award for Medical Scientists and is a Rita Allen Foundation and an Irma T. Hirschl scholar. S.J.E. is an investigator with the Howard Hughes Medical Institute.

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B.A., A.S. and S.J.E. conceived experimental design and conducted data analysis. F.D.S. and R.W.K. carried out GESS analysis. The manuscript was prepared by B.A. and S.J.E. and edited by A.S., F.D.S. and R.W.K.

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Correspondence to Stephen J. Elledge.

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Adamson, B., Smogorzewska, A., Sigoillot, F. et al. A genome-wide homologous recombination screen identifies the RNA-binding protein RBMX as a component of the DNA-damage response. Nat Cell Biol 14, 318–328 (2012). https://doi.org/10.1038/ncb2426

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