Recruitment of RBM6 to DNA Double-Strand Breaks Fosters Homologous Recombination Repair

Abstract

DNA double-strand breaks (DSBs) are highly toxic lesions that threaten genome integrity and cell survival. To avoid harmful repercussions of DSBs, a wide variety of DNA repair factors are recruited to execute DSB repair. Previously, we demonstrated that RBM6 splicing factor facilitates homologous recombination (HR) of DSB by regulating alternative splicing-coupled nonstop-decay of the HR protein APBB1/Fe65. Here, we describe a splicing-independent function of RBM6 in promoting HR repair of DSBs. We show that RBM6 is recruited to DSB sites and PARP1 activity indirectly regulates RBM6 recruitment to DNA breakage sites. Deletion mapping analysis revealed a region containing five glycine residues within the G-patch domain that regulates RBM6 accumulation at DNA damage sites. We further ascertain that RBM6 interacts with Rad51, and this interaction is attenuated in RBM6 mutant lacking the G-patch domain (RBM6^del(G-patch)). Consequently, RBM6^del(G-patch) cells exhibit reduced levels of Rad51 foci after ionizing radiation. In addition, while RBM6 deletion mutant lacking the G-patch domain has no detectable effect on the expression levels of its splicing targets Fe65 and Eya2, it fails to restore the integrity of HR. Altogether, our results suggest that RBM6 recruitment to DSB promotes HR repair, irrespective of its splicing activity.

HIGHLIGHTS

• PARP1 activity indirectly regulates RBM6 recruitment to DNA damage sites.

• Five glycine residues within the G-patch domain of RBM6 are critical for its recruitment to DNA damage sites, but dispensable for its splicing activity.

• RBM6 G-patch domain fosters its interaction with Rad51 and promotes Rad51 foci formation following irradiation.

• RBM6 recruitment to DSB sites underpins HR repair.

Keywords:

• RBM6
• double-strand breaks
• homologous recombination repair
• G-patch domain
• PARP1
• Rad51

SUPPLEMENTARY MATERIAL

Supplemental data for this article can be accessed online at https://doi.org/10.1080/10985549.2023.2187105

ACKNOWLEDGMENTS

We thank Laila Bishara for her substantial help in performing the ChIP-qPCR experiments and analyzing the data. We thank our lab members for their fruitful discussion of the manuscript.

AUTHOR CONTRIBUTIONS

S.W.A. performed the experiments in Fig. 1A–E, Fig. 2, Fig.3, and Fig. 4A–B, Supplementary Fig. S2, wrote the experimental procedures and helped in writing the manuscript. M.M.D. performed the experiments described in Fig. 1F–H, Fig 2A, H–I, Fig. 3G–H, and Fig. 4C–G and prepared the graphs of Fig. 2E,G and Fig. 3E and wrote the experimental procedures and helped editing the manuscript. F.E.M. performed the experiments described in Supplementary Fig. S1, and Fig. 3G–H and helped in the analyzing the results and editing the manuscript. I.A. helped in preparing the experiments described in Fig. 3A–E. N.A. conceived the study, planned the experiments and wrote the original draft.

FUNDING AND ADDITIONAL INFORMATION

Research in the Ayoub lab is supported by grants from the Israel Science Foundation (2511/18), ISF-NSFC fund (# 2511/18), Israel Cancer association (20200080), Israel Cancer Research fund (# 00151). M.M.D is supported by the Neubauer Research Fellowships Program. F.E.M. is supported by the Clore Scholars Program. N.A. is supported by the Neubauer Family foundation.

DATA AVAILABILITY STATEMENT

Data is available at: Recruitment of RBM6 to DNA double-strand breaks fosters homologous recombination repair – Mendeley Data.