Recruitment of RBM6 to DNA double-strand breaks fosters homologous recombination repair

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 by either non-homologous end joining (NHEJ), homologous
recombination (HR), or alternative end-joining pathways. Previously, we demonstrated that
RBM6 splicing factor facilitates 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 rapidly and transiently recruited
to DSB sites in a PARP1-dependent manner. Furthermore, we demonstrate that RBM6 doesn’t
undergo ADP-ribosylation nor binds poly (ADP-ribose) moieties, suggesting that PARP1
activity indirectly regulates RBM6 recruitment to DNA breakage sites. Deletion mapping
analysis revealed a region containing 5 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 as compared to control cells. 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.

DNA双链断裂（DSBs）是极具毒性的损伤，对基因组完整性和细胞存活构成威胁。为避免DSBs的有害影响，多种DNA修复因子被招募以通过非同源末端连接（NHEJ）、同源重组（HR）或替代末端连接途径执行DSBs的修复。先前，我们证明RBM6剪接因子通过调控HR蛋白APBB1/Fe65的剪接偶联的非终止降解，促进了DSBs的HR。在此，我们描述了RBM6在促进DSBs HR修复中的剪接独立功能。我们展示RBM6以PARP1依赖性方式迅速且短暂地被招募至DSB位点。此外，我们证明RBM6不经历ADP核糖基化也不与聚（ADP核糖）基团结合，表明PARP1活性间接调节RBM6向DNA断裂位点的招募。缺失映射分析揭示了一个包含5个甘氨酸残基的区域位于G-盒结构域内，该区域调控RBM6在DNA损伤位点的积累。我们进一步证实RBM6与Rad51相互作用，且在缺乏G-盒结构域的RBM6突变体（RBM6 del(G-patch)）中这种相互作用被削弱。因此，与对照细胞相比，RBM6 del(G-patch)细胞在电离辐射后表现出较低的Rad51焦斑水平。此外，尽管缺乏G-盒结构域的RBM6缺失突变体对其剪接靶标Fe65和Eya2的表达水平没有可检测的影响，但它未能恢复HR的完整性。总体而言，我们的研究结果提示RBM6向DSB的招募促进HR修复，无论其剪接活性如何。