A dual role of RBM42 in modulating splicing and translation of CDKN1A/p21 during DNA damage response.

p53-mediated cell cycle arrest during DNA damage is dependent on the induction of p21 protein, encoded by the CDKN1A gene. p21 inhibits cyclin-dependent kinases required for cell cycle progression to guarantee accurate repair of DNA lesions. Hence, fine-tuning of p21 levels is crucial to preserve genomic stability. Currently, the multilayered regulation of p21 levels during DNA damage is not fully understood. Herein, we identify the human RNA binding motif protein 42 (RBM42) as a regulator of p21 levels during DNA damage. Genome-wide transcriptome and interactome analysis reveals that RBM42 alters the expression of p53-regulated genes during DNA damage. Specifically, we demonstrate that RBM42 facilitates CDKN1A splicing by counteracting the splicing inhibitory effect of RBM4 protein. Unexpectedly, we also show that RBM42, underpins translation of various splicing targets, including CDKN1A. Concordantly, transcriptome-wide mapping of RBM42-RNA interactions using eCLIP further substantiates the dual function of RBM42 in regulating splicing and translation of its target genes, including CDKN1A. Collectively, our data show that RBM42 couples splicing and translation machineries to fine-tune gene expression during DNA damage response.

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. 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 (RBM6del(G-patch)). Consequently, RBM6del(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.HIGHLIGHTSPARP1 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.

CDYL1-dependent decrease in lysine crotonylation at DNA double-strand break sites functionally uncouples transcriptional silencing and repair.

Previously, we showed that CDYL1 is recruited to DNA double-strand breaks (DSBs) to promote homologous recombination (HR) repair and foster transcriptional silencing. However, how CDYL1 elicits DSB-induced silencing is not fully understood. Here, we identify a CDYL1-dependent local decrease in the transcriptionally active marks histone lysine crotonylation (Kcr) and crotonylated lysine 9 of H3 (H3K9cr) at AsiSI-induced DSBs, which correlates with transcriptional silencing. Mechanistically, we reveal that CDYL1 crotonyl-CoA hydratase activity counteracts Kcr and H3K9cr at DSB sites, which triggers the eviction of the transcription elongation factor ENL and fosters transcriptional silencing. Furthermore, genetic inhibition of CDYL1 hydratase activity blocks the reduction in H3K9cr and alleviates DSB-induced silencing, whereas HR efficiency unexpectedly remains intact. Therefore, our results functionally uncouple the repair and silencing activity of CDYL1 at DSBs. In a broader context, we address a long-standing question concerning the functional relationship between HR repair and DSB-induced silencing, suggesting that they may occur independently.

RBM6 splicing factor promotes homologous recombination repair of double-strand breaks and modulates sensitivity to chemotherapeutic drugs.

RNA-binding proteins regulate mRNA processing and translation and are often aberrantly expressed in cancer. The RNA-binding motif protein 6, RBM6, is a known alternative splicing factor that harbors tumor suppressor activity and is frequently mutated in human cancer. Here, we identify RBM6 as a novel regulator of homologous recombination (HR) repair of DNA double-strand breaks (DSBs). Mechanistically, we show that RBM6 regulates alternative splicing-coupled nonstop-decay of a positive HR regulator, Fe65/APBB1. RBM6 knockdown leads to a severe reduction in Fe65 protein levels and consequently impairs HR of DSBs. Accordingly, RBM6-deficient cancer cells are vulnerable to ATM and PARP inhibition and show remarkable sensitivity to cisplatin. Concordantly, cisplatin administration inhibits the growth of breast tumor devoid of RBM6 in mouse xenograft model. Furthermore, we observe that RBM6 protein is significantly lost in metastatic breast tumors compared with primary tumors, thus suggesting RBM6 as a potential therapeutic target of advanced breast cancer. Collectively, our results elucidate the link between the multifaceted roles of RBM6 in regulating alternative splicing and HR of DSBs that may contribute to tumorigenesis, and pave the way for new avenues of therapy for RBM6-deficient tumors.

NELF complex fosters BRCA1 and RAD51 recruitment to DNA damage sites and modulates sensitivity to PARP inhibition.

The negative elongation factor (NELF) is a four-subunit protein complex (NELF-E, NELF-A, NELF-B and NELF-C/D) that negatively regulates transcription elongation of RNA polymerase II (Pol II). Interestingly, upregulation of NELF-E subunit promotes hepatocellular carcinoma (HCC) and pancreatic cancer. In addition, we have previously shown that NELF complex fosters double-strand break (DSB)-induced transcription silencing and promotes homology-directed repair (HDR). However, the mechanisms underlying NELF-E regulation of HDR of DSBs remain unknown. Here, we show that NELF-E interacts with BRCA1 and promotes its recruitment to laser-microirradiated sites and facilitates ionizing radiation-induced foci (IRIF) of BRCA1 in HCC cells (Hep3B). The reduction in BRCA1 IRIF is accompanied by decreased RAD51 IRIF. A corollary to this, NELF-E-deficient Hep3B cells exhibit defective HDR of chromosomal DSBs induced by CRISPR-Cas9 system. Consequently, the disruption of NELF complex integrity, by NELF-E downregulation, sensitizes Hep3B cells to PARP inhibition. Altogether, our results suggest that NELF promotes HDR by facilitating BRCA1 and RAD51 IRIF formation and identify NELF complex as a novel synthetic lethal partner of PARP1.

Transcriptional Regulation at DSBs: Mechanisms and Consequences.

Defective double-strand break (DSB) repair leads to genomic instabilities that may augment carcinogenesis. DSBs trigger transient transcriptional silencing in the vicinity of transcriptionally active genes through multilayered processes instigated by Ataxia telangiectasia mutated (ATM), DNA-dependent protein kinase (DNA-PK), and poly-(ADP-ribose) polymerase 1 (PARP1). Novel factors have been identified that ensure DSB-induced silencing via two distinct pathways: direct inhibition of RNA Polymerase II (Pol II) mediated by negative elongation factor (NELF), and histone code editing by CDYL1 and histone deacetylases (HDACs) that catalyze H3K27me3 and erase lysine crotonylation, respectively. Here, we highlight major advances in understanding the mechanisms underlying transcriptional silencing at DSBs, and discuss its functional implications on repair. Furthermore, we discuss consequential links between DSB-silencing factors and carcinogenesis and discuss the potential of exploiting them for targeted cancer therapy.