RESUMO
Long non-coding RNAs (lncRNAs) are emerging regulators of genomic stability and human disease. However, the molecular mechanisms by which nuclear lncRNAs directly contribute to DNA damage responses remain largely unknown. Using RNA antisense purification coupled with quantitative mass spectrometry (RAP-qMS), we found that the lncRNA BGL3 binds to PARP1 and BARD1, exhibiting unexpected roles in homologous recombination. Mechanistically, BGL3 is recruited to DNA double-strand breaks (DSBs) by PARP1 at an early time point, which requires its interaction with the DNA-binding domain of PARP1. BGL3 also binds the C-terminal BRCT domain and an internal region (amino acids 127-424) of BARD1, which mediates interaction of the BRCA1/BARD1 complex with its binding partners such as HP1γ and RAD51, resulting in BRCA1/BARD1 retention at DSBs. Cells depleted for BGL3 displayed genomic instability and were sensitive to DNA-damaging reagents. Overall, our findings underscore the biochemical versatility of RNA as a mediator molecule in the DNA damage response pathway, which affects the accumulation of BRCA1/BARD1 at DSBs.
Assuntos
Proteína BRCA1/metabolismo , Quebras de DNA de Cadeia Dupla , Dano ao DNA , Complexos Multiproteicos/metabolismo , RNA Longo não Codificante/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteína BRCA1/genética , Células HEK293 , Humanos , Células MCF-7 , Complexos Multiproteicos/genética , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Domínios Proteicos , RNA Longo não Codificante/genética , Proteínas Supressoras de Tumor/genética , Ubiquitina-Proteína Ligases/genéticaRESUMO
Protein methylation plays important roles in DNA damage response. To date, proteome-wide profiling of protein methylation upon DNA damage has been not reported yet. In this study, using HILIC affinity enrichment combined with MS analysis, we conducted a quantitative analysis of the methylated proteins in HEK293T cells in response to IR treatment. In total, 235 distinct methylation sites responding to IR treatment were identified, and 38% of them were previously unknown. Multiple RNA-binding proteins were differentially methylated upon DNA damage stress. Furthermore, we identified 14 novel methylation sites in DNA damage response-related proteins. Moreover, we validated the function of PARP1 K23 methylation in repairing IR-induced DNA lesions. K23 methylation deficiency sensitizes cancer cells to radiation and HU-induced replication stress. In addition, PARP1 K23 methylation participates in the resolution of stalled replication forks by regulating PARP1 binding to damaged forks. Taken together, this study generates a data resource for global protein methylation in response to IR-induced DNA damage and reveals a critical role of PARP1 K23 methylation in DNA repair.