The splicing factor XAB2 interacts with ERCC1-XPF and XPG for R-loop processing

Abstract

RNA splicing, transcription and the DNA damage response are intriguingly linked in mammals but the underlying mechanisms remain poorly understood. Using an in vivo biotinylation tagging approach in mice, we show that the splicing factor XAB2 interacts with the core spliceosome and that it binds to spliceosomal U4 and U6 snRNAs and pre-mRNAs in developing livers. XAB2 depletion leads to aberrant intron retention, R-loop formation and DNA damage in cells. Studies in illudin S-treated cells and Csbm/m developing livers reveal that transcription-blocking DNA lesions trigger the release of XAB2 from all RNA targets tested. Immunoprecipitation studies reveal that XAB2 interacts with ERCC1-XPF and XPG endonucleases outside nucleotide excision repair and that the trimeric protein complex binds RNA:DNA hybrids under conditions that favor the formation of R-loops. Thus, XAB2 functionally links the spliceosomal response to DNA damage with R-loop processing with important ramifications for transcription-coupled DNA repair disorders.

R-loops trigger the release of cytoplasmic ssDNAs leading to chronic inflammation upon DNA damage

“…S5C). Our analysis revealed the presence of ssDNAs originating from the tRA-inducible genes Rarb and Stra6 ( 33 ) in tRA/Illudin S–treated PPCs compared to untreated controls, indicating that cytoplasmic ssDNAs are generated by cotranscriptional R-loops. The β -actin gene was used as a control because it is a tRA-nonresponsive gene that was previously shown to accumulate R-loops ( 34 , 35 ).…”

“…5D and fig. S5E; as indicated) ( 33 , 37 , 38 ). Our finding that, in Ercc1 −/− cells, the XPF DRIP signals are lower than those of XPG supports previous observations indicating that XPF is unstable in the absence of ERCC1 ( 39 , 40 ).…”

“…However, the light-dependent removal of UV-induced CPDs from the genome of CPD-photolyase transgenic cells rapidly eliminates the great majority of cytosolic ssDNAs, indicating that cytoplasmic ssDNAs are of nuclear origin. We and others have recently shown that R-loops are increasingly formed in cells that are defective in DNA repair or RNA processing ( 47 – 49 )—leading to DNA breaks ( 50 ), transcription stalling, and DDR activation—and that XPG and XPF are recruited on RNA:DNA hybrids under conditions that promote R-loop formation ( 33 , 50 ). A central aspect of these findings is that the great majority of ssDNAs in the cytoplasm originate from DNA damage–driven R-loops.…”

XAB2 promotes Ku eviction from single-ended DNA double-strand breaks independently of the ATM kinase

“…Maréchal et al have discussed the possibility that PRP19, through its ability to associate with the transcription machinery and recognize RPA-coated ssDNA, functionally links DDR, transcription, and RNA processing ( 75 ). As our data suggest that XAB2 promotes processing events at the RPA-ssDNA platform upon replication fork collapse, and XAB2 has recently been involved in R-loop processing during development ( 89 ), we propose that the recognition and/or processing of RPA-coated ssDNA generated during DNA or RNA transactions represent a common feature of the splicing factors that promote DNA repair.…”

“…Our data indicate that the XAB2 pathway operates in parallel to the ATM-CtIP-MRE11 axis (Figure 7 ). XAB2 has been shown to interact with several DNA repair factors ( 58 , 89 ). The physical interaction detected between XAB2 and Ku together with the observed recruitment of XAB2 in close proximity to γH2AX and RAD51 foci strongly suggest that XAB2 operates by a direct mechanism to promote Ku eviction at seDSBs.…”