Genotoxic stress causes the accumulation of DNA‐dependent protein kinase catalytic subunit phosphorylated at serine 2056 at nuclear speckles and alters pre‐mRNA alternative splicing

RNA splicing has emerged as a critical player in the DNA damage response (DDR). However, the underlying mechanism(s) by which pre‐mRNA splicing is coordinately regulated by genotoxic stress has remained largely unclear. Here, we show that a DDR factor, DNA‐dependent protein kinase (DNA‐PK), participates in the modulation of pre‐mRNA splicing in the presence of DNA double‐strand break (DSB)‐induced genotoxic stress. Through indirect immunostaining, we made the surprising discovery that DNA‐PK catalytic subunits (DNA‐PKcs) autophosphorylated at serine 2056 (S2056) accumulate at nuclear speckles (dynamic nuclear structures that are enriched with splicing factors), following their dissociation from DSB lesions. Inactivation of DNA‐PKcs, either using a small molecule inhibitor or by RNA interference, alters alternative splicing of a set of pre‐mRNAs in A549 cells treated with the topoisomerase II inhibitor mitoxantrone, indicative of an involvement of DNA‐PKcs in modulating pre‐mRNA splicing following genotoxic stress. These findings indicate a novel physical and functional connection between the DNA damage response and pre‐mRNA splicing, and enhance our understanding of how mRNA splicing is involved in the cellular response to DSB lesions.