Graphical Abstract Highlights d DNA damage is sufficient to trigger a global alternative splicing (AS) response to UV d Photolyase-mediated elimination of the DNA lesions (CPDs) abolishes the AS response d RNAPII is the target, but not a sensor, of the signaling cascade initiated by CPDs d GG-NER and ATR participate in the control of RNAPII phosphorylation and AS by UV In Brief Muñ oz et al. find that UV-induced DNA damage is the main determinant affecting gene expression in response to UV in

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... in cells. DNA repair and the subsequent activation of ATR modulate RNAPII phosphorylation and alternative splicing patterns specifically in keratinocytes. SUMMARY We have previously found that UV irradiation promotes RNA polymerase II (RNAPII) hyperphosphorylation and subsequent changes in alternative splicing (AS). We show now that UV-induced DNA damage is not only necessary but sufficient to trigger the AS response and that photolyase-mediated removal of the most abundant class of pyrimidine dimers (PDs) abrogates the global response to UV. We demonstrate that, in keratinocytes, RNAPII is the target, but not a sensor, of the signaling cascade initiated by PDs. The UV effect is enhanced by inhibition of gap-filling DNA synthesis, the last step in the nucleotide excision repair pathway (NER), and reduced by the absence of XPE, the main NER sensor of PDs. The mechanism involves activation of the protein kinase ATR that mediates the UV-induced RNAPII hyperphosphorylation. Our results define the sequence UV-PDs-NER-ATR-RNAPII-AS as a pathway linking DNA damage repair to the control of both RNAPII phosphorylation and AS regulation.