Major roles for pyrimidine dimers, nucleotide excision repair, and ATR in the alternative splicing response to UV irradiation

Abstract

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.A.R.K. and M.J.M. received support from the Agencia Nacional de Promoción Científica y Tecnológica of Argentina (PICT-2011 1617, PICT-2014 2582), the Universidad de Buenos Aires (UBACYT 20020130100152BA), and the Alberto J. Roemmers Foundation. G.D. was supported by Marie Curie International Outgoing Fellowship within the EU Seventh Framework Programme for Research and Technological Development (FP7/2007-2013) under grant agreement 275632. B.J.B. is supported by grants from the Canadian Institutes of Health Research (FDN 148434). M.I. is supported by grants from the European Research Council (ERC-StG-LS2- 637591) and from Ministerio de Economía y Competitividad (BFU2014-55076-P; MINECO). A.T.-M. is supported by an FPI-SO fellowship from the Spanish Ministry of Economy and Competitiveness. M.F. is supported by the Associazione Italiana per la Ricerca sul Cancro (AIRC) and Telethon-Italy. G.B. is recipient of a fellowship from the University of Milan. S.L. is an employee of IFOM Cell Biology Unit. M.J.M., L.E.G., and A.R.K. are career investigators and N.N.M. and A.E.C.B. are recipients of a graduate student fellowship from the Consejo Nacional de Investigaciones Científicas y Técnicas of Argentina (CONICET). A.R.K. is a Senior International Research Scholar of the Howard Hughes Medical Institute