The Ewing Sarcoma protein (EWS) regulates DNA damage-induced alternative splicing

Alternative splicing allows cells to expand the coding potential of the genome and to respond to many different external stimuli and /or stresses. The Ewing Sarcoma protein EWS is a member of the TET (TLS/EWS/TAF15) family of DNA and RNA binding proteins, with proposed roles in transcription and RNA processing. Here we report that EWS knockdown generates changes in alternative splicing of genes related to DNA repair and DNA damage signalling pathways, including ABL1, CHEK2 and MAP4K2. Chromatin immunoprecipitations, cross-link RNA immunoprecipitation (CLIP) and mobility shift experiments documented that EWS binds to its splicing targets in vitro and in vivo, at both DNA and RNA levels. EWS HITS-CLIP revealed an enrichment of EWS clusters in the 5’ splice sites of constitutive and alternative exons, containing a GGGTG motif. Interestingly, upon irradiation of cells with low intensity UV light, EWS transiently translocates into sub-nuclear compartment, co-localizing with nucleolin and losing affinity for its pre-mRNA targets. Concomitantly, UV light treatment induced changes in alternative splicing that parallel those induced by EWS knockdown. In line with this, upon UV light treatment we observed a reduction of c-ABL protein expression that parallels those induced by EWS knock down in non-irradiated cells. Consistent with the functional relevance of EWS-mediated regulation for DNA damage response, EWS knock down results in reduced cell viability and proliferation upon UV irradiation, an effect attenuated by increased expression of c-ABL. Our results suggest that EWS re-localization upon UV irradiation is part of a new mechanism of cellular response acting as a negative feedback to inhibit excessive amplification of the DNA damage signal in response to mild genotoxic stress.