Splicing controls the ubiquitin response during DNA double-strand break repair.
Although evidence that splicing regulates DNA repair is accumulating, the underlying mechanism(s) remain unclear. Here, we report that short-term inhibition of pre-mRNA splicing by spliceosomal inhibitors impairs cellular repair of DNA double-strand breaks. Indeed, interference with splicing as little as 1 h prior to irradiation reduced ubiquitylation of damaged chromatin and impaired recruitment of the repair factors WRAP53β, RNF168, 53BP1, BRCA1 and RAD51 to sites of DNA damage. Consequently, splicing-deficient cells exhibited significant numbers of residual γH2AX foci, as would be expected if DNA repair is defective. Furthermore, we show that this is due to downregulation of the E3 ubiquitin ligase RNF8 and that re-introduction of this protein into splicing-deficient cells restores ubiquitylation at sites of DNA damage, accumulation of downstream factors and subsequent repair. Moreover, downregulation of RNF8 explains the defective repair associated with knockdown of various splicing factors in recent genome-wide siRNA screens and, significantly, overexpression of RNF8 counteracts this defect. These discoveries reveal a mechanism that may not only explain how splicing regulates repair of double-strand breaks, but also may underlie various diseases caused by deregulation of splicing factors, including cancer.
Department of Oncology-Pathology, Cancer Centrum Karolinska (CCK), Karolinska Institutet, Stockholm 17176, Sweden.
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Pederiva
Department of Oncology-Pathology, Cancer Centrum Karolinska (CCK), Karolinska Institutet, Stockholm 17176, Sweden.
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Böhm
Department of Oncology-Pathology, Cancer Centrum Karolinska (CCK), Karolinska Institutet, Stockholm 17176, Sweden.
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Julner
Department of Oncology-Pathology, Cancer Centrum Karolinska (CCK), Karolinska Institutet, Stockholm 17176, Sweden.
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Farnebo
D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair / Q000235:genetics*; D045643:DNA Repair Enzymes / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D015536:Down-Regulation / Q000235:genetics; D055785:Gene Knockdown Techniques; D015894:Genome, Human; D006367:HeLa Cells; D006801:Humans; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism; D012326:RNA Splicing / Q000235:genetics*; D000072260:RNA Splicing Factors / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases
D004268:DNA-Binding Proteins; D012322:RNA Precursors; D000072260:RNA Splicing Factors; D012333:RNA, Messenger; C486521:RNF8 protein, human; D025801:Ubiquitin; D044767:Ubiquitin-Protein Ligases; D045643:DNA Repair Enzymes