Abstract
Altered expression of the multifunctional protein WRAP53β (WD40 encoding RNA Antisense to p53), which targets repair factors to DNA double-strand breaks and factors involved in telomere elongation to Cajal bodies, is linked to carcinogenesis. While loss of WRAP53β function has been shown to disrupt processes regulated by this protein, the consequences of its overexpression remain unclear. Here we demonstrate that overexpression of WRAP53β disrupts the formation of and impairs the localization of coilin to Cajal bodies. At the same time, the function of this protein in the repair of DNA double-strand breaks is enhanced. Following irradiation, cells overexpressing WRAP53β exhibit more rapid clearance of phospho-histone H2AX (γH2AX), and more efficient homologous recombination and non-homologous end-joining, in association with fewer DNA breaks. Moreover, in these cells the ubiquitylation of damaged chromatin, which is known to facilitate the recruitment of repair factors and subsequent repair, is elevated. Knockdown of the ubiquitin ligase involved, ring-finger protein 8 (RNF8), which is recruited to DNA breaks by WRAP53β, attenuated this effect, suggesting that overexpression of WRAP53β leads to more rapid repair, as well as improved cell survival, by enhancing RNF8-mediated ubiquitylation at DNA breaks. Our present findings indicate that WRAP53β and RNF8 are rate-limiting factors in the repair of DNA double-strand breaks and raise the possibility that upregulation of WRAP53β may contribute to genomic stability in and survival of cancer cells.
Highlights
IntroductionWe previously identified the RNA produced from the WRAP53 (WD40 encoding RNA Antisense to p53) gene as an antisense transcript (WRAP53α) that stabilizes the tumor suppressor p53.1 In addition, this gene encodes the WRAP53β protein ( referred to as WRAP53 or WDR79 or TCAB1), which does not regulate p53 but instead is involved in the regulation of telomere elongation and repair of DNA double-strand breaks by recruiting telomerase to nuclear Cajal bodies and the repair factor RNF8 to these break, respectively.[2,3] The role played by WRAP53β in the repair of DNA double-strand breaks is independent of p53, as WRAP53β regulates DNA repair in cells that lack p53 expression.[3,4] WRAP53β directs coilin, the survival of motor neuron (SMN) protein and small Cajal body-associated (sca) RNAs to Cajal bodies.[2,5,6]
The WRAP53β protein is highly enriched in Cajal bodies, and to examine whether this localization is altered upon overexpression, the total protein lysate from human U-2 osteosarcoma (U2OS) cancer cells that stably overexpress Flag-tagged WRAP53β was analyzed with both rabbit α-WRAP53-C2 antibody, which detects both the nuclear and cytoplasmic forms, and the mouse monoclonal α-WDR79 clone 1F12, which detects the nuclear variant, and is the only antibody that can be used to visualize this protein in repair foci.[3,4]
No Cajal bodies were observed in the cells overexpressing WRAP53β, where this protein and coilin were distributed throughout the nucleoplasm (Figure 1b), in agreement with previous findings.[5]
Summary
We previously identified the RNA produced from the WRAP53 (WD40 encoding RNA Antisense to p53) gene as an antisense transcript (WRAP53α) that stabilizes the tumor suppressor p53.1 In addition, this gene encodes the WRAP53β protein ( referred to as WRAP53 or WDR79 or TCAB1), which does not regulate p53 but instead is involved in the regulation of telomere elongation and repair of DNA double-strand breaks by recruiting telomerase to nuclear Cajal bodies and the repair factor RNF8 to these break, respectively.[2,3] The role played by WRAP53β in the repair of DNA double-strand breaks is independent of p53, as WRAP53β regulates DNA repair in cells that lack p53 expression.[3,4] WRAP53β directs coilin, the survival of motor neuron (SMN) protein and small Cajal body-associated (sca) RNAs to Cajal bodies.[2,5,6]. Loss of nuclear WRAP53β or singlenucleotide polymorphisms in the WRAP53 gene is correlated with shorter survival of patients with head and neck, breast and ovarian cancer.[4,10,11,12,13,14,15] In addition, attenuated expression of this protein correlates with disruption of the DNA damage response in ovarian tumors,[4] as well as with resistance of head and neck cancer to radiotherapy.[14] altered DNA repair may be the underlying cause of cancers associated with abnormalities in WRAP53β and influence the response of such tumors to treatment. We examined the potential influence of such overexpression on the DNA damage response
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