Overexpression of the scaffold WD40 protein WRAP53ß enhances the repair of and cell survival from DNA double-strand breaks.

Altered expression of the multifunctional protein WRAP53ß (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 WRAP53ß function has been shown to disrupt processes regulated by this protein, the consequences of its overexpression remain unclear. Here we demonstrate that overexpression of WRAP53ß 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 WRAP53ß 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 WRAP53ß, attenuated this effect, suggesting that overexpression of WRAP53ß leads to more rapid repair, as well as improved cell survival, by enhancing RNF8-mediated ubiquitylation at DNA breaks. Our present findings indicate that WRAP53ß and RNF8 are rate-limiting factors in the repair of DNA double-strand breaks and raise the possibility that upregulation of WRAP53ß may contribute to genomic stability in and survival of cancer cells.

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 WRAP53ß, 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.

Downregulation of the cancer susceptibility protein WRAP53ß in epithelial ovarian cancer leads to defective DNA repair and poor clinical outcome.

Alterations in the scaffold protein WRAP53ß have previously been linked to carcinogenesis and, in particular, associated with an increased risk for epithelial ovarian cancer. Here, we investigated the pathogenic impact and prognostic significance of WRAP53ß in connection with epithelial ovarian cancer and examined the underlying mechanisms. We find that reduced expression of WRAP53ß in ovarian tumors correlated with attenuated DNA damage response and poor patient survival. Furthermore, in ovarian cancer cell lines, WRAP53ß was rapidly recruited to DNA double-strand breaks, where it orchestrated the recruitment of repair factors involved in homologous recombination and non-homologous end joining, including RNF168, 53BP1, BRCA1 and RAD51. Mechanistically, WRAP53ß accomplishes this by facilitating the necessary ubiquitinylation at DNA breaks. Finally, we demonstrate that loss of WRAP53ß significantly impairs the repair of DNA double-strand breaks, resulting in their accumulation. Our findings establish WRAP53ß as a regulator of homologous recombination and non-homologous end joining repair in ovarian cancer cells, suggesting that loss of this protein contributes to the development and/or progression of ovarian tumors. Moreover, our current observations identify the nuclear levels of WRAP53ß as a promising biomarker for the survival of patients with ovarian cancer.

WRAP53 promotes cancer cell survival and is a potential target for cancer therapy.

We previously identified WRAP53 as an antisense transcript that regulates the p53 tumor suppressor. The WRAP53 gene also encodes a protein essential for Cajal body formation and involved in cellular trafficking of the survival of motor neuron complex, the telomerase enzyme and small Cajal body-specific RNAs to Cajal bodies. Here, we show that the WRAP53 protein is overexpressed in a variety of cancer cell lines of different origin and that WRAP53 overexpression promotes cellular transformation. Knockdown of the WRAP53 protein triggers massive apoptosis through the mitochondrial pathway, as demonstrated by Bax/Bak activation, loss of mitochondrial membrane potential and cytochrome c release. The apoptosis induced by WRAP53 knockdown could moreover be blocked by Bcl-2 overexpression. Interestingly, human tumor cells are more sensitive to WRAP53 depletion as compared with normal human cells indicating that cancer cells in particular depends on WRAP53 expression for their survival. In agreement with this, we found that high levels of WRAP53 correlate with poor prognosis of head and neck cancer. Together these observations propose a role of WRAP53 in carcinogenesis and identify WRAP53 as a novel molecular target for a large fraction of malignancies.