Antisense transcription is a widespread phenomenon in the mammalian genome. It is thought to play a role in regulation of gene expression, but its exact functional significance is largely unknown. We have identified a natural antisense transcript of p53, designated Wrap53, that regulates endogenous p53 mRNA levels and further induction of p53 protein by targeting the 5' untranslated region of p53 mRNA. siRNA knockdown of Wrap53 results in significant decrease in p53 mRNA and suppression of p53 induction upon DNA damage. Conversely, overexpression of Wrap53 increases p53 mRNA and protein levels. Blocking of potential Wrap53/p53 RNA hybrids reduces p53 levels nearly as efficiently as Wrap53 knockdown, strongly suggesting that Wrap53 regulates p53 via Wrap53/p53 RNA interaction. Furthermore, induction of Wrap53 sensitizes cells for p53-dependent apoptosis. This discovery not only reveals a regulatory pathway for controlling p53, but also proposes a general mechanism for antisense-mediated gene regulation in human cells.

The WRAP53 protein regulates the formation and maintenance of Cajal bodies (nuclear sub-organelles), as well as directs the recruitment of nuclear factors to Cajal bodies.

The WD40 domain-containing protein WRAP53b (WD40 encoding RNA antisense to p53; also referred to as WDR79/TCAB1) controls trafficking of splicing factors and the telomerase enzyme to Cajal bodies, and its functional loss has been linked to carcinogenesis, premature aging, and neurodegeneration. Here, we identify WRAP53b as an essential regulator of DNA double-strand break (DSB) repair. WRAP53b rapidly localizes to DSBs in an ATM-, H2AX-, and MDC1-dependent manner. We show that WRAP53b targets the E3 ligase RNF8 to DNA lesions by facilitating the interaction between RNF8 and its upstream partner, MDC1, in response to DNA damage. Simultaneous binding of MDC1 and RNF8 to the highly conserved WD40 scaffold domain of WRAP53b facilitates their interaction and accumulation of RNF8 at DSBs. In this manner, WRAP53b controls proper ubiquitylation at DNA damage sites and the downstream assembly of 53BP1, BRCA1, and RAD51. Furthermore, we reveal that knockdown of WRAP53b impairs DSB repair by both homologous recombination (HR) and nonhomologous end-joining (NHEJ), causes accumulation of spontaneous DNA breaks, and delays recovery from radiation-induced cell cycle arrest. Our findings establish WRAP53b as a novel regulator of DSB repair by providing a scaffold for DNA repair factors.

No abstract

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.