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The mechanisms involved in the p53-dependent control of gene expression following DNA damage have not been completely elucidated. Here, we show that the p53 C terminus associates with factors that are required for the ultraviolet (UV)-induced inhibition of the mRNA 3' cleavage step of the polyadenylation reaction, such as the tumor suppressor BARD1 and the 3' processing factor cleavage-stimulation factor 1 (CstF1). We found that p53 can coexist in complexes with CstF and BARD1 in extracts of UV-treated cells, suggesting a role for p53 in mRNA 3' cleavage following DNA damage. Consistent with this, we found that p53 inhibits 3' cleavage in vitro and that there is a reverse correlation between the levels of p53 expression and the levels of mRNA 3' cleavage under different cellular conditions. Supporting these results, a tumor-associated mutation in p53 not only decreases the interaction with BARD1 and CstF, but also decreases the UV-induced inhibition of 3' processing, all of which is restored by wild-type-p53 expression. We also found that p53 expression levels affect the polyadenylation levels of housekeeping genes, but not of p21 and c-fos genes, which are involved in the DNA damage response (DDR). Here, we identify a novel 3' RNA processing inhibitory function of p53, adding a new level of complexity to the DDR by linking RNA processing to the p53 network.

The BARD1 N-terminal RING domain binds BRCA1 while the BARD1 C-terminal ankyrin and tandem BRCT repeat domains bind CstF-50 to modulate mRNA processing and RNAP II stability in response to DNA damage. Here we characterize the BARD1 structural biochemistry responsible for CstF-50 binding. The crystal structure of the BARD1 BRCT domain uncovers a degenerate phosphopeptide binding pocket lacking the key arginine required for phosphopeptide interactions in other BRCT proteins. Small angle X-ray scattering together with limited proteolysis results indicates that ankyrin and BRCT domains are linked by a flexible tether and do not adopt a fixed orientation relative to one another. Protein pull-down experiments utilizing a series of purified BARD1 deletion mutants indicate that interactions between the CstF-50 WD-40 domain and BARD1 involve the ankyrin-BRCT linker but do not require ankyrin or BRCT domains. The structural plasticity imparted by the ANK-BRCT linker helps to explain the regulated assembly of different protein BARD1 complexes with distinct functions in DNA damage signaling including BARD1-dependent induction of apoptosis plus p53 stabilization and interactions. BARD1 architecture and plasticity imparted by the ANK-BRCT linker are suitable to allow the BARD1 C-terminus to act as a hub with multiple binding sites to integrate diverse DNA damage signals directly to RNA polymerase.

The mRNA polyadenylation factor CstF interacts with the BRCA1-associated protein BARD1, and this interaction represses the nuclear mRNA polyadenylation machinery in vitro. Given the suspected role of BRCA1/BARD1 in DNA repair, we tested whether inhibition of mRNA processing is linked to DNA damage. Strikingly, we found that 3' cleavage in extracts from cells treated with hydroxyurea or ultraviolet light was strongly, but transiently, inhibited. Although no changes were detected in CstF, BARD1, and BRCA1 protein levels, increased amounts of a CstF/BARD1/BRCA1 complex were detected. Supporting the physiological significance of these results, a previously identified tumor-associated germline mutation in BARD1 (Gln564His) reduced binding to CstF and abrogated inhibition of polyadenylation. Together these results indicate a link between mRNA 3' processing and DNA repair and tumor suppression.

Polyadenylation of messenger RNA precursors requires a complex protein machinery that is closely integrated with the even more complex transcriptional apparatus. Here a polyadenylation factor, CstF-50 (cleavage stimulation factor), is shown to interact in vitro and in intact cells with a nuclear protein of previously unknown function, BRCA1-associated RING domain protein (BARD1). The BARD1-CstF-50 interaction inhibits polyadenylation in vitro. BARD1, like CstF-50, also interacts with RNA polymerase II. These results indicate that BARD1-mediated inhibition of polyadenylation may prevent inappropriate RNA processing during transcription, perhaps at sites of DNA repair, and they reveal an unanticipated integration of diverse nuclear events.