The BARD1-CstF-50 Interaction Links mRNA 3′ End Formation to DNA Damage and Tumor Suppression

Abstract

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.

p53 inhibits mRNA 3′ processing through its interaction with the CstF/BARD1 complex

“…BARD1 can also interact with the 3 0 processing factor CstF1, inhibiting mRNA 3 0 processing and linking it to the DNA damage response (DDR) (Kleiman and Manley, 1999). Interestingly, the Gln564His mutation of BARD1 also reduces the binding of CstF1 to BARD1, interfering with the role of BARD1 in mRNA 3 0 processing (Kleiman and Manley, 2001). Taken together, these studies suggest a functional interaction between BARD1, CstF1 and p53 under DNA-damaging conditions.…”

“…Nuclear extracts preparation and immunoblotting analysis After UV treatment, NEs were prepared from harvested cells as described (Kleiman and Manley, 2001). Sixty micrograms of each NE was analyzed by immunoblotting with antibodies against BARD1 (H-300, Santa Cruz Biotechnology, Santa Cruz, CA, USA), p53 (SC-126, Santa Cruz), CstF2 (generously provided by Dr Manley, Columbia University, New York, NY, USA), CstF1 (10064-2-AP, Protein Tech Group, Chicago, IL, USA) and Topoisomerase II (Topo II, SC-25330, Santa Cruz).…”

“…The beads were recovered by centrifugation and treated at 4 1C with 50 mg of RNase A/ml for 10 min. Immunoprecipitations were carried out as described (Kleiman and Manley, 2001), except that washing was with buffer A (1 Â phosphate-buffered saline: 137 mM NaCl, 3 mM KCl, 10 mM Na 2 HPO 4 , 1.8 mM KH 2 PO 4 , 0.01% Nonidet P-40, 0.5 mM phenylmethylsulfonyl fluoride and 0.04% bovine serum albumin). Aliquots of pellets and supernatants were analyzed by sodium dodecyl sulfatepolyacrylamide gel electrophoresis and immunoblotting as described above.…”

“…It has been described that the induction of p53 expression upon ultraviolet (UV) treatment is associated with changes in the levels of total poly(A) þ mRNA McKay and Ljungman, 1999). Interestingly, this p53-associated changes in poly(A) þ RNA levels might also be functionally related to the UV-induced inhibition of mRNA polyadenylation (Kleiman and Manley, 2001). As mRNA poly(A) tails are important for the regulation of mRNA stability, it is possible that these changes of poly(A) þ mRNA levels might represent another mechanism of p53-mediated control of gene expression.…”

“…The assembly of the cleavage/polyadenylation machinery requires specific signal sequences in the mRNA precursor as well as interactions of a large number of protein factors (reviewed by Mandel et al, 2008). It has been shown that the regulation of mRNA 3 0 end formation can have significant roles in cancer (Kleiman and Manley, 2001;Topalian et al, 2001;Scorilas, 2002;Rozenblatt-Rosen et al, 2009). Most importantly, alternative mRNA cleavage and polyadenylation changes the length of the 3 0 -untranslated region and regulates gene expression of different mRNAs in cancer cells (Mayr and Bartel 2009;Singh et al, 2009) and during cell differentiation (Sandberg et al, 2008;Zlotorynski and Agami, 2008;Ji et al, 2009).…”