BRCA1/BARD1 inhibition of mRNA 3' processing involves targeted degradation of RNA polymerase II - PubMed (original) (raw)

BRCA1/BARD1 inhibition of mRNA 3' processing involves targeted degradation of RNA polymerase II

Frida E Kleiman et al. Genes Dev. 2005.

Abstract

Mammalian cells exhibit a complex response to DNA damage. The tumor suppressor BRCA1 and associated protein BARD1 are thought to play an important role in this response, and our previous work demonstrated that this includes transient inhibition of the pre-mRNA 3' processing machinery. Here we provide evidence that this inhibition involves proteasomal degradation of a component necessary for processing, RNA polymerase II (RNAP II). We further show that RNAP IIO, the elongating form of the enzyme, is a specific in vitro target of the BRCA1/BARD1 ubiquitin ligase activity. Significantly, siRNA-mediated knockdown of BRCA1 and BARD1 resulted in stabilization of RNAP II after DNA damage. In addition, inhibition of 3' cleavage induced by DNA damage was reverted in extracts of BRCA1-, BARD1-, or BRCA1/BARD1-depleted cells. We also describe corresponding changes in the nuclear localization and/or accumulation of these factors following DNA damage. Our results support a model in which a BRCA1/BARD1-containing complex functions to initiate degradation of stalled RNAP IIO, inhibiting the coupled transcription-RNA processing machinery and facilitating repair.

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Figures

Figure 1.

Role of protein turnover and RNAP II phosphorylation in UV-induced inhibition of 3′ processing. (A) An inhibitor of the proteasome rescues UV-induced inhibition of 3′ cleavage. HeLa cells were treated with UV irradiation and allowed to recover in the presence (lanes 1_–_5) or absence (lanes 6_–_10) of the proteasomal inhibitor MG132 for the times indicated. NEs prepared from these cells were used in cleavage reactions with an SV40 late (top) or adenovirus L3 (bottom) pre-mRNA. Positions of pre-mRNA and the 5′ and 3′ cleavage products are indicated. (B) The CTD-kinase inhibitor H7 blocks UV-induced inhibition of 3′ cleavage. HeLa cells were treated with UV irradiation and allowed to recover in the absence (lanes 1_–_5) or presence (lanes 6_–_10) of H7 for the times indicated. Cells were also treated with H7 only (lanes 11_–_14). NEs prepared from these cells were used in 3′ cleavage reactions as above. Positions of the SV40 pre-mRNA and 5′ and 3′ cleavage products are indicated. RNAP IIO, RNAP IIA, and actin protein levels in NEs from treated cells were monitored by Western blotting. Proteins were detected by immunoblotting with antibodies against a nonphosphorylated CTD epitope of RNAP II LS (8WG16) and actin.

Figure 2.

RNAP II reverse the inhibitory effect of DNA damage on 3′ processing. (A) Activation of cleavage by the addition of GST-CTD. NEs active (0-h UV treatment) and inactive (5-h UV treatment) for 3′ cleavage were preincubated with no addition (lanes 1,2,5,6) or with increasing amounts (100 and 200 ng) of recombinant GST-CTD (lanes 3,4,7,8). The cleavage reactions were performed in the absence (lanes 1,3_–_5,7,8) or presence (lanes 2,6) of creatine phosphate. Positions of pre-mRNA and the 5′ and 3′ cleavage products are indicated. (B) Activation of cleavage by the addition of RNAP IIO. NEs inactive (5-h UV treatment) for 3′ cleavage were preincubated with no addition (lane 1) or with increasing amounts (25, 50, and 100 ng) of purified RNAP IIO (lanes 2_–_4). Positions of pre-mRNA and the 5′ and 3′ cleavage products are indicated.

Figure 3.

RNAP IIO but not RNAP IIA is ubiquitinated by BRCA1/BARD1. (A) RNAP IIO was incubated with E1, E2, His-HA-Ub, and a purified heterodimer comprised of truncated BRCA1 and full-length BARD1 (ΔBRCA1/BARD1-wt) as indicated (lanes 1_–_3,5). Lane 4 shows a reaction containing the mutant heterodimer ΔBRCA1/BARD1-C61G. Reactions were terminated and proteins were separated by SDS-PAGE and analyzed by immunoblotting with anti-RNAP II LS and anti-Ub antibodies in the top and lower panels, respectively. Positions of the RNAP IIO and RNAP IIA forms are indicated on the left, and the polyubiquitinated forms of RNAP IIO and molecular-weight markers are indicated on the right. (B) Kinetics of RNAP IIO ubiquitination by ΔBRCA1/BARD1-wt. Ubiquitination reactions were performed and analyzed as in A, except that the incubation times were as indicated. (C) Ubiquitination of RNAP IIO with a heterodimeric complex comprised of full-length BRCA1 and BARD1. Reactions were performed and analyzed as in A. (D) ΔBRCA1/BARD1-wt stimulates polyubiquitination of RNAP IIO but not RNAP IIA. Ubiquitination reactions were performed as above except in presence of either RNAP IIO or RNAP IIA. (E) ΔBRCA1/BARD1 does not stimulate ubiquitination of either GST-CTD or phosphorylated GST-CTD. Ubiquitination reactions were done with nonphosphorylated (lanes 1_–_3) or in vitro phosphorylated (lanes 4_–_6) GST-CTD as in panel A. Positions of the nonphosphorylated and phosphorylated GST-CTD proteins are indicated on the left. (F) Concentration and purity of RNAP IIO and RNAP IIA (lanes_1,2_; only the largest subunits are shown), GST-CTD and pGST-CTD (lanes 3,4), and the heterodimeric complexes comprised of full-length BARD1 and truncated wild-type and C61G BRCA1 (lanes 5,6) were monitored by silver or Coomassie blue staining following SDS-PAGE. Positions of the three largest RNAP II subunits (IIa, IIo, and IIc) are indicated on the left, and positions of molecular-weight markers are indicated on the right.(G) Schematic diagrams of BRCA1 (1863 amino acids) and BARD1 (777 amino acids) showing the N-terminal RING domains (BRCA1 23–76, BARD1 49–100), the ankyrin repeats (BARD1 427–525), and C-terminal BRCT domains.

Figure 4.

siRNA knockdown of both BRCA1/BARD1 expression abolishes UV-induced degradation of RNAP II. (A) Protein levels of BRCA1, BARD1, actin, and RNAP II in NEs prepared from cells subjected to control (cont), BRCA1, BARD1, and BRCA1/BARD1 siRNA and UV irradiation. Nonirradiated and UV-irradiated samples were prepared 48 h after the addition of the siRNAs. Irradiated cells were allowed to recover 2 h after exposure to UV doses of 10 Jm–2. Panels depict blots using antibodies against BRCA1, BARD1, RNAP IIO (H5), RNAP II (N-20), and actin. Protein concentrations were equalized by immunostaining with antibodies against actin. The actin and the RNAP II blots correspond to the same gel. The positions of each protein are indicated in the corresponding panel. (B) An inhibitor of the proteasome prevents UV-induced degradation but not ubiquitination of RNAP II in siRNA-treated cells. siRNA-treated cells were irradiated with UV and allowed to recover in the presence of the proteasomal inhibitor MG132 for 2 h. BRCA1, BARD1, RNAP II, RNAP IIO, and actin protein levels in NEs from these cells were analyzed by Western blot as above.

Figure 5.

siRNA knockdown of both BRCA1/BARD1 abolishes the UV-induced inhibition of polyadenylation. (A) NEs were prepared from cells subjected to control, BRCA1, BARD1, and BRCA1/BARD1 siRNA treatment, and then used in cleavage reactions with the SV40 late substrate RNA. Positions of pre-mRNA and the 5′ and 3′ cleavage products are indicated. (B) NEs were prepared and used in processing reactions as in A. Reactions contained no addition (lanes 1_–_8), 10 ng full-length BRCA1/BARD1 (lanes 9_–_16), or 7 ng truncated BRCA1 (1–304)/BARD1 (1–202) complex (lanes 17_–_24).

Figure 6.

UV treatment transiently disperses BRCA1/BARD1 nuclear foci, RNAP IIO dots, and cleavage bodies, but not SC35 speckles. Asynchronous MCF7 cells were treated with UV and were fixed in 2% formaldehyde after the UV treatment at the times indicated. Fixed cells were immunostained with a BRCA1 mAb (MS110), BARD1 polyclonal antibody (669D), CstF-64 mAb, RNAP IIO LS mAb (H5), and an SC35 mAb. Immunostaining with monoclonal or polyclonal antibodies was visualized by using a goat anti-mouse or anti-rabbit IgG-fluorescein, respectively.

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BRCA1/BARD1 inhibition of mRNA 3' processing involves targeted degradation of RNA polymerase II - PubMed (original) (raw)