BRCA1 Interacts with Poly(A)-binding Protein
2006; Elsevier BV; Volume: 281; Issue: 34 Linguagem: Inglês
10.1074/jbc.m602176200
ISSN1083-351X
AutoresÉva Dizin, Céline Gressier, Clémence Magnard, Hind Ray, Didier Décimo, Théophile Ohlmann, Nicole Dalla Venezia,
Tópico(s)Genetically Modified Organisms Research
ResumoBRCA1 has been implicated in a number of cellular processes, including transcription regulation, DNA damage repair, cell cycle control, and apoptosis. We identified poly(A)-binding protein 1 (PABP) as a novel BRCA1-interacting protein in a yeast two-hybrid screen and confirmed the interaction by in vitro assays and coimmunoprecipitation in mammalian cells. Endogenous interaction between BRCA1 and PABP was also observed. This interaction was abolished by BRCA1 cancer-associated mutations, suggesting that it may be physiologically relevant. Deletion mapping demonstrated that the RNA recognition motifs 1–4 region of PABP is required to mediate the interaction with BRCA1. To understand the biological function of the BRCA1-PABP complex, we sought to determine whether BRCA1 is a modulator of translation. We showed here that inhibition of endogenous BRCA1 using a small interfering RNA-based approach decreased protein synthesis. Conversely, overexpression of BRCA1 activated translation. Using a RNA transfection approach, we clearly showed that BRCA1 modulates translation, independently of any transcriptional activity. The data presented here suggest that BRCA1 modulates protein synthesis via its interaction with PABP, providing a novel mechanism by which BRCA1 may exert its tumor suppressor function. BRCA1 has been implicated in a number of cellular processes, including transcription regulation, DNA damage repair, cell cycle control, and apoptosis. We identified poly(A)-binding protein 1 (PABP) as a novel BRCA1-interacting protein in a yeast two-hybrid screen and confirmed the interaction by in vitro assays and coimmunoprecipitation in mammalian cells. Endogenous interaction between BRCA1 and PABP was also observed. This interaction was abolished by BRCA1 cancer-associated mutations, suggesting that it may be physiologically relevant. Deletion mapping demonstrated that the RNA recognition motifs 1–4 region of PABP is required to mediate the interaction with BRCA1. To understand the biological function of the BRCA1-PABP complex, we sought to determine whether BRCA1 is a modulator of translation. We showed here that inhibition of endogenous BRCA1 using a small interfering RNA-based approach decreased protein synthesis. Conversely, overexpression of BRCA1 activated translation. Using a RNA transfection approach, we clearly showed that BRCA1 modulates translation, independently of any transcriptional activity. The data presented here suggest that BRCA1 modulates protein synthesis via its interaction with PABP, providing a novel mechanism by which BRCA1 may exert its tumor suppressor function. The breast cancer susceptibility gene breast cancer 1 (BRCA1) acts as a tumor suppressor gene (1Miki Y. Swensen J. Shattuck-Eidens D. Futreal P.A. Harshman K. Tavtigian S. Liu Q. Cochran C. Bennett L.M. Ding W et al.Science. 1994; 266: 66-71Crossref PubMed Scopus (5211) Google Scholar). Germ-line mutations of BRCA1 are found in about 40% of patients with inherited breast cancer and up to 90% of families with breast and ovarian cancer (2Couch F.J. Weber B.L. Hum. Mutat. 1996; 8: 8-18Crossref PubMed Scopus (269) Google Scholar). The decreased BRCA1 expression found in approximately one-third of sporadic breast tumors may also play a role in these cancers (3Thompson M.E. Jensen R.A. Obermiller P.S. Page D.L. Holt J.T. Nat. Genet. 1995; 9: 444-450Crossref PubMed Scopus (542) Google Scholar, 4Magdinier F. Ribieras S. Lenoir G.M. Frappart L. Dante R. Oncogene. 1998; 17: 3169-3176Crossref PubMed Scopus (119) Google Scholar). Several lines of approach tend to define the biochemical function of BRCA1 protein. BRCA1 has thus far been involved in regulation of cell cycle checkpoints, apoptosis, DNA damage repair, transcription, and ubiquitination (5Venkitaraman A.R. Cell. 2002; 108: 171-182Abstract Full Text Full Text PDF PubMed Scopus (1362) Google Scholar). Although the implication of BRCA1 in these cellular processes has been demonstrated, the exact mechanism of BRCA1 function remains unclear. The BRCA1 protein contains a RING finger at its N-terminal region (1Miki Y. Swensen J. Shattuck-Eidens D. Futreal P.A. Harshman K. Tavtigian S. Liu Q. Cochran C. Bennett L.M. Ding W et al.Science. 1994; 266: 66-71Crossref PubMed Scopus (5211) Google Scholar), two nuclear export signals near its N terminus (6Rodriguez J.A. Henderson B.R. J. Biol. Chem. 2000; 275: 38589-38596Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 7Thompson M.E. Robinson-Benion C.L. Holt J.T. J. Biol. Chem. 2005; 280: 21854-21857Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar), two nuclear localization signals in the central portion of the protein (8Chen Y. Farmer A.A. Chen C.F. Jones D.C. Chen P.L. Lee W.H. Cancer Res. 1996; 56: 3168-3172PubMed Google Scholar), and a tandem of two BRCT domains at its C-terminal region (9Koonin E.V. Altschul S.F. Bork P. Nat. Genet. 1996; 13: 266-268Crossref PubMed Scopus (358) Google Scholar, 10Callebaut I. Mornon J.P. FEBS Lett. 1997; 400: 25-30Crossref PubMed Scopus (483) Google Scholar, 11Bork P. Hofmann K. Bucher P. Neuwald A.F. Altschul S.F. Koonin E.V. FASEB J. 1997; 11: 68-76Crossref PubMed Scopus (656) Google Scholar). The majority of cancer-associated BRCA1 mutations affect the BRCT tandem, resulting in truncated products lacking one or two BRCT domains (2Couch F.J. Weber B.L. Hum. Mutat. 1996; 8: 8-18Crossref PubMed Scopus (269) Google Scholar). These findings together with the observation that deletion of the Brca1 BRCT domains is responsible for tumor development in mice (12Ludwig T. Fisher P. Ganesan S. Efstratiadis A. Genes Dev. 2001; 15: 1188-1193Crossref PubMed Scopus (110) Google Scholar) demonstrate that BRCT domains play a central role in the BRCA1 tumor suppressor function. Several proteins interact with this region and collaborate functionally with BRCA1. For example, BRCA1 interacts with proteins implicated in transcriptional regulation such as RNA polymerase holoenzyme, CtIP (13Scully R. Anderson S.F. Chao D.M. Wei W. Ye L. Young R.A. Livingston D.M. Parvin J.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5605-5610Crossref PubMed Scopus (417) Google Scholar, 14Wong A.K. Ormonde P.A. Pero R. Chen Y. Lian L. Salada G. Berry S. Lawrence Q. Dayananth P. Ha P. Tavtigian S.V. Teng D.H. Bartel P.L. Oncogene. 1998; 17: 2279-2285Crossref PubMed Scopus (135) Google Scholar, 15Yu X. Wu L.C. Bowcock A.M. Aronheim A. Baer R. J. Biol. Chem. 1998; 273: 25388-25392Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar), and histone deacetylases (16Yarden R.I. Brody L.C. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4983-4988Crossref PubMed Scopus (283) Google Scholar), with BACH1 implicated in response to DNA damage (17Cantor S.B. Bell D.W. Ganesan S. Kass E.M. Drapkin R. Grossman S. Wahrer D.C. Sgroi D.C. Lane W.S. Haber D.A. Livingston D.M. Cell. 2001; 105: 149-160Abstract Full Text Full Text PDF PubMed Scopus (536) Google Scholar) and with acetyl-CoA carboxylase α implicated in fatty acid synthesis (18Magnard C. Bachelier R. Vincent A. Jaquinod M. Kieffer S. Lenoir G.M. Venezia N.D. Oncogene. 2002; 21: 6729-6739Crossref PubMed Scopus (80) Google Scholar). In this study we further explored new functions of BRCA1 through its BRCT tandem using the yeast two-hybrid approach. We show that BRCA1 interacts with poly(A)-binding protein 1 (PABP), 5The abbreviations used are: PABP, poly(A)-binding protein 1; CHX, cycloheximide; GST, glutathione S-transferase; RRM, RNA recognition motif; eIF4G, eukaryotic translation initiation factor 4G; PAIP, PABP-interacting protein. 5The abbreviations used are: PABP, poly(A)-binding protein 1; CHX, cycloheximide; GST, glutathione S-transferase; RRM, RNA recognition motif; eIF4G, eukaryotic translation initiation factor 4G; PAIP, PABP-interacting protein. a highly conserved protein involved in mRNA stabilization and translation (19Gray N.K. Coller J.M. Dickson K.S. Wickens M. EMBO J. 2000; 19: 4723-4733Crossref PubMed Scopus (191) Google Scholar, 20Grosset C. Chen C.Y. Xu N. Sonenberg N. Jacquemin-Sablon H. Shyu A.B. Cell. 2000; 103: 29-40Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar, 21Copeland P.R. Wormington M. RNA. 2001; 7: 875-886Crossref PubMed Scopus (89) Google Scholar) and recently described as a canonical translation initiation factor (22Kahvejian A. Svitkin Y.V. Sukarieh R. M'Boutchou M.N. Sonenberg N. Genes Dev. 2005; 19: 104-113Crossref PubMed Scopus (356) Google Scholar). We first demonstrated that BRCA1 binds to PABP in vitro as well as in vivo. Notably, this interaction is disrupted by germ line BRCA1 mutations that affect the BRCT repeats. We then provide evidence showing that BRCA1 can stimulate translation, supporting a model in which control of translation would be mediated in part via the BRCA1-PABP interaction. Chemicals—The primary antibodies used in this study were as follows. Mouse monoclonal antibody to human BRCA1 (OP92) was purchased from Oncogene Research Products, mouse monoclonal antibody to actin was from ICN Biochemicals, mouse monoclonal antibody to Myc (anti-Myc) from Roche Applied Science, and the mouse monoclonal antibody against the synthetic V5 epitope was from Invitrogen. Mouse monoclonal antibodies (as ascites fluids) against human PABP (clone 10E10) were gifts from Gideon Dreyfuss (University of Pennsylvania) (23Gorlach M. Burd C.G. Dreyfuss G. Exp. Cell Res. 1994; 211: 400-407Crossref PubMed Scopus (213) Google Scholar). Polyclonal antibody to P300 (N-15) was from Santa Cruz Biotechnology. Secondary antibodies used were peroxidase-conjugated anti-mouse immunoglobulin (Amersham Biosciences). Cycloheximide (CHX) was purchased from Sigma. Bacterial Expression Constructs—The pGEX-BRCT plasmid and the pGEX-BRCT mutants GST-BRCT-A1709E, GST-BRCT-P1749R, GST-BRCT-R1835X, and GST-BRCT-Y1853X were generated as previously described (18Magnard C. Bachelier R. Vincent A. Jaquinod M. Kieffer S. Lenoir G.M. Venezia N.D. Oncogene. 2002; 21: 6729-6739Crossref PubMed Scopus (80) Google Scholar). The pGEX-BRCT-N plasmid expressing the N-terminal region of BRCA1 was generated by PCR amplification of nucleotides 1–661 of BRCA1 using the cDNA of BRCA1 cloned in pCDNA3β as the template and the following primers: 5′-GCGAATTCATGGATTTATCTGCTCTTCG-3′ and 5′-GCGTCGACAGAATCCAAACTGATTTC-3′. The PCR product was cloned into the EcoRI/SalI sites of pGEX-4T-1 (Amersham Biosciences). The construct was verified by DNA sequencing. Mammalian Expression Constructs—The pCDNA3β plasmid expressing human BRCA1 full-length protein and the N-terminal hemagglutinin-tagged pCDNA3β plasmid expressing the trun-cated mutant Y1853X were previously described (24Scully R. Chen J. Plug A. Xiao Y. Weaver D. Feunteun J. Ashley T. Livingston D.M. Cell. 1997; 88: 265-275Abstract Full Text Full Text PDF PubMed Scopus (1313) Google Scholar). The pCDNA-PABP plasmid was generated by PCR amplification of PABP cDNA using the pGEM1-PABP construct as a template (25Grange T. de Sa C.M. Oddos J. Pictet R. Nucleic Acids Res. 1987; 15: 4771-4787Crossref PubMed Scopus (142) Google Scholar) and the primers 5′-GCTCTAGAGAGATGAACCCCAGTGCCCC-3′ and 5′-GAGCGGCCGCAACAGT'TGGAACACCGGTGGC-3′. The resultant product was inserted into the XbaI/NotI sites of pCDNA3.1/Myc-His (Invitrogen). To construct plasmids encoding PABP partial sequences, the PABP coding regions (numbers correspond to amino acids) RNA recognition motifs (RRMs) 1 and 2 (1–189), RRM3 and -4 (189–368), RRM1–4 (1–368), C1 (369–494), C2 (494–633), and C1+C2 (369–633) were PCR-amplified using pCDNA-PABP as template and the primers 5′-GCTCTAGAGAGATGAACCCCAGTGCCCC-3′ and 5′-GAGCGGCCGCTTCTTTTGCCCTAGCTCCAAG-3′, 5′-GCTCTAGAAACATGGAATTCACCAATGTTTACATCAAG-3′ and 5′-GAGCGGCCGCTTTGCGCTGAGCTAAAGCTAC-3′, 5′-GCTCTAGAGAGATGAACCCCAGTGCCCC-3′ and 5′-GAGCGGCCGCTTTGCGCTGAGCTAAAGCTAC-3′,5′-GCTCTAGAAACATGGAAGAGCGCCAGGCTCACC-3′ and 5′-GAGCGGCCGCTGCAGCTGCAGGACGTG-3′, 5′-GCTCTAGAAACATGGCAGCCGCTGCAGCTACTC-3′ and 5′-GAGCGGCCGCAACAGTTGGAACACCGGTGGC-3′,5′-GCTCTAGAAACATGGAAGAGCGCCAGGCTCACC-3′ and 5′-GAGCGGCCGCAACAGT'TGGAACACCGGTGGC-3′, respectively. The constructs were verified by DNA sequencing. The PABP-interacting protein 1 (PAIP1) sequence was PCR-amplified from human cDNA library using the primers 5′-GCTCTAGAAGCATGTCGGACGGTTTCG-3′ and 5′-GGGGTACCCTGTTTTCGCTTACGCTCTG-3′. The cDNA fragment was subcloned into the XbaI/KpnI sites of pCDNA3.1/Myc-His (Invitrogen) and sequenced. Renilla luciferase cDNA sequence was PCR-amplified from the pRL-CMV vector (Promega) using the primers 5′-CGGGATCCTACTTCGAAAGTTTATGATCCAG-3′ and 5′-TCCCCCGGGTTGTTCATTTTTGAGAACTCGC-3′, digested with BamHI and SmaI (PCR added restriction sites), and cloned into the multiple cloning region of the monocistronic plasmid derived from pGEM-2 (Promega), between the T7 promoter and poly(A) stretch. The structure of the resulting DNA construct, named PGEM-Renil, was verified by restriction enzyme digestion and sequencing. The luciferase sequence is under control of the T7 RNA polymerase promoter for in vitro transcription. Yeast Expression Constructs—The pGBT9-BRCT bait plasmid was generated by PCR amplification of the region spanning amino acids 1583–1812 of Brca1 using pUHD10.3-Brca1 construct (26Bachelier R. Dalla Venezia N. Mazoyer S. Frappart L. Lenoir G.M. Vincent A. Int. J. Cancer. 2000; 88: 519-524Crossref PubMed Scopus (11) Google Scholar) as a template and the primers 5′-GCGAATTCACATCTTCAGAAGAAAGAGC-3′ and 5′-GCGTCGACTTAATCATTGGAGTCTTGTGG-3′. The resultant product was inserted into the EcoRI/SalI sites of pGBT9 (Clontech) and fully sequenced. The L40 strain (Invitrogen) was cotransformed with pGBT9-BRCT and a Gal4 transactivation domain-tagged 11-day-old mouse embryo cDNA library (Clontech). Yeasts were plated on a Leu/Trp/His amino acid-depleted DOB medium containing 1 mm 3-aminotriazol. Transformants were tested for β-galactosidase activity using a yeast colony-filter assay. Positive (blue) colonies were isolated, and the β-galactosidase assay was repeated. Plasmids of these colonies were recovered and used to transform an Electromax DH10B bacterial strain. The prey plasmids amplified in bacteria were retransformed with pGBT9 or pGBT9-BRCT into L40 strain and analyzed by DNA sequencing. HBL100 human epithelial mammary cells were maintained in Dulbecco's modified Eagle's medium containing 1 g/liter glucose supplemented with 10% fetal calf serum, 100 μg/ml streptomycin, and 100 units/ml penicillin. Bosc human embryonic kidney cells were maintained in Dulbecco's modified Eagle's medium containing 4.5 g/liter glucose (or 1 g/liter glucose for metabolic labeling experiments) supplemented with 10% fetal calf serum, 100 μg/ml streptomycin, and 100 units/ml of penicillin. Bosc cells were plated at 4 × 106 cells per 10-cm-diameter dish 24 h before transfection. Cells were transfected with 4 μg of BRCA1 expressing vector (pCDNA3β) and 20 μl of ExGen 500 (Euromedex) following the supplier's procedure. Forty-eight hours after transfection cells were processed for immunoblotting or immunoprecipitation. For metabolic labeling experiments Bosc cells were plated at 4 × 105 cells per plate in a 6-well flask 24 h before transfection. Cells were transfected with 1 μg of pCDNA3.1 control plasmid or pCDNA3β plasmid expressing BRCA1 and 10 μl of ExGen 500 (Euromedex). Where pCDNA-PAIP1 plasmid was used, cells were transfected with 2 μg of plasmid and 20 μl of ExGen. Forty-eight hours after transfection cells were processed for metabolic labeling. Cells were lysed in lysis buffer A (20 mm Hepes-KOH pH 7.2, 100 mm KCl, 1 mm dithiothreitol, 0.5 mm EDTA, 0.5% Nonidet P-40, 10% glycerol) supplemented with protease inhibitor (Complete EDTA-free, Roche Applied Science). Protein concentrations were determined by the Bradford procedure (Bio-Rad). When immunoprecipitation was performed on cellular cytoplasmic and nuclear fractions, the fractions were separated as follows. Cells were pelleted, washed, and suspended in hypotonic buffer (10 mm HEPES, pH 8, 10 mm KCl, 1.5 mm MgCl2) supplemented with protease inhibitor (Complete EDTA-free, Roche Applied Science) for 15 min at 4 °C. An equal volume of hypotonic buffer containing 1% Nonidet P-40 was added and incubated an additional 5 min at 4 °C. After centrifugation (3000 rpm, 5 min), the supernatant was collected (cytoplasmic fraction), and the pellet was resuspended in hypertonic buffer (2 mm HEPES, pH 8, 450 mm KCl, 1.5 mm MgCl2, 25% glycerol) supplemented with protease inhibitor. After incubation for 30 min at 4 °C, the supernatant (nuclear fraction) was collected by centrifugation (13,000 rpm, 5 min). Lysates containing 2 mg of protein were precleared by stirring with 100 μl of protein G-Sepharose for 1 h at 4°C. After centrifugation immunoprecipitation was performed with the precleared lysate and 2 μg of antibody for 3 h at 4°C. Thirty microliters of protein G-Sepharose were added and incubated for 30 min at 4 °C. After centrifugation, beads were washed 3 times with lysis buffer A, and proteins were eluted by heating at 95 °C for 5 min in SDS-loading buffer and 100 mm dithiothreitol. Proteins were subjected to SDS-PAGE and blotted onto polyvinylidene difluoride membranes (Immobilon-P, Millipore). Membranes were blocked in Tris-buffered saline solution containing 0.05% Tween 20 and 5% nonfat milk and incubated with primary antibodies. Horseradish peroxidase-conjugated secondary antibodies (Amersham Biosciences) were used for detection of immunoreactive proteins by chemiluminescence (ECL, Amersham Biosciences). Cells were lysed in lysis buffer A supplemented with protease inhibitor (Complete EDTA-free, Roche Applied Science). One mg of protein was incubated for 1 h with 100 μl of 50% glutathione-Sepharose beads solution to eliminate nonspecific interactions. After a centrifugation of 2 min at 5000 rpm, supernatants were incubated with 5 μg of GST or GST-BRCT proteins and 50 μl glutathione-Sepharose beads for 3 h at 4°C. Protein complexes were washed three times with buffer A. Protein complexes were released by heating at 95 °C in SDS-loading buffer and 100 mm dithiothreitol. Proteins were analyzed by immunoblotting assay as described above. The presence of GST fusion proteins was examined by Coomassie Blue staining. Where indicated, lysates were treated with RNase I (0.1 mg/ml) for 1 hat room temperature. When the GST pulldown assay was performed on in vitro translated proteins, 2μg of GST or GST-BRCT proteins were incubated with 10 μl of [35S]methionine-labeled protein previously synthesized in a reticulocyte lysate-coupled transcription/translation system (Promega). Binding assays were carried out as above. Induced bacterial extracts containing GST, GST-BRCT, and GST-BRCA1-N (containing the N-terminal region of BRCA1) were subjected to SDS-PAGE and transferred to polyvinylidene difluoride membrane. Membranes were blocked in Tris-buffered saline solution containing 0.05% Tween 20 and 5% nonfat milk and incubated for 2 h with 40 μl of [35S]methionine-labeled in vitro translated protein in 3 ml of Tris-buffered saline solution containing 0.05% Tween 20 and 2% nonfat milk. Membranes were washed four times, dried, and radiographed. The siRNA duplexes were purchased from Proligo (Paris, France) and provided as a purified and annealed duplexes. The sequences of these siRNAs were Si-BRCA1-1 (5′-AAGGAACCUGUCUCCACAAAGUU-3′), Si-BRCA1-2) (5′-AAGGAACCACGAUCCACAAAGUU-3′), and Si-control (5′-AACACGAUGUGACAGUGAUAUUU-3′). The DNA sequences were subjected to a BLAST analysis. For transfection, HBL100 cells were plated at 4 × 105 cells per plate in a 6-well flask 24 h before transfection. Cells were transfected with 0.125 nmol of siRNA and 2 μl of Lipofectamine 2000 (Invitrogen) using the protocol of the supplier. 72 h after transfection cells were processed for immunoblotting or metabolic labeling. After transfection of the cells with siRNAs or plasmids, cells were incubated for 1 h with 3 ml of methionine-depleted medium. Then cells were pulse-labeled in 1 ml of methionine-depleted medium supplemented with 20 μCi of [35S] methionine for 1 h. Cells were washed with phosphate-buffered saline and harvested, and cytoplasmic extracts were prepared as follows. Cells were first treated in lysis buffer B (10 mm Hepes-KOH pH 8, 1.5 mm MgCl2, 10 mm KCl) supplemented with protease inhibitor (Complete EDTA free, Roche Applied Science) for 15 min and subsequently incubated with buffer B containing 0.5% Nonidet P-40 for 5 min. Cells were briefly centrifuged, and protein concentration was determined by Bradford procedure (Bio-Rad). 10–20 μg of protein were precipitated with 10% trichloroacetic acid. Precipitates were washed with 5% trichloroacetic acid, and radioactivity was determined by scintillation counting. Values were normalized for sample protein contents. When newly synthesized proteins were subjected to SDS-PAGE, cells were lysed in buffer A and analyzed (50 μg of protein) using 6% SDS-PAGE, and labeled proteins were visualized by autoradiography. For assessing total protein translation after treatment with CHX, cells were pretreated for 2 h with CHX (10 μg/ml) in Dulbecco's modified Eagle's medium, then starved, labeled (treatment with CHX continued during the methionine starvation and the radioactive pulse), and harvested as described above. For in vitro transcription DNA was linearized at the EcoRI site. Synthesis of capped transcripts was carried out with the bacteriophage T7 RNA polymerase as previously described (27Ohlmann T. Rau M. Morley S.J. Pain V.M. Nucleic Acids Res. 1995; 23: 334-340Crossref PubMed Scopus (79) Google Scholar). GTP concentration was reduced to 0.32 mm, and m7GpppG RNA capping analog (Invitrogen) was added at a concentration of 1.3 mm. The reaction was carried at 37 °C for 2 h. The resulting mRNAs were treated with DNase, purified from nonincorporated nucleotides by gel filtration chromatography with RNeasy kit (Qiagen) and precipitated by ethanol. The integrity of the RNAs was checked by electrophoresis on 1% agarose gels, and their concentrations were measured by spectrophotometry. Radiolabeled mRNAs were synthesized as described above in the presence of 10 μCi of [32P]UTP. For in vitro synthesis of unadenylated luciferase mRNAs, the plasmid DNA was linearized at the SmaI restriction site. Bosc cells were plated at 2 × 105 cells per plate in a 12-well flask 24 h before transfection. Cells were transfected with 1 μg of pCDNA3.1 control plasmid or pCDNA3β plasmid expressing BRCA1, Y1853X, or PAIP1 and 5 μl of ExGen 500 (Euromedex). Twenty-four hours after transfection, cells were transfected with 1 μg of in vitro synthesized RNA and 4 μl of DMRIE-C (Invitrogen). Cells were exposed to the RNA/DM-RIE-C mixture for 4 h, the medium was changed to fresh, serum-containing Dulbecco's modified Eagle's medium, and incubation was continued for an additional 6 h. Cell lysis and the analysis of translational products from transfected mRNA template were done using the Renilla luciferase assay (Promega). Values were normalized for samples protein contents. Cells were transfected with 1 μg of radiolabeled RNAs as described above. 6 h post-transfection, RNAs were isolated with TRIzol reagent (Invitrogen), and 5 μg of total RNAs were loaded on a guanidine thiocyanate-denaturing agarose gel as described (28Goda S.K. Minton N.P. Nucleic Acids Res. 1995; 23: 3357-3358Crossref PubMed Scopus (110) Google Scholar) and submitted to autoradiography. Results are expressed as the mean ± S.D. Statistical analysis was performed with a two-tailed paired Student's t test. Identification of PABP as a BRCA1 BRCT Tandem Interacting Protein—To identify proteins that interact with the tandem of two BRCT domains of BRCA1, we used the two-hybrid system. The mouse BRCA1 residues 1583–1812 were fused to the Gal4 DNA binding domain to generate the bait construct pGBT9-BRCT. The pGBT9-BRCT demonstrated a minimal transactivation activity, which was suppressed by 3-aminotriazol when used at a concentration of 1 mm. A screen of 106 transformants of an 11-day-old mouse embryo cDNA library (29Durfee T. Becherer K. Chen P.L. Yeh S.H. Yang Y. Kilburn A.E. Lee W.H. Elledge S.J. Genes Dev. 1993; 7: 555-569Crossref PubMed Scopus (1295) Google Scholar) yielded 273 His+ colonies. 115 β-galactosidase-positive clones were isolated. Sequence analysis of the interacting clones showed that one of them encoded amino acids 205–406 of Pabp. The protein sequence of the two BRCT domains of mouse Brca1 share 75 and 58% identity with those of human BRCA1, respectively (10Callebaut I. Mornon J.P. FEBS Lett. 1997; 400: 25-30Crossref PubMed Scopus (483) Google Scholar), and the protein sequence of mouse Pabp is highly conserved with that of human PABP. Therefore, we examined whether the interaction between BRCA1 and PABP was conserved in human cells. To test whether human full-length PABP directly binds to the BRCT domains of human BRCA1, a Far Western analysis was performed. The fusion proteins GST, GST-BRCT, and GST-BRCA1-N (containing the N-terminal region of BRCA1) were probed with [35S]methionine-labeled PABP. A specific hybridization was obtained with GST-BRCT only (Fig. 1A). As another approach, a GST pulldown assay was performed on Bosc cell lysates. The specific retention of PABP was observed with GST-BRCT but not with the control GST (Fig. 1B). Therefore, BRCT repeats of BRCA1 interact with full-length PABP in vitro. Because PABP associates with mRNA, it was possible that BRCA1 binds to PABP via RNA. Treatment of the cell lysates with RNase I before GST pulldown shows that the association is not mediated by RNA (Fig. 1B). To address the functional importance of the BRCT-PABP interaction, we investigated the effect of four germ line BRCA1 mutations located in BRCT domains on in vitro binding with PABP (Fig. 1C). GST pulldown assays were performed on Bosc cell lysates. As shown in Fig. 1D, the two single amino acid substitutions (A1708E and P1749R) and the two truncating mutations (R1835X and Y1853X) abolished the interaction with PABP. These results demonstrate that tumorigenic lesions of the BRCT domains prevent the association of BRCA1 with PABP, suggesting that the BRCA1-PABP interaction may be of physiologic relevance. Interaction of BRCA1 with PABP in Mammalian Cells—To determine whether BRCA1 could interact with PABP in human cells, lysates from Bosc cells transiently expressing BRCA1 were immunoprecipitated with anti-PABP antibody and further analyzed by immunoblotting using anti-BRCA1 antibody. As shown in Fig. 2A, BRCA1 coprecipitated with PABP. The negative control, an unrelated mouse monoclonal antibody anti-V5, was unable to coprecipitate BRCA1. Anti-PABP immunoprecipitation of lysates from Bosc cells transiently expressing the BRCA1 Y1853X mutant failed to coprecipitate BRCA1 (Fig. 2B). Western blot analysis confirmed that BRCA1 and the Y1853X mutant were expressed at equivalent levels after transfection (Figs. 2, A and B). This reaffirms the physiologic relevance of the interaction of BRCA1 with PABP via its BRCT tandem. To further examine whether endogenous BRCA1 and PABP form a complex, we performed coimmunoprecipitation analysis in untransfected epithelial mammary HBL100 cells. Immunoprecipitation of PABP resulted in the coprecipitation of BRCA1, whereas no bands were observed in negative control (Fig. 2C). In reciprocal experiments, anti-BRCA1 antibody coprecipitated PABP, but not the negative control (Fig. 2D). Therefore, BRCA1 and PABP form an endogenous complex in vivo. To determine whether BRCA1 and PABP interact in the nucleus or the cytoplasm, Bosc cells overexpressing BRCA1 were fractionated into nuclear and cytoplasmic fractions. The fractionation of these cells was checked with antibodies raised against nuclear P300 and cytoplasmic actin. An immunoprecipitation assay using anti-PABP antibody resulted in the coprecipitation of BRCA1 in the cytoplasmic fraction, whereas no band was observed in the nuclear fraction (Fig. 2E). An immunoprecipitation assay using an unrelated monoclonal antibody anti-V5 was used as control. Therefore, BRCA1 interacts with PABP in the cytoplasm of cells. PABP Associates with BRCA1 through Its RRM—To map the interaction domain of BRCA1 on PABP, GST pulldown assays were performed on [35S]methionine-labeled fragments of PABP. As shown in Fig. 3, the three N-terminal fragments RRM1 and -2, RRM3 and -4, and RRM1–4 clearly bound to the BRCT domains, whereas the C-terminal fragments C1, C2, and C1+C2 of PABP failed to bind. GST did not bind to any of these fragments, and GST-BRCT strongly bound to full-length PABP. It is noteworthy that the intensity of the signals obtained in Fig. 3C (RRM1–4, RRM1 and -2, and RRM3 and -4) are rather proportional to that of the radiolabeled products shown in Fig. 3B, suggesting that the BRCT domains interact with PABP through RRM1 and -2 and RRM3 and -4 with a nearly comparable efficiency. Therefore, BRCA1 interacts through its BRCT domains with the N-terminal region of PABP encompassing the four RRM motifs. This finding suggests that BRCA1 could participate in regulation of mRNA translation. Indeed, previous studies indicated that the RRM motifs of PABP interact with 3′ mRNA poly(A) tail (30Deo R.C. Bonanno J.B. Sonenberg N. Burley S.K. Cell. 1999; 98: 835-845Abstract Full Text Full Text PDF PubMed Scopus (403) Google Scholar), eukaryotic translation initiation factor 4G (eIF4G) (31Imataka H. Gradi A. Sonenberg N. EMBO J. 1998; 17: 7480-7489Crossref PubMed Scopus (464) Google Scholar), PAIP1 (32Craig A.W. Haghighat A. Yu A.T. Sonenberg N. Nature. 1998; 392: 520-523Crossref PubMed Scopus (324) Google Scholar), and PAIP2 (33Khaleghpour K. Svitkin Y.V. Craig A.W. DeMaria C.T. Deo R.C. Burley S.K. Sonenberg N. Mol. Cell. 2001; 7: 205-216Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar) and, therefore, are crucial
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