Isolation of XAB2 Complex Involved in Pre-mRNA Splicing, Transcription, and Transcription-coupled Repair
2007; Elsevier BV; Volume: 283; Issue: 2 Linguagem: Inglês
10.1074/jbc.m706647200
ISSN1083-351X
AutoresIsao Kuraoka, Shinsuke Ito, Tadashi Wada, Mika Hayashida, Lily Lee, Masafumi Saijo, Yoshimichi Nakatsu, Megumi Matsumoto, Tsukasa Matsunaga, Hiroshi Handa, Jun Qin, Yoshihiro Nakatani, Kiyoji Tanaka,
Tópico(s)CRISPR and Genetic Engineering
ResumoNucleotide excision repair is a versatile repair pathway that counteracts the deleterious effects of various DNA lesions. In nucleotide excision repair, there is a transcription-coupled repair (TCR) pathway that focuses on DNA damage that blocks RNA polymerase IIo in transcription elongation. XAB2 (XPA-binding protein 2), containing tetratricopeptide repeats, has been isolated by virtue of its ability to interact with xeroderma pigmentosum group A protein (XPA). Moreover, XAB2 has been shown to interact with Cockayne syndrome group A and B proteins (CSA and CSB) and RNA polymerase II, as well as XPA, and is involved in TCR and transcription. Here we purified XAB2 as a multimeric protein complex consisting of hAquarius, XAB2, hPRP19, CCDC16, hISY1, and PPIE, which are involved in pre-mRNA splicing. Knockdown of XAB2 with small interfering RNA in HeLa cells resulted in a hypersensitivity to killing by UV light and a decreased recovery of RNA synthesis after UV irradiation and regular RNA synthesis. Enhanced interaction of XAB2 with RNA polymerase IIo or XPA was observed in cells treated with DNA-damaging agents, indicating DNA damage-responsive activity of the XAB2 complex. These results indicated that the XAB2 complex is a multifunctional factor involved in pre-mRNA splicing, transcription, and TCR. Nucleotide excision repair is a versatile repair pathway that counteracts the deleterious effects of various DNA lesions. In nucleotide excision repair, there is a transcription-coupled repair (TCR) pathway that focuses on DNA damage that blocks RNA polymerase IIo in transcription elongation. XAB2 (XPA-binding protein 2), containing tetratricopeptide repeats, has been isolated by virtue of its ability to interact with xeroderma pigmentosum group A protein (XPA). Moreover, XAB2 has been shown to interact with Cockayne syndrome group A and B proteins (CSA and CSB) and RNA polymerase II, as well as XPA, and is involved in TCR and transcription. Here we purified XAB2 as a multimeric protein complex consisting of hAquarius, XAB2, hPRP19, CCDC16, hISY1, and PPIE, which are involved in pre-mRNA splicing. Knockdown of XAB2 with small interfering RNA in HeLa cells resulted in a hypersensitivity to killing by UV light and a decreased recovery of RNA synthesis after UV irradiation and regular RNA synthesis. Enhanced interaction of XAB2 with RNA polymerase IIo or XPA was observed in cells treated with DNA-damaging agents, indicating DNA damage-responsive activity of the XAB2 complex. These results indicated that the XAB2 complex is a multifunctional factor involved in pre-mRNA splicing, transcription, and TCR. DNA carrying genetic information is continuously exposed to exogenous and endogenous DNA-damaging agents. The DNA damage interferes with DNA replication, transcription, and cell cycle progression and leads to mutations and cell death, which may cause cancer, inborn diseases, and aging (1Hoeijmakers J.H. Nature. 2001; 411: 366-374Crossref PubMed Scopus (3137) Google Scholar, 2Lindahl T. Wood R.D. Science. 1999; 286: 1897-1905Crossref PubMed Scopus (1278) Google Scholar). However, a wide variety of DNA lesions, such as ultraviolet light-induced photo-lesions, intra-strand cross-links, and bulky adducts induced by various carcinogens and mutagens, are eliminated by nucleotide excision repair (NER) 5The abbreviations used are:NERnucleotide excision repairTCRtranscription-coupled repairXPAxeroderma pigmentosum group A proteinXPxeroderma pigmentosumsiRNAsmall interfering RNACSCockayne syndromeRNAP IIRNA polymerase IITFIIHtranscription factor IIHTPRtetratricopeptide repeatPMSFphenylmethylsulfonyl fluorideRTreverse transcriptionRSRNA synthesisRRSRNA synthesis after UV irradiationsnRNAsmall nuclear RNAGGRglobal genome repair. 5The abbreviations used are:NERnucleotide excision repairTCRtranscription-coupled repairXPAxeroderma pigmentosum group A proteinXPxeroderma pigmentosumsiRNAsmall interfering RNACSCockayne syndromeRNAP IIRNA polymerase IITFIIHtranscription factor IIHTPRtetratricopeptide repeatPMSFphenylmethylsulfonyl fluorideRTreverse transcriptionRSRNA synthesisRRSRNA synthesis after UV irradiationsnRNAsmall nuclear RNAGGRglobal genome repair. (3Wood R.D. Mitchell M. Sgouros J. Lindahl T. Science. 2001; 291: 1284-1289Crossref PubMed Scopus (1106) Google Scholar). NER is well conserved from Escherichia coli to mammals (4Sancar A. Annu. Rev. Biochem. 1996; 65: 43-81Crossref PubMed Scopus (962) Google Scholar, 5Prakash S. Prakash L. Mutat. Res. 2000; 451: 13-24Crossref PubMed Scopus (283) Google Scholar) and consists of the consecutive steps of damage recognition, dual incisions on either side of the damage, excision of 24–32 oligonucleotides containing the damage, gap filling by repair DNA synthesis using the error-free strand as a template, and ligation (6Araujo S.J. Tirode F. Coin F. Pospiech H. Syvaoja J.E. Stucki M. Hubscher U. Egly J.M. Wood R.D. Genes Dev. 2000; 14: 349-359Crossref PubMed Google Scholar, 7Guzder S.N. Habraken Y. Sung P. Prakash L. Prakash S. J. Biol. Chem. 1995; 270: 12973-12976Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). It has been shown that NER operates via the following two pathways: global genome repair (GGR) and transcription-coupled repair (TCR). GGR can act on DNA lesions at any location in the genome, whereas TCR is involved in a rapid removal of the lesions on the transcribed strand and a resumption of transcription (1Hoeijmakers J.H. Nature. 2001; 411: 366-374Crossref PubMed Scopus (3137) Google Scholar, 8Svejstrup J.Q. Nat. Rev. Mol. Cell Biol. 2002; 3: 21-29Crossref PubMed Scopus (306) Google Scholar). nucleotide excision repair transcription-coupled repair xeroderma pigmentosum group A protein xeroderma pigmentosum small interfering RNA Cockayne syndrome RNA polymerase II transcription factor IIH tetratricopeptide repeat phenylmethylsulfonyl fluoride reverse transcription RNA synthesis RNA synthesis after UV irradiation small nuclear RNA global genome repair. nucleotide excision repair transcription-coupled repair xeroderma pigmentosum group A protein xeroderma pigmentosum small interfering RNA Cockayne syndrome RNA polymerase II transcription factor IIH tetratricopeptide repeat phenylmethylsulfonyl fluoride reverse transcription RNA synthesis RNA synthesis after UV irradiation small nuclear RNA global genome repair. The biological importance of NER in humans has been suggested by studies of autosomal recessive human genetic disorders as follows: xeroderma pigmentosum (XP), Cockayne syndrome (CS), and trichothiodystrophy, in which NER activity is impaired (9Lehmann A.R. Biochimie (Paris). 2003; 85: 1101-1111Crossref PubMed Scopus (407) Google Scholar). XP patients are hypersensitive to sunlight and show an increased incidence of UV-induced skin cancers (9Lehmann A.R. Biochimie (Paris). 2003; 85: 1101-1111Crossref PubMed Scopus (407) Google Scholar). Although CS patients are sensitive to sunlight, they have no predisposition to sunlight-induced skin cancer but instead show severe developmental and neurological abnormalities as well as premature aging (10Nance M.A. Berry S.A. Am. J. Med. Genet. 1992; 42: 68-84Crossref PubMed Scopus (628) Google Scholar). Seven NER-deficient complementation groups have been identified in XP (XP-A to XP-G) and two in CS (CS-A and CS-B) (3Wood R.D. Mitchell M. Sgouros J. Lindahl T. Science. 2001; 291: 1284-1289Crossref PubMed Scopus (1106) Google Scholar). In addition, XP-B patients and certain patients with XPD or XPG show features of CS in addition to symptoms of XP (XP-B/CS, XP-D/CS, and XP-G/CS). Recently, we reported that XPG stabilizes TFIIH. Mutations in XPG found in cells of patients with XP-G/CS result in the dissociation of CAK and XPD from the core TFIIH. As a consequence, the phosphorylation and transactivation of nuclear receptors were disturbed in XP-G/CS cells. These results indicated that the features of CS in XP-G/CS are because of abnormal transcriptional activation by an unstable TFIIH (11Ito S. Kuraoka I. Chymkowitch P. Compe E. Takedachi A. Ishigami C. Coin F. Egly J.M. Tanaka K. Mol. Cell. 2007; 26: 231-243Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). In human cells, XPC/HR23B (12Sugasawa K. Ng J.M. Masutani C. Iwai S. van der Spek P.J. Eker A.P. Hanaoka F. Bootsma D. Hoeijmakers J.H. Mol. Cell. 1998; 2: 223-232Abstract Full Text Full Text PDF PubMed Scopus (742) Google Scholar) and UV-DDB (13Groisman R. Polanowska J. Kuraoka I. Sawada J. Saijo M. Drapkin R. Kisselev A.F. Tanaka K. Nakatani Y. Cell. 2003; 113: 357-367Abstract Full Text Full Text PDF PubMed Scopus (589) Google Scholar) are involved in the GGR-specific damage-recognition step, whereas in TCR, blockage of RNA polymerase II (RNAP II) at the DNA damage site on the transcribed strand is thought to trigger a TCR reaction (14Inukai N. Yamaguchi Y. Kuraoka I. Yamada T. Kamijo S. Kato J. Tanaka K. Handa H. J. Biol. Chem. 2004; 279: 8190-8195Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 15Mei Kwei J.S. Kuraoka I. Horibata K. Ubukata M. Kobatake E. Iwai S. Handa H. Tanaka K. Biochem. Biophys. Res. Commun. 2004; 320: 1133-1138Crossref PubMed Scopus (54) Google Scholar, 16Donahue B.A. Yin S. Taylor J.S. Reines D. Hanawalt P.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8502-8506Crossref PubMed Scopus (310) Google Scholar). Following the damage-recognition step in each pathway, NER factors, including transcription factor IIH (TFIIH), XPG, XPA, and replication protein A, are recruited to the lesion, leading to the local unwinding of the DNA double helix. TFIIH, including XPB and XPD helicases, plays a critical role in the formation of this open complex. Two structure-specific endonucleases, XPF-ERCC1 and XPG, subsequently introduce single-stranded breaks on the 5′ and 3′ sides of the lesion, respectively, leading to excision of the oligonucleotides containing the damage. We have performed yeast two-hybrid screening with XPA protein as bait and isolated a cDNA encoding a novel tetratricopeptide repeat (TPR) protein consisting of 855 amino acids and designated as XAB2 (XPA-binding protein 2) (17Nakatsu Y. Asahina H. Citterio E. Rademakers S. Vermeulen W. Kamiuchi S. Yeo J.P. Khaw M.C. Saijo M. Kodo N. Matsuda T. Hoeijmakers J.H.J. Tanaka K. J. Biol. Chem. 2000; 275: 34931-34937Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). It has been shown that XAB2 also interacts with the TCR-specific factors CSA, CSB, and RNA polymerase II and that the microinjection of anti-XAB2 antibodies into normal human cells specifically inhibited the recovery of RNA synthesis after UV irradiation as well as transcription but not UV-induced unscheduled DNA synthesis, and that XAB2 knock-out mice show preimplantation lethality (18Yonemasu R. Minami M. Nakatsu Y. Takeuchi M. Kuraoka I. Matsuda Y. Higashi Y. Kondoh H. Tanaka K. DNA Repair. 2005; 4: 479-491Crossref PubMed Scopus (31) Google Scholar). A recent in vivo cross-linking and chromatin immunoprecipitation study showed that XAB2 binds to RNA polymerase II in a CSA- and CSB-dependent manner (19Fousteri M. Vermeulen W. van Zeeland A.A. Mullenders L.H. Mol. Cell. 2006; 23: 471-482Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar). All these results have indicated that XAB2 is involved in TCR as well as transcription but not in GGR. It is suggested that the TPR domains play a role in intra- and inter-molecular protein interactions. Therefore, we purified XAB2 as a multimeric protein complex from extracts of HeLa cells expressing FLAG-XAB2 fusion protein using anti-FLAG antibody beads. The XAB2 complex consists of hAquarius (IBP160), XAB2 (hSYF1), hPRP19, CCDC16, hISY1, and PPIE, which are known to be involved in pre-mRNA splicing, and bound to RNA but not DNA, indicating that this complex functions in pre-mRNA splicing. The down-regulation of XAB2 expression in normal human cells resulted in hypersensitivity to killing by UV light, and it decreased the rate of recovery of RNA synthesis after UV irradiation, nascent RNA synthesis, and pre-mRNA splicing. Moreover, in the cells treated with DNA-damaging agents, the association of XAB2 with RNA polymerase IIo (elongation mode) and XPA was enhanced. The XAB2 complex is a multifunctional factor involved in transcription, pre-mRNA splicing, and TCR. Purification of XAB2 Complex—HeLa cells stably expressing N-terminally FLAG-tagged XAB2 cDNA were prepared as described previously (13Groisman R. Polanowska J. Kuraoka I. Sawada J. Saijo M. Drapkin R. Kisselev A.F. Tanaka K. Nakatani Y. Cell. 2003; 113: 357-367Abstract Full Text Full Text PDF PubMed Scopus (589) Google Scholar, 20Nakatani Y. Ogryzko V. Methods Enzymol. 2003; 370: 430-444Crossref PubMed Scopus (229) Google Scholar). FLAG-XAB2 complexes were purified by immunoprecipitation with anti-FLAG antibody from cell extracts. FLAG-XAB2-containing fractions were further purified using Superose 6 HR10/30 (Amersham Biosciences) with buffer A containing 20 mm Tris-HCl (pH 8.0), 150 mm NaCl, 10% glycerol, 0.1% Tween 20, 10 mm 2-mercaptoethanol, and 5 mm MgCl2. The fractions containing XAB2 complexes were collected and loaded onto a MiniQ column (Amersham Biosciences). The bound proteins were eluted with KCl buffer (50 mm to 1 m) containing 10 mm Tris-HCl (pH 8.0), 10% glycerol, 0.1% Tween 20, 10 mm 2-mercaptoethanol, and 5 mm MgCl2. The fractions containing the FLAG-XAB2 complex were pooled and dialyzed into buffer containing 20 mm Tris-HCl (pH 7.3), 100 mm KCl, 20% glycerol, 0.2 mm EDTA, 10 mm 2-mercaptoethanol, and 0.25 mm phenylmethylsulfonyl fluoride (PMSF). The FLAG-XAB2 complex was stored at –80 °C. Mass Spectrometric Analysis—The identification of proteins with mass spectrometry was conducted as described previously (21Wang Y. Cortez D. Yazdi P. Neff N. Elledge S.J. Qin J. Genes Dev. 2000; 14: 927-939Crossref PubMed Scopus (95) Google Scholar). Antibodies—Anti-XAB2, anti-hPrp19, and anti-CCDC16 rabbit polyclonal antibodies were raised against peptides corresponding to amino acids 811–838 of XAB2, to amino acids 182–208 of hPrp19, and to amino acids 93–115 of CCDC16, respectively. Anti-hAquarius, hISY1, and PPIE rabbit antibodies were raised against the recombinant His-hAquarius (amino acids 1308–1421), GST-hISY1, and GST-PPIE proteins (amino acids 67–164), respectively. These antibodies were affinity-purified using a protein A-Sepharose column. Anti-FLAG monoclonal antibody (M2) was purchased from Sigma. Anti-RNAP II polyclonal antibodies (C-21 and N-20) were from Santa Cruz Biotechnology. Anti-RNAP II monoclonal antibodies (8WG16 and H5) were from Berkeley Antibody. Immunoprecipitation and GST Pulldown Assay—For the coimmunoprecipitation analysis, extracts of HeLa cells stably expressing FLAG-tagged XAB2 protein were prepared as described previously (13Groisman R. Polanowska J. Kuraoka I. Sawada J. Saijo M. Drapkin R. Kisselev A.F. Tanaka K. Nakatani Y. Cell. 2003; 113: 357-367Abstract Full Text Full Text PDF PubMed Scopus (589) Google Scholar, 17Nakatsu Y. Asahina H. Citterio E. Rademakers S. Vermeulen W. Kamiuchi S. Yeo J.P. Khaw M.C. Saijo M. Kodo N. Matsuda T. Hoeijmakers J.H.J. Tanaka K. J. Biol. Chem. 2000; 275: 34931-34937Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). The extracts (500 μl) were incubated with either anti-PPIE, hISY1, CCDC16, hPRP19, or hAquarius antibody (1 μg) at 4 °C for 2 h, and the mixtures were further incubated with protein G-Sepharose beads (Amersham Biosciences) for precipitation at 4 °C for 2 h. After the beads had been washed with NETN buffer (50 mm Tris-HCl (pH 7.8), 150 mm NaCl, 1 mm EDTA, 1% Nonidet P-40, 1 mm PMSF, 10 mm 2-mercaptoethanol, and a protease inhibitor mixture (complete, Roche Diagnostics)), the proteins bound to the beads were eluted by boiling them in SDS sample buffer. For the co-immunoprecipitation experiments in Fig. 1E, lane 8, and Fig. 2, B and C, HeLa cells (8 × 105) were transfected with FLAG-tagged hPRP19 or a series of FLAG-tagged XAB2 in the expression vector pCAGGS (3 μg) using PolyFect (Qiagen) according to the manufacturer's instructions. Twenty four hours after transfection, cells were collected and washed twice with phosphate-buffered saline (PBS). Cell extracts were prepared as described previously (13Groisman R. Polanowska J. Kuraoka I. Sawada J. Saijo M. Drapkin R. Kisselev A.F. Tanaka K. Nakatani Y. Cell. 2003; 113: 357-367Abstract Full Text Full Text PDF PubMed Scopus (589) Google Scholar, 17Nakatsu Y. Asahina H. Citterio E. Rademakers S. Vermeulen W. Kamiuchi S. Yeo J.P. Khaw M.C. Saijo M. Kodo N. Matsuda T. Hoeijmakers J.H.J. Tanaka K. J. Biol. Chem. 2000; 275: 34931-34937Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). The extracts were incubated with anti-FLAG antibody (M2)-conjugated agarose (Sigma) and rotated at 4 °C for 2 h. After the agarose had been washed with NETN buffer, bound proteins were eluted with NETN buffer containing 0.5 mg/ml FLAG peptide (Sigma).FIGURE 2Regions of XAB2 and PPIE needed for interactions with other subunits.A, schematic representation of a series of deletion mutants of FLAG-XAB2. For example, FLAG-XAB2-(1–734) has a deletion in the C-terminal region encompassing amino acid residues 735–855. B, production of deletion mutants of FLAG-XAB2. FLAG-XAB2 cDNAs with various deletions were expressed in HeLa cells, and the mutant FLAG-XAB2 proteins were detected by immunoblotting using anti-FLAG antibody (M2). The band indicated with an asterisk is a nonspecific band. C, interactions of various mutants of XAB2 with other subunits. The proteins that bound the mutant FLAG-XAB2 were analyzed by immunoblotting using anti-PPIE (diluted 1/5000), anti-hISY1 (1/1000), anti-CCDC16 (1/5000), anti-hPRP19 (1/10,000), and anti-hAquarius (1/5000). D, schematic representation of GST-PPIE with various deletions. A gray box and a black box in PPIE indicate an RNA recognition motif and a peptidy-prolyl cis-trans isomerase (Pro_isomerase) domain, respectively. For example, GST-PPIE-(67–164) has deletions in the PPIE region encompassing amino acid residues 1–66 and 165–301. The XAB2-binding region in PPIE is shown according to the results in E. E, in vitro pulldown of XAB2 with GST-PPIE. In vitro translated XAB2 was used for the GST-PPIE pulldown assay. The bound proteins were analyzed by immunoblotting with anti-XAB2 antibodies. As a control (lane 1), 1/25 of the in vitro translated XAB2 protein used for the binding assay was loaded onto the SDS-polyacrylamide gel.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In Fig. 5A, FLAG-tagged XAB2 complexes were incubated with RNA polymerase II at 4 °C for 3 h and then rotated at 4 °C for 2 h with either anti-RNA polymerase II beads (C-21) (Santa Cruz Biotechnology) or anti-FLAG M2 beads (Sigma). After the beads had been washed with NETN buffer, the bound proteins were eluted by boiling in SDS sample buffer. In Fig. 5B, extracts of HeLa cells expressing FLAG-tagged XAB2 protein were incubated with anti-FLAG M2-agarose and rotated at 4 °C for 2 h. After the agarose had been washed with NETN buffer, the bound proteins were eluted with NETN buffer containing 0.5 mg/ml FLAG peptide (Sigma). For the co-immunoprecipitation analysis in Fig. 5F, 293 cells stably expressing N-terminally 3xFLAG-His-tagged XPA were prepared using a pcDNA5/FRT/V5-His TOPO TA expression kit (Invitrogen) according to the manufacturer's instructions. HeLa cells stably expressing FLAG-His-tagged RPB3 of RNA polymerase II were prepared as described previously (22Hasegawa J. Endou M. Narita T. Yamada T. Yamaguchi Y. Wada T. Handa H. J. Biochem. (Tokyo). 2003; 133: 133-138Crossref PubMed Scopus (9) Google Scholar). Cell extracts were prepared using NTN buffer (50 mm Tris-HCl (pH 7.8), 150 mm NaCl, 1% Nonidet P-40, 1 mm PMSF, 10 mm 2-mercaptoethanol, and a protease inhibitor mixture (Nacalai)). After incubation at 4 °C for 15 min, the lysates were centrifuged at 15,000 rpm for 30 min. The resulting supernatants were incubated with nickel-agarose (Qiagen) and rotated at 4 °C for 2 h. After the agarose had been washed with NTN buffer, proteins were eluted with NTN buffer containing 20 mm imidazole. The samples were incubated with anti-FLAG antibody (M2)-conjugated agarose (Sigma) and rotated at 4 °C for 1 h. After the agarose had been washed with NETN buffer, proteins were eluted with NETN buffer containing 0.5 mg/ml of FLAG peptide for RNAP II or 3xFLAG peptide (Sigma) for XPA. The GST-pulldown assay was done as described (17Nakatsu Y. Asahina H. Citterio E. Rademakers S. Vermeulen W. Kamiuchi S. Yeo J.P. Khaw M.C. Saijo M. Kodo N. Matsuda T. Hoeijmakers J.H.J. Tanaka K. J. Biol. Chem. 2000; 275: 34931-34937Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar, 23Nagai A. Saijo M. Kuraoka I. Matsuda T. Kodo N. Nakatsu Y. Mimaki T. Mino M. Biggerstaff M. Wood R.D. Seijbers A. Hoeijmakers J.H.J. Tanaka K. Biochem. Biophys. Res. Commun. 1995; 211: 960-966Crossref PubMed Scopus (54) Google Scholar). Construction of Truncated XAB2 and PPIE Expression Plasmids—Expression constructs of the FLAG-tagged XAB2 cDNA with a C-terminal deletion in the mammalian expression vector pCAGGS were generated with the QuickChange mutagenesis kit (Stratagene) using oligonucleotides that contain a stop codon at the indicated sites. The truncation mutant of XAB2 (1–20 and 578–855) was made by restriction digestion of the full-length XAB2 cDNA with PpuMI. The N-terminal and C-terminal deletion mutants of XAB2 cDNA were generated by a PCR-based method. GST-PPIE proteins with various deletions were made as follows. PPIE cDNAs (3–301 and 67–164) were amplified by RT-PCR. PPIE cDNAs (3–81, 83–236, and 238–301) were obtained by restriction digestion of PPIE cDNAs (3–301) with MscI, and PPIE cDNAs (3–152 and 152–301) by restriction digestion with NaeI. These PPIE cDNAs were inserted into pGEX-6p (Amersham Biosciences). All the constructs were checked by DNA sequencing. Gel Mobility Shift Assays—DNA probes (202 bp) for gel mobility shift assays were generated by PCR amplification of pBluescript SK(–) (Stratagene) using the primers (reverse primer) 5′-GGAAACAGCTATGACCATG-3′ and (M13–20 primer) 5′-GTAAAACGACGGCCAGT-3′ in the presence of an NTP mixture containing [α-32P]CTP (Amersham Biosciences). RNA probes were synthesized from the DNA probe using T7 RNA polymerase (Toyoba) in the presence of an rNTP mixture containing [α-32P]UTP (Amersham Biosciences). Both probes were purified in a nondenatured 6% polyacrylamide gel. RNA and DNA competitors for the gel mobility shift competition assays were generated by the same methods in the presence of cold rNTP and NTP mixtures, respectively. The XAB2 complex was incubated with the RNA or DNA probe in binding buffer (10 μl) containing 10 mm Tris-HCl (pH 8.0), 50 mm KCl, 10% glycerol, 0.5 mm EDTA, 0.5 mm dithiothreitol, and 6 mm MgCl2 for 30 min at 4 °C. After the incubation, the binding reaction mixtures were separated on a nondenatured 6% polyacrylamide gel in 0.5× TBE buffer. The dried gels were then analyzed with a FUJIFILM BAS 2500 bio-image analyzer. For the supershift assays using anti-FLAG antibody, the reaction mixtures were incubated with 2.45 μg of monoclonal anti-FLAG antibody for 5 min at 4 °C before being loaded onto the polyacrylamide gel. For RNA binding assays, the XAB2 complex was incubated with either glutathione-Sepharose, poly(A)-Sepharose, or poly(U)-Sepharose (Amersham Biosciences) in binding buffer containing 10 mm Tris-HCl (pH 8.0), 50 mm KCl, 10% glycerol, 0.5 mm EDTA, 0.5 mm dithiothreitol, 6 mm MgCl2, and 100 μg/ml bovine serum albumin for 30 min at 4 °C. After the incubation, unbound XAB2 complex was removed by washing the Sepharose beads three times with NETN buffer containing 100 μg/ml bovine serum albumin. Bound fractions were then separated by SDS-PAGE and analyzed by Western blotting using antibodies against each subunit of the XAB2 complex. Knockdown of XAB2 by siRNA—The XAB2 siRNA duplex mixtures used in the experiment in Fig. 4A were purchased from Dharmacon. The siRNAs were transfected into HeLa cells using Oligofectamine (Invitrogen) according to the manufacturer's instructions. Cy3-labeled Luciferase GL2 duplex (Dharmacon) was employed as control siRNA. The XAB2 siRNAs used in the experiment in Fig. 4D were purchased from Dharmacon. The XAB2 target sequences are as follows: XAB2 siRNA-1 (5′-AAGCCCAGGCUCAAUCAGCUAUA-3′) and XAB2 siRNA-2 (5′-AACAACUGUCAUGAGAGGGCCUU-3′). All the siRNA experiments were carried out 48 h after transfection. RT-PCR Assay for Pre-mRNA Splicing—RNA was extracted from siRNA-transfected cells with an RNA purification kit (Qiagen) and then treated with RNase-free DNase I (Takara) as recommended by the supplier. Purified RNAs were employed to synthesize PCR products with a cMaster RT-PCR system and RT kit (Eppendorf), and primers for the amplification of Bcl-x cDNA were described previously (31Mercatante D.R. Bortner C.D. Cidlowski J.A. Kole R. J. Biol. Chem. 2001; 276: 16411-16417Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 32Mercatante D.R. Kole R. Biochim. Biophys. Acta. 2002; 1587: 126-132Crossref PubMed Scopus (38) Google Scholar). Recovery of RNA Synthesis after UV Irradiation and Nascent RNA Synthesis—Cells (2 × 105) were inoculated into 35-mm Petri dishes. After a 24-h incubation, cells were washed with PBS and treated with UV irradiation at 10 J/m2. After another 24-h incubation, cells were washed with PBS and labeled for 1 h in Dulbecco's modified Eagle's medium containing [3H]uridine (10 μCi/ml) to quantify the recovery of RNA synthesis after UV irradiation. After the labeling with [3H]uridine, 20 μl of 10 mg/ml sodium azide (NaN3) was added to 1 ml of cell culture, and the cells were washed twice with PBS containing NaN3 (200 μg/ml). The cells were suspended in 200 μl of sterilized water and then frozen. After the cells were thawed at room temperature, 400 μl of 1.2% SDS was added to the cell suspension, which was then incubated for 30 min at room temperature. After the addition of 400 μl of water, the cell lysates were collected into sample tubes and incubated for 30 min on ice. One milliliter of 10% trichloroacetic acid, 0.1 m sodium pyrophosphate (NaPPi) was added into each sample tube and mixed. After incubation for 30 min on ice, these mixtures were transferred onto GF/C glass microfiber filters (Whatman). The filters were washed with 5% trichloroacetic acid, 0.05 m NaPPi, then with ethanol, and dried. The filters were transferred into vials, and scintillation solution was added. Radioactivity was measured with an LS 6500 liquid scintillation counter (Beckman Coulter). The radioactivity of the unirradiated cells was indicated as RNA synthesis (RS), whereas the percentage of radioactivity in the UV-irradiated cells divided by that in the unirradiated cells was indicated as recovery of RNA synthesis after UV irradiation (RRS). Immunofluorescence Microscopy—Cells grown on coverslips were washed with PBS and fixed with PBS containing 0.2% Triton X-100 and 2% formaldehyde at room temperature for 10 min and then incubated in acetone for 5 min at –20 °C. The samples were blocked with PBS containing 5% normal goat serum at room temperature for 30 min. Cells were washed three times with PBS for 5 min, and incubated with anti-XAB2 antibody or antibody indicated in the legends for Fig. 5 in blocking buffer at room temperature for 1 h. Cells were then washed three times with PBS for 5 min, incubated with Alexa Fluor-conjugated antibody (diluted 1:500; Molecular Probes) in PBS at room temperature for 1 h, and washed three times with PBS. The samples were examined with a MRC-1024 fluorescence microscopy system (Bio-Rad). Colony-forming Assay—Cells were inoculated into 10-cm dishes at a density of 1000 cells per dish. After a 6-h incubation, cells were washed with PBS and UV-irradiated at 0, 4, and 12 J/m2. The cells were then incubated for 2–3 weeks. The colonies that formed were fixed with 10% formalin, stained with 0.1% crystal violet, and counted under a binocular microscope. Purification of XAB2 Protein Complex—XAB2 is a TPR protein of 855 amino acids containing three stretches of acidic residues (Fig. 1A). Mutational and structural studies of some TPR proteins suggested that TPR domains play a role in intra- and inter-molecular protein interactions (24Das A.K. Cohen P.W. Barford D. EMBO J. 1998; 17: 1192-1199Crossref PubMed Scopus (709) Google Scholar, 25Lamb J.R. Tugendreich S. Hieter P. Trends Biochem. Sci. 1995; 20: 257-259Abstract Full Text PDF PubMed Scopus (550) Google Scholar). Consistent with these reports, our gel filtration analysis revealed that XAB2 is included in the ∼0.5-MDa fraction (data not shown). Therefore, we purified XAB2 as a multimeric protein complex from nuclear extract of HeLa cells stably expressing FLAG-XAB2 fusion protein. First, the FLAG-XAB2 complex was isolated by immunoprecipitation with anti-FLAG antibody. The protein complex was further purified by gel filtration and MiniQ column chromatography (Fig. 1B). Silver staining of the main peak fractions of Superose 6 indicated that XAB2 was co-migrated with p160, p57, p50, p42, and p35 (Fig. 1C, fractions 12–14). Silver staining of the peak fraction of MiniQ revealed that XAB2 is part of a protein complex consisting of six subunits (Fig. 1D, left panel). Mass spectrometric analysis of these subunits revealed that they are hAquarius (IBP160: p160), XAB2 (hSYF1: p100), hPRP19 (hPSO4: p57), CCDC16 (p50), hISY1 (p42), and peptidyl-prolyl cis-trans isomerase E (PPIE: p35) (Table 1). The hAquarius is an intron-binding protein (IBP160), which has been reported as the key factor
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