Báo cáo Y học: LRP130, a protein containing nine pentatricopeptide repeat motifs, interacts with a single-stranded cytosine-rich sequence of mouse hypervariable minisatellite Pc-1 docx

LRP130, a protein containing nine pentatricopeptide repeat motifs, interacts with a single-stranded cytosine-rich sequence of mouse hypervariable minisatellite Pc-1 Naoto Tsuchiya, Hirokazu Fukuda, Takashi Sugimura, Minako Nagao and Hitoshi Nakagama Biochemistry Division, National Cancer Center Research Institute, Chuo-ku, Tokyo, Japan Recently, we have identified and purified minisatellite DNA binding proteins (MNBPs) that bind to the mouse hyper- variable minisatellite Pc-1, from NIH3T3 cells. This study describes the isolation and characterization of a mouse leu- cine-rich protein (mLRP130) as one of the MNBPs that binds to the C-rich strand of Pc-1. The mLRP130 cDNA was demonstrated to encode a polypeptide of 1306 amino- acid residues with a deduced molecular mass of 137 kDa, and the mLRP130 mRNA is detected in various organs, including heart, brain, liver, skeletal muscle, kidneys and testes. The mLRP130 protein has nine copies of pentatrico- peptide repeat (PPR) motifs that are considered to serve as protein–protein interactions. Two forms of the mLRP130 protein were detected in NIH3T3 cells with an approximate molecular mass of 140 kDa (mLRP130) and 100 kDa (mLRP130 der ), and were detected mainly in nuclear and cytoplasmic fractions, respectively. Immunofluorescence microscopic analysis demonstrated dominant localization of mLRP130 at the perinuclear region, and also in the nucleus and cytoplasm with dot- or squiggle-like staining. The immunoprecipitated mLRP130 bound to the single- stranded d(CTGCC) 8 , but not to its complementary G-rich strand of d(GGCAG) 8 or double-stranded form. Possible biological roles of mLRP130 are discussed in association with the stability of minisatellite DNA sequences. Keywords: minisatellite DNA; C-rich sequence; mLRP130; PPR motif. Vertebrate genomes contain hypervariable tandem repeats with short nucleotides (5–100 bp), called a minisatellite (MN) or variable number of tandem repeat (VNTR) [1,2]. MNs are known to be recombination hot spots in meiotic cells, but generally stable in somatic cells [3]. However, alterations of repeat numbers of MNs, MN mutations, were frequently found in several types of tumors in humans [4–7] and in experimental animals [8,9], and also in cultured cells after treatment with various carcinogens, or c- and UV- irradiations [10–12]. Several MNs have been shown to be implicated in predisposition to human disorders. The insulin-VNTR, which is located at the 5¢ region of the insulin gene (INS), is suggested to be associated with insulin- dependent diabetes mellitus (IDDM) [13,14]. The expression of the HRAS1 gene was also affected by the polymorphism of Ha-ras-VNTR, which lies in the 3¢ downstream region of the gene, whose mutant alleles are considered to be involved in the increased risk of cancers [15–18]. Mouse MN Pc-1 is composed of tandem repeats of d(GGCAG) with locus-specific flanking sequences, and the mutation rate of the Pc-1 in meiotic cells was 15% per gamete, while that in somatic cells was 2–3% per population doubling, indicating that the Pc-1 locus is maintained relatively stable in somatic cells [19,20]. By means of DNA fingerprint analysis using the mouse MN Pc-1 fragment as a probe, we previously demonstrated that MN sequences become highly unstable in NIH3T3 cells by treatment with the tumor promoter, okadaic acid, a specific inhibitor of serine/threonine protein phosphatases, and also in fibroblast derived from severe combined immunodeficiency (SCID) mice, in which a catalytic subunit of DNA-dependent protein kinase (DNA-PKs) is impaired [21,22]. Although the precise mechanisms for the induction of MN mutations still remains unresolved, these findings led us to the possibility that maintenance of MN integrity is partly regulated by DNA-PK, and changes in the phosphorylation status in cellular proteins could cause the induction of MN mutations at Pc-1 or other related loci with repetitive sequences similar to Pc-1. In order to gain further insights into the molecular mechanisms underlying the induction of MN mutations, we attempted to isolate MN Pc-1 binding proteins, (MNBPs) from NIH3T3 cells treated with okadaic acid, and six MNBPs, MNBP-pA, -pB, -pD, -pE, -pF and -pG, have been isolated [23]. pA and pB bound to the G-rich strand of the MN Pc-1, d(GGCAG) 8 , which forms an intramolecular tetraplex structure and causes DNA synthesis arrest in vitro [24]. Four other proteins, pD, pE, pF and pG, bound to its complementary C-rich strand [23]. These MNBPs were Correspondence to H. Nakagama, Biochemistry Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo, 104-0045, Japan. Fax: + 81 3 3542 2530, Tel.: + 81 3 3547 5239, E-mail: hnakagam@gan2.res.ncc.go.jp Abbreviations: MNBP, minisatellite DNA binding protein; mLRP, mouse leucine-rich protein; PPR, pentatricopeptide repeat; TPR, tetratricopeptide repeat; MN, minisatellite; VNTR, variable number of tandem repeat; IDDM, insulin-dependent diabetes mellitus; SCID, severe combined immunodeficiency; DNA-PKc, DNA-dependent protein kinase; DMEM, Dulbecco’s modified Eagle’s medium; WCL, whole cell lysate; FITC, fluorescein isothiocyanate; EMSA, electro- phoretic mobility shift assay; PPR, pentatricopeptide repeat. (Received 22 February 2002, accepted 29 April 2002) Eur. J. Biochem. 269, 2927–2933 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02966.x different from previously isolated minisatellite binding proteins, MSBPs, in their molecular sizes and DNA binding properties [25–27]. In this study, isolation and character- ization of a cDNA for one of the C-rich strand binding MNBP, pE, were carried out. Nucleotide and amino-acid sequence analyses demonstrated that pE is a partial fragment of the mouse homologue of human leucine-rich protein 130 (mLRP130). Another C-rich strand binding MNBP, pF, was also revealed to be the mLRP130. Human LRP130 was initially isolated as a highly expressed transcript from the hepatoblastoma cell line, HepG2 [28]. However, its biological functions, the expression profile of its mRNA, are still uncharacterized. In this study, mLRP130 was demonstrated to bind to the C-rich single- stranded DNA sequences in a sequence-specific manner, and its biological roles in association with the induction of MN mutations were discussed. MATERIALS AND METHODS Cloning of a cDNA encoding mLRP130 MNBPs were purified from 2 · 10 9 NIH3T3 cells treated with 7.5 n M okadaic acid, as described previously [23]. The resulting partial polypeptide sequences were determined according to the methods described by Shinohara et al.[29]. Five polypeptide sequences of C-rich binding MNBP-pE were determined by Edman degradation under a contract with APRO Science Co. Ltd (Tokushima, Japan). Database searches enabled us to determine that pE could be a partial fragment of the mouse homologue of the human leucine- rich protein 130, LRP130. Polypeptide sequences of another C-rich binding MNBP also revealed that pF is a homologue of LRP130. Therefore, pE and pF were considered to be encoded by the same gene, and isolation of the full-length cDNA for pE was carried out as follows. Total RNAs from NIH3T3 cells were prepared by the method of Chomczynski & Sacchi using Torizol reagent (Gibco BRL) [30]. One microgram of total RNA was annealed with an oligo(dT) primer and the first strand cDNAs were synthesized with avian myeblastoma virus (AMV) reverse transcriptase (Takara Biomedicals) at 45 °C for 60 min. Partial cDNA fragments for the mLRP130 were amplified with a set of degenerated primers, 5¢-TAT/CTTT/CCAT/CCAA/GT/ CTNC/AGNGA-3¢ and 5¢-GCA/GTCNGCNCG/TT/CT GCCAA/GTC-3¢, sequences designed from the identified polypeptide sequences of pF. PCR products were subcloned into a pCRII (Invitrogen) vector and sequenced. A 1.2-kbp cDNA fragment, pE Cl 2, had an open reading frame that included three of five polypeptide sequences determined from purified pE and two partial peptide sequences determined from pF. To isolate a full-length mLRP130 cDNA, 2 · 10 6 plaques of an NIH3T3 cDNA library in kZAP II (Stratagene) were screened by plaque hybridiza- tion [31] using pE Cl 2 as a probe. A probe was labeled with [a- 32 P]dCTP (Amersham Pharmacia Biotech) by random primer labeling as described by Feinberg & Vogelstein [32]. Isolated cDNA clones were subcloned into pBluescript (Stratagene). Sequence reactions were performed using a laser dye-labeled primer and Thermo Sequenase DNA polymerase (Amersham Pharmacia Biotech) by the chain termination method [31], and nucleotide sequences were determined on an Li-COR DNA Sequencer (Model 4200). Alignment of multiple nucleotide sequences and determin- ation of the consensus sequence were achieved with GENE- WORKS software (Intelligenetics). A homology search of the databases was performed by the BLAST program [33]. Northern blot analysis Multiple Tissue Northern Blots (Clontech) containing 2 lg of poly(A) + RNA per lane were hybridized with 32 P-labeled mLRP130 cDNA fragments in a buffer consisting of 6 · NaCl/P i /EDTA (1 · NaCl/P i /EDTA consists of 150 m M NaCl, 10 m M NaH 2 PO 4 and 1 m M EDTA, pH 7.4), 50% formamide, 5 · Denhardt’s solution, 0.2% SDS, 100 lgÆmL )1 of sonicated and heat-denatured salmon sperm DNA, at 42 °C for 12 h. cDNA fragments of mLRP130 of both 5¢ (nucleotides 1–2374), and 3¢ regions (nucleotides 3174–4388) were labeled as described above. After hybridization, membranes were washed twice with 2 · NaCl/Cit/0.1% SDS (1 · NaCl/Cit is 15 m M sodium citrate and 150 m M NaCl) for 15 min at room temperature, then with 0.1 · NaCl/Cit/0.1% SDS twice for 30 min at 60 °C, and exposed to X-ray films at )80 °C. Preparation of polyclonal antibodies Polyclonal antibodies for both N- and C-terminal regions of mLRP130 were generated by immunizing rabbits with syn- thetic peptides selected from deduced amino-acid sequence. Peptide sequences used for generation of antibodies were as follows. F-N peptide: VYLQNEYKFSPTDFLAK (amino acids 84–100), corresponding to an E-1 peptide fragment, and F-C peptide: TAKNLKLDDLFLKRYA (amino acids 1258–1273). The IgG fractions were obtained using protein G Sepharose from the antisera, and were purified by Sepharose column coupled with each synthetic peptide used as the immunogen. Eluates were used as aF-N and aF-C antibodies against N- and C-terminal regions, respectively. Immunoblot analysis Immunoblot analysis was carried out as described previ- ously [34]. Briefly, cell extracts were loaded and separated on a 7.5% SDS/polyacrylamide gel, and then transferred to an immobilon P membrane (Millipore). The membrane was washed with NaCl/Tris, and incubated with 5% nonfat skim milk/NaCl/Tris/0.05% Tween 20 (NaCl/Tris/Tween) for 60 min at 37 °C. The membrane was incubated with aF-N or aF-C for 12 h at 4 °C. After incubation, the membrane was washed three times with NaCl/Tris/Tween and incubated with horseradish peroxidase-conjugated anti- (rabbit IgG) Ig for 2 h at room temperature. The protein bands were detected with an ECL plus kit (Amersham Pharmacia Biotech). Cell fractionation Approximately 1 · 10 6 NIH3T3 cells, which were propa- gated on 10-cm Petri dishes in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactiva- ted fetal bovine serum, were washed with ice-cold NaCl/P i twice, detached by adding 0.25% trypsin, and collected by centrifugation at 650 g for 10 min. Collected cells were 2928 N. Tsuchiya et al. (Eur. J. Biochem. 269) Ó FEBS 2002 washed with NaCl/P i three times, suspended in a 5 cell-pack vol. of hypotonic buffer, consisting of 10 m M Hepes (pH 7.4), 5 m M KCl, 1.5 m M MgCl 2 ,1m M dithiothreitol, 0.2 l M phenylmethanesulfonyl fluoride, 3 lgÆmL )1 leupep- tin and protease inhibitor cocktail tablets (Roche Molecular Biochemicals) and centrifuged as above. Final cell pellets were resuspended in the same volume of hypotonic buffer and homogenized with a Dounce homogenizer. These homogenates were used as a whole cell lysate (WCL), and cytoplasmic and nuclear fractions were further separated by centrifuging the WCL at 1000 g for 10 min The supernatant was used as the cytoplasmic fraction, and nuclear pellets were washed three times with ice-cold NaCl/P i to remove the residual cytoplasmic proteins. An anti-(a-tubulin) Ig (ICN Biomedicals) was used to visualize a-tubulin, which is a marker for the cytoplasmic fraction. Indirect immunofluorescence microscopy NIH3T3 cells grown on a collagen type I coated dish (Aashi Techno Glass) were washed with NaCl/P i and fixed with prechilled methanol at )20 °C for 20 min. Cells were first treated with an antibody, aF-N or normal rabbit IgG, then incubated at room temperature for 60 min, and further incubated with fluorescein isothiocyanate (FITC)-conjugat- ed anti-(rabbit IgG) Ig as the secondary antibody for 60 min, then mounted with 80% glycerol containing 2.5% of 1,4-diazabicyclo-(2,2,2)-octane. Cells were examined by fluorescence microscopy (Olympus). Immunoprecipitation To prepare cell extracts, 1 · 10 7 NIH3T3 cells were harvested and washed with NaCl/P i , lysed with a 5 cell- pack vol. of lysis buffer consisting of 50 m M Tris/HCl (pH 8.0), 140 m M NaCl, 1 m M EDTA, 1 m M EGTA, 1m M dithiothreitol, 0.2 l M phenylmethanesulfonyl fluor- ide, 3 lgÆmL )1 leupeptin and 1% NP-40, and rotated at 4 °C for 1 h. For immunoprecipitation, 5 lgofaF-N, or normal rabbit IgG as a negative control, were added to 200 lL of cell extract prepared as described above. Samples were incubated at 4 °C for 2 h and further incubated for 1 h with 50 lL of 50% slurry of protein A Sepharose. After washing with the lysis buffer, immunoprecipitates were eluted from protein A Sepharose by adding the F-N peptide at a concentration of 20 ngÆlL )1 , and an aliquot of eluate was used for electrophoretic mobility shift assay. Electrophoretic mobility shift assay (EMSA) DNA-binding activities of mLRP130 were analyzed by EMSA as described previously [35]. The single-stranded oligonucleotide sequences used for EMSA are as follows; C8: 5¢-dGAT(CTGCC) 8 -3¢,C5:5¢-d(CTGCC) 5 -3¢,C5-Pc-2: 5¢-d(CCTGCC) 5 -3¢,C5-gg:d(CGGCC) 5 -3¢,G8:5¢-dTCG (GGCAG) 8 -3¢. C5gg has single base substitutions, T fi G, in the repeat sequence of C5. DS12 is a double-stranded form of d(GGCAG) 12 [24]. Single-stranded oligonucleotides were end-labeled with [c- 32 P]ATP by T4 polynucleotide kinase, and a double-stranded oligonucleotide was labeled with [a- 32 P]dCTP by fill-in reaction with a Klenow fragment [31]. The standard reaction mixture (10 lL) contained 2 lL of 5 · binding buffer (50 m M Tris/HCl, pH 7.5, 500 m M KCl, 5 m M dithiothreitol, 1 m M EDTA, 1 mgÆmL )1 BSA and 38% glycerol), 10 m M MgCl 2 , 100–500 fmol of a labeled probe and 2 lL of eluate, as described above. These mixtures were incubated at 25 °C for 20 min, and the reaction was stopped by adding 2 lL of dye solution, composed of 0.25% bromophenol blue, 0.25% xylene cyanol and 30% glycerol. DNA–protein complexes were separated on an 8% nondenaturing polyacrylamide gel in 0.5 · Tris/borate/EDTA buffer (45 m M Tris/borate, pH 8.0, and 1 m M EDTA), and analyzed with a Bio-Image Analyzer, BAS2000 (Fuji Photo Film). RESULTS Isolation of mLRP130 cDNA To isolate cDNA for the 100 kDa MNBP-pE, large-scale purification of the pE was performed as described previ- ously [23], and five polypeptide sequences were determined. These sequences showed high homology with the internal amino-acid sequences of human LRP130. Unexpectedly, polypeptide sequences of another MNBP, pF, also showed high homology to the internal amino-acid sequences of human LRP130, although none of the analyzed peptides of pE and pF were the same. These results guided us to the possibility that pE and pF were produced from the same gene. Thus, cloning of mouse LRP130 (mLRP130) cDNA was carried out by using the sequence informations of both pE and pF, as described in Materials and methods. A 1.2-kbp partial cDNA fragment, pE Cl 2, whose deduced amino-acid sequence included three of five determined peptide sequences, E-3, E-4 and E-5, was isolated by RT-PCR using a degenerated primer set designed from pF polypeptide sequences, F-1 and F-2 (underlined in Fig. 1). Furthermore, a total of 26 cDNA clones were isolated by screening of an NIH3T3 cDNA library using pECl2 as a probe, and the complete nucleotide sequence was deter- mined by sequencing three overlapping clones. The entire cDNA, being 4571 bp in size, contained an ORF of 3918 bp between nucleotides 471 and 4388. A polyadenylated signal was noted at nucleotides 4540–4545, and an in-frame upstream stop codon located at nucleotides 453–455 (data not shown). The nucleotide sequence of the mLRP130 will appear in GenBank/EMBL/DDBJ with the accession number AB027124. As shown in Fig. 1, the coding region encodes a protein of 1306 amino-acid residues with a deduced molecular mass of 137 kDa. Five (E-1 to E-5) and two (F-1 and F-2) polypeptide sequences derived from pE and pF, respectively, are indicated in Fig. 1. These results suggested the possibility that pF is a full length mLRP130, and pE is produced by cleavage of the C-terminal region of mLRP130. The amino-acid sequence of mLRP130 showed 75.2% identity to human LRP130 (data not shown). Furthermore, a unique structural motif called a pentatrico- peptide repeat (PPR) motif, a 35 amino-acid sequence found in plant and yeast proteins [36], was dispersed nine times throughout the entire protein (boxed in Fig. 1). Expression of mLRP130 mRNA No expression of LRP130 mRNA has been previously reported in rat normal tissues using human cDNA as a probe [28]. To clarify the expression of mLRP130 mRNA in Ó FEBS 2002 Single-stranded C-rich DNA binding protein, LRP130 (Eur. J. Biochem. 269) 2929 mouse tissue, Northern blot analysis of various organs of mice was performed using 5¢ region of mLRP130 cDNA as a probe. A single species of 4.7 kb mRNA was expressed strongly in heart, liver, and kidneys, and weakly in brain, skeletal muscle and testes, and not detectable in spleen and lungs. The mLRP130 mRNA was also detected, but weakly, in all the embryonal stages examined, and a slight increase was observed in accordance with the embryonal develop- ment (Fig. 2). Hybridization with the 3¢ probe produced the same results (data not shown). Subcellular localization of mLRP130 in NIH3T3 cells Nuclear and cytoplasmic fractions were prepared from NIH3T3 cells and analyzed by immunoblot analysis. As shown in Fig. 3A, a major band with a molecular mass of  140 kDa was detected by aF-N in WCL (lane 1), as well as a minor band of  100 kDa. The size of the former was almost equivalent to the predicted molecular mass of mLRP130 (137 kDa). The smaller molecular mass protein, which was tentatively named as mLRP130 derivative (mLRP130 der ), was detected in the cytoplasmic fraction (Fig. 3A, lane 2). In contrast, mLRP130 was found in the nuclear fraction by aF-N (lane 3) and aF-C (lane 6). Interestingly, mLRP130 der (a shorter sequence) was not detected by aF-C (lanes 4 and 5), suggesting it may be the C-terminal truncated form of mLRP130. Coincidentally, thesizeofmLRP130 der was equivalent to that of pE [23], and both mLRP130 and mLRP130 der were found to bind to single-stranded C-rich sequence, C8, by South-Western analysis (data not shown). Taken together, the available data suggest that mLRP130 der and mLRP130 may correspond to pE and pF, respectively. In addition, a band with a molecular mass of  140 kDa was detected by aF-C in the cytoplasmic fraction (lane 5). Another isoform of mLRP130, having a different N-terminal region, could be present in NIH3T3 cells. Further studies should be conducted to exclude the possibility that a different species cross-reacted with aF-C specifically or nonspecifically. Subcellular localization of mLRP130 in NIH3T3 cells was also analyzed immunohistochemically using aF-N (Fig. 3B). Strong staining of mLRP130 was detected at the perinuclear region, and also in the cytoplasm with dot- or squiggle-like staining pattern. Weaker staining was also observed in nuclei. A similar staining pattern was not observed by normal rabbit IgG (data not shown). MLRP130 binds to the single-stranded C-rich sequence of MN Pc-1 Cell fractionation analysis suggested that the full-length mLRP130 functions within nuclei. To analyze the DNA binding activity of cellular mLRP130, immunoprecipita- tion was performed using aF-N. As shown in Fig. 4A, the mLRP130 was detected in the immunoprecipitated frac- tion by aF-N (lane 3), but not by normal rabbit IgG (lane 2). The same band was also detected by aF-C in the immunoprecipitated fraction by aF-N (Fig. 4A, lower panel, lanes 3). The precipitate of mLRP130 der was hardly detectable by aF-N, and the DNA-binding activity of mLRP130 was carried out using immunoprecipitated mLRP130 as follows: mLRP130 was eluted from the immunoprecipitated fraction by incubation with F-N peptide, as described in Materials and methods. Aliquots of eluate were subjected to EMSA to determine DNA binding activity using C8, G8 and DS12 probes. A DNA– mLRP130 complex was formed with the single-stranded C-rich sequence, C8 (Fig. 4B, lane 3). The DNA–protein complex was not detected by the single-stranded G-rich sequence, G8, and double-stranded form, DS12 (lanes 6 Fig. 2. Expression of mLRP130 mRNA. Left and right panels are Northern blots for mouse normal tissues and embryonal stages, respectively. Northern blots containing 2 lg of poly(A) + RNA per lane were analyzed by hybridization with the 5¢ region of 32 P-labeled mLRP130 cDNA as described in Materials and methods. Arrowheads indicate a band of  4.7-kb mLRP130 mRNA. The molecular sizes are indicated on the left of each panel. Fig. 1. Deduced amino-acid sequence of mLRP130. The amino-acid sequence of mLRP130 was deduced from its cDNA sequence, and numbered on the right. Peptide sequences, E-1 to E-5, determined from purified pE, and, F-1 and F-2, from pF are indicated bold italic letters. Nine PPR motifs found in mLRP130 are boxed. A 1.2-kb pE Cl2 initially isolated by RT-PCR is underlined. Peptide sequences used for the generation of antibodies are dotted on the sequences. 2930 N. Tsuchiya et al. (Eur. J. Biochem. 269) Ó FEBS 2002 and 9). No DNA–protein complex was detected using eluate of the immunoprecipitate by normal rabbit IgG (lanes 2, 5 and 8). Sequence specificity for DNA binding Sequence specificity of mLRP130 for DNA binding was analyzed using various oligonucleotides as competitors. The DNA–protein complex was formed by incubation of mLRP130 with a 32 P-labeled C8 probe (Fig. 5, lane 3). Formation of this complex was undetectable when a 10-fold molar excess of unlabeled C8 was added (lane 4). C5 Pc-2 demonstrated a similar binding activity to C8 (lanes 10–12). In contrast, the binding activity to C5 is 10-fold less than that of C8 and C5Pc-2 (lanes 7–9). No binding was observed with C5gg or poly(dA) (lanes 13–15 and 16–18, respectively). DISCUSSION This study described the cloning and characterization of a cDNA encoding a mLRP130 from NIH3T3 cells as a C-rich binding MNBP. We have previously identified four C-rich binding MNBPs from the nuclear extract of NIH3T3 cells, Fig. 3. Subcellular localization of mLRP130 in NIH3T3 cells. (A) Cell fractionation analysis. Left and right panels indicate the immunoblot analysis using aF-N and aF-C, respectively. The positions of the protein standard are indicated on the left. Closed and open arrowheads show the bands of mLRP130 and mLRP130 der , respectively. a-tubulin was used as a marker for cytoplasmic extraction. The same membrane was used for immunoblot analysis with aF-N, aF-C and anti-(a-tubulin) Ig. Lanes 1 and 4, whole cell lysate; lanes 2 and 5, cytoplasmic fraction; lanes 3 and 6, nuclear fraction. (B) Indirect immunofluorescence microscopy. NIH3T3 cells were fixed with prechilled methanol and labeled with aF-N, and then visualized with FITC conjugated anti-(rabbit IgG) Ig as a secondary antibody. Cells were examined with fluorescence microscopy (·40). Fig. 4. mLRP130 binds to the single-stranded C-rich sequence of MN Pc-1. (A) Upper and lower panels are immunoblot analyses using aF-N and aF-C following immunoprecipitation of cell extracts from NIH3T3 cells using a aF-N or normal rabbit IgG as a control, res- pectively. Positions of protein molecular weight marker (Bio-Rad) are shown on the left of each panel. Closed and open arrowheads indicate the mLRP130 and mLRP130 der bands, respectively. Lane 1, cell extract; lane 2, immunoprecipitant (ipp)withnormalrabbitIgG;lane 3, ipp with aF-N. (B) EMSA using immunoprecipitated mLRP130. 32 P-Labeled probes (C8, G8 and DS12) were incubated with mLRP130, and the DNA-protein complex was analyzed by EMSA as described in Materials and methods. Arrowheads indicate a DNA– protein complex and each unbound probe, respectively. Lanes 1–3, 4–6 and 7–9 were results of EMSA with C8, G8 and DS12 probes, respectively. Lanes 1, 4 and 7, without protein; lanes 2, 5 and 8, ipp with normal rabbit IgG; lanes 3, 6 and 9, ipp with aF-N. Fig. 5. DNA binding activities of mLRP130 in the presence of various oligonucleotides. Immunoprecipitated mLRP130 and 32 P-labeled C8 were incubated with or without various concentrations of unlabeled oligonucleotides, and the DNA-protein complex was analyzed by EMSA. Three concentrations, 10-, 50- and 100-fold molar excess of unlabeled competitors, are indicated at the top. Arrowheads show a DNA–protein complex and unbound probe, respectively. Lane 1, without protein; lane 2, ipp with normal rabbit IgG; lane 3, ipp with aF-N without competitors; and with competitors, lanes 4, 5 and 6, with C8; lanes 7, 8 and 9, with C5; lanes 10, 11 and 12, with C5Pc-2; lanes 13, 14 and 15, with C5gg; lanes 16, 17 and 18, with poly(dA). Ó FEBS 2002 Single-stranded C-rich DNA binding protein, LRP130 (Eur. J. Biochem. 269) 2931 and two of them, pE and pF, have a molecular mass of 100 and 130 kDa, respectively [23]. An amino-acid sequence of mLRP130 contained all of the peptide sequences of pE and pF. Furthermore, mLRP130 shows the same DNA binding properties to that of the previously characterized pE [23]. We therefore hypothesized that pE and pF are mLRP130 der and mLRP130, respectively, the former representing a truncated form of the C-terminal region produced by post- translational processing. However, more detailed studies, including determination of the cleavage site of the mLRP130 and analysis of the DNA binding activity of the cleaved form, are needed to confirm that mLRP130 der and pE is the same protein produced by processing of mLRP130. Subcellular localization of mLRP130 was analyzed by immunofluorescence microscopy and cell fractionation analysis. The localization of mLRP130 is remarkable at the perinuclear region, with some in the nuclei, and in the cytoplasm with dot- and squiggle-like staining patterns. The cytoplasmic staining is likely to be ascribed to mLRP130 der , from cell fractionation analysis, and its association with cytoplasmic structural protein or organelles could be suggested. This is one of the possible explanations why mLRP130 der was not immunoprecipitated efficiently by aF-N. On the other hand, mLRP130 was detected mainly in the nuclear fraction. The different subcellular localization of mLRP130 and mLRP130 der may reflect the distinct biolo- gical functions of this protein between nuclei and cytoplasm. Human LRP130 was first identified in the hepatoblastoma cell line HepG2 as a highly expressed transcript [28], and its expression was detected strongly in liver as well as heart and kidneys examined in this study. The mLRP130 contains nine copies of PPR motifs, and the first four PPR motifs lie at the N-terminal region in tandem. Compositions of amino-acid residues and the predicted secondary structure of the PPR motif are very similar to that of the tetratrico- peptide repeat (TPR) motif [36]. TPR motifs are presented in a variety of proteins with variable repeat numbers, and are known to be essential motifs for protein–protein interaction in, for example, some phosphatases or cell cycle regulated proteins [37]. PPR motifs could also take the tertiary structure similar to that of TPR motifs, and serve as a protein–protein interaction domain of mLRP130. Iden- tification of proteins interacting with the PPR motifs is to be conducted in future experiments to clarify the biological functions of PPR motifs in mLRP130. In vitro DNA binding assay showed that immunopre- cipitated mLRP130 binds to single-stranded C-rich sequence of MN Pc-1, but not to its complementary sequence. Although the possibility that the immunopre- cipitated fraction contains mLRP130 interacting proteins could not be completely excluded, no other protein bands were detected in the immunoprecipitated fraction when stained with Coomassie Brilliant Blue after SDS/PAGE analysis (data not shown). Therefore, DNA binding activity is attributed to the presence of mLRP130 itself. mLRP130 did not bind to the double-stranded form, or to C5gg, indicating that mLRP130 binds to the single- stranded C-rich sequence in a sequence-dependent man- ner. These results led us to the hypothesis that mLRP130 efficiently binds to the single-stranded region of repetitive sequences composed of d(CCTXCC) n .Furthermore,it appears to be likely that a long C-rich sequence or a long tandem repeat allele was required for the DNA binding activity of mLRP130. It has been known that G-rich repetitive sequences, including telomere and insulin– VNTR, form the unusual DNA structure like G-quartet [38,39]. Additionally, formation of the I-tetraplex structure by the C-rich strand of the telomere and insulin-VNTR has also been reported [38,40]. Recently, we have reported that the G-rich sequence of MN Pc-1 forms an intra- molecular G-quartet structure, and DNA synthesis arrests at repeat sequences in vitro [24]. These unusual structures might be one of the dominant causes for induction of MN mutations. Although the biological functions of mLRP130 could not be fully elucidated, its binding to the single- stranded C-rich region of chromosomes, which is exposed by formation of the unusual structure of its complement- ary G-rich strand, could affect the stability of G-rich repetitive sequence loci. ACKNOWLEDGEMENTS This work was supported by Grant-in Aid for Cancer Research from the Ministry of Health, Labour and Welfare of Japan, by Grant-in Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan. Peptide synthesis and production of peptide antibodies was carried out by IBL Co. Ltd. N. T. is a recipient of Research Resident Fellowship from Foundation for Promotion of Cancer Research in Japan. REFERENCES 1. Jeffreys, A.J., Wilson, V. & Thein, S.L. (1985) Hypervariable ÔminisatelliteÕ regions in human DNA. Nature 314, 67–73. 2. Jeffreys, A.J. 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