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• W2092312800 abstract "Recent studies indicate that the balance between cell survival and proapoptotic signals determines which cells commit to life or death. We have shown that the balance between follicle-stimulating hormone and prolactin determines differentiation or apoptosis in 7th generation spermatogonia during newt spermatogenesis; however, the molecular mechanisms specifying their fate are poorly understood. Here we show that the newt RNA-binding protein (nRBP) plays a critical role in determining their fate. nRBP was identified as a clone whose mRNA is decreased by prolactin, resulting in the reduction of the protein, which is otherwise expressed predominantly in the spermatogonia. nRBP protein associated with the mRNA for newt programmed cell death protein 4 (nPdcd4) at the 3′-untranslated region. nRBP reduction increased nPdcd4 mRNA but decreased its protein. In a cell-free system, cytoplasmic extracts containing reduced amounts of nRBP and nPdcd4 protein induced apoptosis, whereas adding nRBP protein to the extracts blocked apoptosis. Furthermore, overexpression of nRBP protected cells from apoptosis, stabilized the chimeric transcript containing the nPdcd4 3′-untranslated region, and accelerated its translation. These data suggest that, in the absence of nRBP, nPdcd4 mRNA is not stabilized and its translation is suppressed, leading to apoptosis in the spermatogonia. Recent studies indicate that the balance between cell survival and proapoptotic signals determines which cells commit to life or death. We have shown that the balance between follicle-stimulating hormone and prolactin determines differentiation or apoptosis in 7th generation spermatogonia during newt spermatogenesis; however, the molecular mechanisms specifying their fate are poorly understood. Here we show that the newt RNA-binding protein (nRBP) plays a critical role in determining their fate. nRBP was identified as a clone whose mRNA is decreased by prolactin, resulting in the reduction of the protein, which is otherwise expressed predominantly in the spermatogonia. nRBP protein associated with the mRNA for newt programmed cell death protein 4 (nPdcd4) at the 3′-untranslated region. nRBP reduction increased nPdcd4 mRNA but decreased its protein. In a cell-free system, cytoplasmic extracts containing reduced amounts of nRBP and nPdcd4 protein induced apoptosis, whereas adding nRBP protein to the extracts blocked apoptosis. Furthermore, overexpression of nRBP protected cells from apoptosis, stabilized the chimeric transcript containing the nPdcd4 3′-untranslated region, and accelerated its translation. These data suggest that, in the absence of nRBP, nPdcd4 mRNA is not stabilized and its translation is suppressed, leading to apoptosis in the spermatogonia. Multicellular organisms maintain tissue homeostasis through response of their cells to extracellular signals that either promote their proliferation and differentiation or induce their death. Evidence is accumulating that extracellular stimuli, such as growth factors and cytokines, operate via complex signal transduction networks that ultimately control cellular fates (1.Janes K.A. Albeck J.G. Gaudet S. Sorger P.K. Lauffenburger D.A. Yaffe M.B. Science. 2005; 310: 1646-1653Crossref PubMed Scopus (453) Google Scholar); however, the complete molecular mechanisms need to be elucidated. One convenient system for studying the molecular mechanisms governing cell survival and death is spermatogenesis. It is mediated not only by cell proliferation and differentiation but also by programmed cell death or apoptosis, culminating in the production of spermatozoa. Apoptosis is necessary for eliminating unwanted cells and adjusting cell numbers in multicellular organisms to ensure tissue homeostasis. In the testis of the Japanese red-bellied newt, Cynops pyrrhogaster, primary spermatogonia proliferate through seven mitotic divisions, and then in the 8th generation the spermatogonia differentiate into primary spermatocytes and initiate meiosis in the spring when the ambient temperature is high; in contrast, in the autumn when the ambient temperature is low, the spermatogonia often undergo apoptosis in the 7th generation, resulting in the cessation of spermatogenesis (2.Abé S.I. Zool. Sci. 2004; 21: 691-704Crossref PubMed Scopus (18) Google Scholar). These findings suggest the existence of molecular mechanisms regulating the fate of 7th generation spermatogonia. We have shown in vitro and in vivo that the cellular fate in the spermatogonia is mainly regulated by changes in the endogenous levels of two peptide hormones secreted from the pituitary gland as follows: follicle-stimulating hormone (FSH) 2The abbreviations used are: FSHfollicle-stimulating hormonePRLprolactinnRBPnewt RNA-binding proteinCIRPcold-inducible RNA-binding protein3′UTR3′-untranslated region3′RACE3′-rapid amplification of cDNA endsRNPribonucleoproteinPARPpoly(ADP-ribose) polymeraseRTreverse transcriptionGFPgreen fluorescent proteinWBWestern blottingIPimmunoprecipitationTUNELterminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labelingDAPI4′,6-diamidino-2-phenylindoleGSTglutathione S-transferaseHAhemagglutininPMSFphenylmethylsulfonyl fluorideDTTdithiothreitolrnRBPrecombinant newt RBPntnucleotidePIPES1,4-piperazinediethanesulfonic acidhGAPDHhuman glyceraldehyde-3-phosphate dehydrogenaseFLfull length. that stimulates spermatogonial proliferation and differentiation into primary spermatocytes, and prolactin that induces apoptosis. When the relative concentration ratio of prolactin to FSH is high, the spermatogonia undergo apoptosis, but when the ratio is low, they survive (3.Yazawa T. Yamamoto K. Kikuyama S. Abé S.I. Gen. Comp. Endocrinol. 1999; 113: 302-311Crossref PubMed Scopus (36) Google Scholar, 4.Yazawa T. Yamamoto T. Abé S.I. Endocrinology. 2000; 141: 2027-2032Crossref PubMed Scopus (35) Google Scholar, 5.Yazawa T. Yamamoto T. Jin Y. Abé S.I. Biol. Reprod. 2002; 66: 14-20Crossref PubMed Scopus (26) Google Scholar). However, little is known about the intracellular events occurring when cells respond to extracellular stimuli that decide their fate. follicle-stimulating hormone prolactin newt RNA-binding protein cold-inducible RNA-binding protein 3′-untranslated region 3′-rapid amplification of cDNA ends ribonucleoprotein poly(ADP-ribose) polymerase reverse transcription green fluorescent protein Western blotting immunoprecipitation terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling 4′,6-diamidino-2-phenylindole glutathione S-transferase hemagglutinin phenylmethylsulfonyl fluoride dithiothreitol recombinant newt RBP nucleotide 1,4-piperazinediethanesulfonic acid human glyceraldehyde-3-phosphate dehydrogenase full length. Newt spermatogonia are an ideal model for analyzing the mechanisms controlling cellular life or death. The germ cells are in close contact with somatic Sertoli cells in a spermatogenic cyst, the smallest unit of the testis. The testis consists of lobules in successive zones arranged along a cephalocaudal axis, in which spermatogenesis proceeds synchronously (2.Abé S.I. Zool. Sci. 2004; 21: 691-704Crossref PubMed Scopus (18) Google Scholar, 6.Callard G.V. Pang P.K.T. Schreibman M.T. Vertebrate Endocrinology: Fundamentals and Biomedical Implications. Academic Press, New York1991: 303-341Google Scholar). Therefore, we can dissect zones containing particular spermatogenic stages and identify the generation of spermatogonia by counting their numbers in a cyst. Such a simple structure of the testis allows us to isolate and characterize the molecules influencing the fate of spermatogonia. This study demonstrated the following: 1) a putative glycine-rich RNA-binding protein (nRBP) disappears in the cytoplasm of 7th generation spermatogonia after prolactin exposure; 2) this loss is implicated in the induction of apoptosis and in the suppression of translation for the newt orthologue of programmed cell death protein 4 (nPdcd4) mRNA, the 3′-untranslated region (UTR) that interacts with nRBP protein; and 3) overexpression of nRBP protects intact cells from apoptosis and accelerates the translation of the chimeric green fluorescent protein (GFP)-nPdcd4 3′UTR mRNA by increasing its stability. These results suggest that the RNA-binding protein functions as an antiapoptotic factor by stabilizing nPdcd4 mRNA and promoting its translation, thereby determining spermatogonial survival. The antigen peptide (FVSEGDGGRLKPESY) for an antibody to human Pdcd4 (Rockland) and the PCR primers were produced in TORAY Research Center Co., Ltd., and Hokkaido System Science Co., Ltd., Japan, respectively. Adult male newts and adult female mice were purchased from Hamamatsu Seibutsu Kyozai Ltd. and Kyudo Co., Ltd., Japan, respectively. All other chemicals were from standard commercial sources, unless otherwise stated. Newts were kept at 7 °C in the dark and then transferred to 22 °C and fed frozen Tubifex. Their testes containing late spermatogonial and early spermatocyte stages were used. Induction of apoptosis in 7th generation spermatogonia was performed by injecting into each newt 25 IU prolactin (Sigma) or the vehicle (saline) as the control. After 48 h, their testes were removed (3.Yazawa T. Yamamoto K. Kikuyama S. Abé S.I. Gen. Comp. Endocrinol. 1999; 113: 302-311Crossref PubMed Scopus (36) Google Scholar, 4.Yazawa T. Yamamoto T. Abé S.I. Endocrinology. 2000; 141: 2027-2032Crossref PubMed Scopus (35) Google Scholar). Except for changes in the expression of mRNA and protein for nRBP and nPdcd4, newts were exposed for various periods from 6 to 48 h after prolactin injection. In some experiments, FSH (0.1 IU; Sigma) was injected into each newt (5.Yazawa T. Yamamoto T. Jin Y. Abé S.I. Biol. Reprod. 2002; 66: 14-20Crossref PubMed Scopus (26) Google Scholar) and then analyzed at 12, 24, and 48 h. Total RNA was extracted from the testes by homogenization in ISOGEN (Nippon Gene) and treated with RNase-free DNase I (Takara). Then cDNA was synthesized using oligo(dT) primers and random hexamers with a reverse transcriptase Superscript III (Invitrogen) according to the respective manufacturer's instructions (7.Eto K. Eda K. Kanemoto S. Abe S.I. Biochem. Biophys. Res. Commun. 2006; 350: 263-271Crossref PubMed Scopus (19) Google Scholar). For microarray analyses, we made microarrays carrying 5321 independent cDNA clones and hybridized them with Cy3- or Cy5-labeled cDNA probes prepared from total RNA (CyScribe First Strand cDNA labeling kit; GE Healthcare), which had been extracted from the testes of newts that were injected with prolactin or the vehicle. Fluorescent signals were quantified by ScanArray 4000 (GE Healthcare), and the data were analyzed using QuantArray software (GE Healthcare). The full-length clone encoding the open reading frame of nRBP and the partial clone encoding that of nPdcd4 were isolated from the cDNA library used in microarray analyses, and their nucleotide sequences were determined using an Applied Biosystems model 310 automated DNA sequencer. For RT-PCR analyses, 5 μg of total RNA or all of the coimmunoprecipitated RNA was treated with DNase I and reverse-transcribed using random hexamers, as described previously (7.Eto K. Eda K. Kanemoto S. Abe S.I. Biochem. Biophys. Res. Commun. 2006; 350: 263-271Crossref PubMed Scopus (19) Google Scholar). 10% of the respective reverse transcription reaction was subjected to PCR as the template with Ex Taq polymerase (Takara). The PCR conditions were as follows: for 30 cycles at 95 °C for 30 s, at 55 °C for 30 s, and at 72 °C for 30 s for nRBP, GFP, and human glyceraldehyde-3-phosphate dehydrogenase (hGAPDH); for 28, 42, or 45 cycles at 95 °C for 30 s, at 55 or 57 °C for 30 s, and at 72 °C for 40 s for nPdcd4; and for 25 cycles at 95 °C for 30 s, at 55 °C for 30 s, and at 72 °C for 30 s for newt elongation factor-1α (nEF-1α). The forward and reverse primers used were as follows: 5′-gat ggt aaa ctc ttc gtg gg-3′ and 5′-gct gtc gta gct gtc tct gta g-3′ for nRBP; 5′-atg ata gtg aag ctg ctg aa-3′ and 5′-aca gaa gct ttc tca aga tg-3′ for nPdcd4; 5′-agc cct aga ctc aat cat cc-3′ and 5′-atc caa cac agg agc gta tc-3′ for nEF-1α; 5′-atg gtg agc aag ggc gag gag ctg-3′ and 5′-tta ctt gta cag ctc gtc cat gcc-3′ for GFP; and 5′-gtc agt ggt gga cct gac ct-3′ and 5′-tga gct tga caa agt ggt cg-3′ for hGAPDH. Each of the amplified DNA fragments (nRBP, 400 bp; nPdcd4, 386 bp; nEF-1α, 550 bp; hGAPDH, 212 bp; and GFP, 719 bp) was analyzed by 1% agarose gel electrophoresis and cloned into pT7Blue vector (Merck). The nucleotide sequence was verified. The RT-PCR reaction mixture without reverse transcriptase was used as the negative control. To express in mammalian cells nRBP protein tagged in the carboxyl terminus with hemagglutinin (HA), the coding region of nRBP cDNA was amplified by PCR for 30 cycles at 95 °C for 30 s, at 55 °C for 30 s, and at 72 °C for 45 s using primers 5′-gtg aat tca tgt cgt ctg atg atg gt-3′ and 5′-cag cgg ccg ctc agg cat agt cag gga cgt cat aag gat agc tgt cgt agc tgt ctc t-3′. The PCR product was digested with EcoRI and NotI and ligated into a mammalian expression vector pME18s (DNAX Institute) (pME18s-nRBP). For construction of the plasmid expressing recombinant nRBP protein (rnRBP) fused at the amino terminus to GST and at the carboxyl terminus to HA (GST-nRBP), the cDNA portion encoding amino acid residues 2–159 was amplified by PCR for 30 cycles at 95 °C for 30 s, at 55 °C for 30 s, and at 72 °C for 45 s using primers 5′-gtg aat ctc gtc tga tga tgg taa ac-3′ and 5′-cag cgg ccg ctc agg cat agt cag gga cgt cat aag gat agc tgt cgt agc tgt ctc t-3′. The PCR products were digested with EcoRI and NotI and ligated into pGEX4T-1 (GE Healthcare), a bacterial expression vector for GST fusion protein. Bacterial cells (Escherichia coli, DH5α strain) were transformed with the expression plasmid for GST-nRBP or GST protein. These proteins were separately expressed in the bacterial cells grown to 0.6 of A600 nm by addition of 0.1 mm isopropyl 1-thio-β-d-galactopyranoside, cultured for a further 3 h, and purified by glutathione-Sepharose (0.5 ml; GE Healthcare) column chromatography according to the manufacturer's instructions. The recombinant proteins, eluted with a buffer containing 10 mm glutathione, 50 mm Tris/HCl, pH 8.0, and 0.2 mm PMSF, were dialyzed against phosphate-buffered saline. To raise a polyclonal antibody against nRBP, GST-nRBP (∼0.2 mg) was injected into each mouse (C57BL/6 strain). The mice received four booster injections at 5-day intervals. The blood was harvested and incubated for 1 h at 37 °C and then overnight at 4 °C. After centrifugation at 1000 × g for 10 min at 4 °C, the supernatant was applied to a glutathione-Sepharose column bound with GST protein to remove its antibodies. The flow-through fraction was used as an anti-nRBP antiserum for Western blotting, immunohistochemistry, and immunoprecipitation. Normal mouse serum was prepared by the same method. For construction of chimeric genes connecting the full-length and mutant 3′UTR of nPdcd4 mRNA with the GFP coding region at the carboxyl terminus, the 3′UTR was cloned by 3′RACE (Invitrogen) according to the manufacturer's protocol. Briefly, 5 μg of total RNA was reverse-transcribed at 42 °C for 50 min using oligo(dT)-containing adapter primer, which was provided by the manufacturer. The 3′UTR cDNA was cloned by sequential PCR with Ex Taq polymerase using the abridged universal amplification primer, which was provided by the manufacturer, and three distinct gene-specific primers, which we designed on the basis of the nucleotide sequence of the putative nPdcd4 open reading frame as follows: 5′-aag ttc tcc atg atg ttg tg-3′ for 35 cycles at 94 °C for 1 min, at 55 °C for 1 min, and at 72 °C for 2 min; and 5′-aca tcc aga ttg atg tcg gg-3′ and 5′-tgg aag ccc cgg ttc atc tg-3′for 35 cycles at 94 °C for 0.5 min, at 55 °C for 0.5 min, and at 72 °C for 1 min, ligated into pT7 blue vector, and thoroughly sequenced. The cDNA portions encoding the full-length 3′UTR (nucleotide (nt) 1–1527, FL) and its 3′-terminal truncated mutants (nt 1–1208, nt 1–814, and nt 1–380), shown schematically in Fig. 6A, were amplified with the original 3′UTR as the template by PCR for 35 cycles at 94 °C for 0.5 min, at 55 °C for 0.5 min, and at 72 °C for 1 min to introduce restriction sites EcoRI and SalI. The forward primer used was 5′-ccg aat tct aac ccg aat ctc gaa cac ac-3′ and the reverse primer: 5′-tgg att gcc acg gtc gac tag tac-3′ for the full-length region; 5′-aag tcg acg caa tcg aga cca gtt ata ac-3′ for nt 1–1208; 5′-aag tcg acc agt gag acc tgc tag gtt ac-3′ for nt 1–814; or 5′-aag tcg aca ctg agc cat tcg tat caa ca-3′ for nt 1–380. Each of the products was subcloned into pEGFP-C2 (Takara), a mammalian expression vector for GFP fusion protein, 3′-terminally after the stop codon following the GFP coding region to generate chimeric gene constructs pEGFP-nPdcd4-3′UTR-FL, -1208, -814, and -380 for expressing the respective GFP/nPdcd4–3′UTR mRNA. Human cervical carcinoma (HeLa) cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum at 37 °C under an atmosphere of 5% CO2. Cells were plated at ∼40% confluency onto 30-mm-diameter dishes, and 1 day later cells were transfected with 1 μg of the expression construct pME18s-nRBP or the empty vector (mock) using 6 μg of Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. The cells were treated for 2.5 h with 1 μm staurosporine or the vehicle (0.05% DMSO) 40 h after transfection. Nuclei were visualized using a fluorescent microscope (BX60; Olympus) by DAPI staining and TUNEL assay. The latter was performed with an in situ apoptosis detection kit (Takara) according to the manufacturer's instructions. For quantification of apoptotic cells, a minimum of 100 cells, each from at least three microscopic fields, was examined for TUNEL-positive nuclei among DAPI-positive ones. The frequency of apoptosis was expressed as a percentage (means ± S.E.) of apoptotic cells obtained from three independent experiments. To test the binding of nRBP protein with the 3′UTR of nPdcd4 mRNA and analyze its effect on the stability and translation of mRNA for the chimeric genes, HeLa cells grown on 30-mm dishes to 60% confluency were transiently transfected with a combination of pME18s-nRBP (1 μg) and either pEGFP-nPdcd4-3′UTR-FL, -1208, -814, -380, or pEGFP-C2 (1 μg, each) using 5 μl of FuGENE (Roche Applied Science) according to the manufacturer's instructions. Cells were harvested 3, 6, 9, and 12 h after transfection, and subjected to immunoprecipitation, RT-PCR, and Western blotting. For inhibition of transcription, 12 h after transfection the cells were treated with actinomycin D (5 μg/ml; Merck) for 1, 2, 4, and 8 h. Testes were fixed in Bouin's fixative, embedded in paraffin wax, and sectioned at 5 μm. Sections were stained with hematoxylin and eosin to examine the apoptotic cells and the spermatogenic stages. For immunohistochemistry of nRBP, testes were fixed in carnoy fixative (ethanol:chloroform:acetic acid = 6:3:1) at room temperature for 1 h, embedded in paraffin wax, and sectioned at 5 μm. Sections were immunostained with a mouse anti-nRBP antiserum (1:100), and developed with the activity of alkaline phosphatase conjugated with a secondary antibody. Testes were homogenized in cell extract buffer (CEB: 50 mm PIPES, pH 7.4, 50 mm KCl, 5 mm EGTA, 2 mm MgCl2, 1 mm DTT, 1 mm PMSF, protease inhibitor mixture (Roche Applied Science)) and lysis buffer (20 mm Tris/HCl, pH 7.5, 100 mm NaCl, 1% Triton X-100, 0.2 mm PMSF, protease inhibitor mixture) at 5-fold volume to weight. HeLa cells were lysed in SDS-PAGE sample buffer (62.5 mm Tris/HCl, pH 6.8, 25% glycerol, 2% SDS, 0.01% bromphenol blue, 5% β-mercaptoethanol) at 150 μl to ∼2 × 105 cells. The extracts (testes, 20 μg of protein; HeLa cells, 10% (v/v) of the total) were subjected to SDS-15% PAGE and Western blotting with antibodies against nRBP (1:200 or 500), Pdcd4 (1:1000), HA tag (1:250 or 500; Santa Cruz Biotechnology), PARP (1:1000; Enzo Life Sciences), GFP (1:1000; BD Biosciences), and β-actin (1:2000; Millipore). Cytoplasmic extracts were prepared from the testes of newts injected with prolactin or the vehicle by the method of Martin et al. (8.Martin S.J. Newmeyer D.D. Mathias S. Farschon D.M. Wang H.G. Reed J.C. Kolesnick R.N. Green D.R. EMBO J. 1995; 14: 5191-5200Crossref PubMed Scopus (241) Google Scholar) with minor modifications. Testes were homogenized in an equal volume of CEB to weight that contained RNase inhibitor (Promega). After centrifugation at 14,000 × g for 15 min at 4 °C, the supernatant was collected as the cytoplasmic extracts. Protein concentrations were determined and adjusted to 10 mg/ml with extraction buffer containing the ATP-regenerating system (EDB: 10 mm HEPES, pH 7.2, 50 mm NaCl, 5 mm EGTA, 2 mm MgCl2, 1 mm DTT, 2 mm ATP, 10 mm phosphocreatine, 50 μg/ml creatine kinase, 1 mm PMSF, protease inhibitor mixture, RNase inhibitor). Dissociated cells from testes were prepared as described (9.Saribek B. Jin Y. Saigo M. Eto K. Abe S.I. Biochem. Biophys. Res. Commun. 2006; 349: 1190-1197Crossref PubMed Scopus (13) Google Scholar). Healthy nuclei were isolated from the dissociated germ cells by solubilization for 5 min on ice with 40 ng/ml digitonin in nuclear isolation buffer (NB: 10 mm PIPES, pH 7.4, 10 mm KCl, 2 mm MgCl2,1 mm DTT, 1 mm PMSF, protease inhibitor mixture, RNase inhibitor). Nuclei were precipitated by centrifugation at 200 × g for 5 min at 4 °C and washed with NB. The nuclear number was determined and adjusted to 105/μl with NB. For cell-free reactions, 10 μl of cytoplasmic extracts (100 μg) were mixed with 1 μl of germ cell nuclei (105) and 2 μl of EDB containing RNase inhibitor, and then incubated for 9 h at 22 °C. To investigate the effect of nRBP protein on prolactin-dependent cell-free apoptosis, cytoplasmic extracts were preincubated for 4 h at 22 °C with 1 μl of GST-nRBP or GST as the control (2 μg, each) and then mixed with nuclei. The optimal temperature and times for incubation of cytoplasmic extracts and nuclei and for that of cytoplasmic extracts and recombinant proteins were determined by examining the effects of varying temperatures and times on the frequency of apoptosis in nuclei. Nuclei were visualized with a fluorescent microscope after DAPI staining and TUNEL assay. For quantification of apoptotic nuclei, a minimum of 100 nuclei, each from at least three microscopic fields, was examined for TUNEL-positive nuclei among DAPI-positive ones. The frequency of apoptosis was expressed as a percentage (means ± S.E.) of the apoptotic nuclei obtained from three independent experiments. For coimmunoprecipitation of endogenous nPdcd4 mRNA with endogenous nRBP protein, testes were dissected from normal newts and homogenized in CEB containing RNase inhibitor but lacking DTT. Cytoplasmic extracts (1 mg of protein) were separated by centrifugation at 10,000 × g for 15 min at 4 °C and precleared for 12 h at 4 °C on protein G-Sepharose beads (GE Healthcare) with constant rotation. After centrifugation at 1,000 × g for 1 min at 4 °C, the supernatants were collected and divided into two tubes (0.5 mg of protein each). They were then incubated with the new beads and 10 μl of either anti-nRBP antiserum or normal mouse serum as the control for 6 h at 4 °C under constant rotation. For immunodepletion of endogenous nRBP protein, GST-nRBP (100 μg) was preincubated for 12 h on ice with 10 μl of anti-nRBP antiserum, and the immunocomplexes were used for immunoprecipitation. Beads were washed three times with CEB containing RNase inhibitor and lacking DTT and three times with RIPA buffer (50 mm Tris/HCl, pH 7.5, 1 mm EDTA, 1 m NaCl, 1% Nonidet P-40, 0.1% SDS, 1 mm PMSF, protease inhibitor mixture, RNase inhibitor) and then resuspended in 300 μl of RIPA buffer. Coimmunoprecipitated RNA was extracted from ⅔ of the beads with ISOGEN by the standard procedure and subjected to RT-PCR using primers of some genes involved in apoptosis, including nPdcd4 as described. The remaining beads were eluted in SDS-PAGE sample buffer for Western blotting with the anti-nRBP antiserum. For coimmunoprecipitation of endogenous nPdcd4 mRNA with exogenous nRBP protein, either GST-nRBP or GST protein (5 μg, each) was incubated for 4 h at 22 °C with cytoplasmic extracts (0.25 mg of protein) prepared from normal testes, and subjected to immunoprecipitation with either an antibody to HA tag of the recombinant protein (1 μg) or normal rabbit IgGs (1 μg) followed by RT-PCR using nPdcd4 primers and Western blotting with the anti-HA antibody. Coimmunoprecipitation of the chimeric gene mRNA with nRBP protein was performed essentially as described above with an antibody to HA tag of the recombinant protein (2 μg) using cytoplasmic extracts (0.5 mg of protein) prepared by CEB containing RNase inhibitor and 0.1% Nonidet P-40 and lacking DTT from the transfectants with a combination of pME18s-nRBP and either pEGFP-nPdcd4–3′UTR-FL, -1208, -814, -380, or the GFP vector. Before immunoprecipitations, total RNA extracted from the cells 12 h after transfection was reverse-transcribed, and PCR was performed for 35 cycles at 95 °C for 30 s, at 55 °C for 30 s, and at 72 °C for 2 min for the chimeric gene mRNA, GFP/nPdcd4–3′UTR-FL (2.2 kbp), -1208 (1.9 kbp), -814 (1.5 kbp), and -380 (1.1 kbp), with the GFP forward primer and each reverse primer of the nPdcd4 full-length and mutant 3′UTR. Two-thirds of the immunoprecipitates were subjected to RT-PCR for the chimeric gene transcripts and the remaining to Western blotting for nRBP with the anti-HA antibody. For immunodepletion of an antibody to human Pdcd4, the antibody solution diluted at 4000-fold (134 μg of protein) was mixed with the antigen peptide (500 μg) dissolved in water. After incubating overnight at 4 °C with rotation, the mixture was used for Western blotting of the extracts prepared from the testes and HeLa cells as a positive control. Data were obtained as the means ± S.E. For statistical comparison, Student's t test was used. p values less than 0.05 were considered to be statistically significant. Protein concentrations were estimated by Coomassie dye binding (Bio-Rad) using bovine serum albumin as a standard. For some experiments, signals detected in the Western blotting and RT-PCR were quantified by densitometry using ImageJ software. When apoptosis was induced following injection of prolactin into newts, DNA fragmentation, cellular shrinkage, and chromatin condensation were observed at 48 h in 7th generation spermatogonia by TUNEL assay and hematoxylin/eosin staining (3.Yazawa T. Yamamoto K. Kikuyama S. Abé S.I. Gen. Comp. Endocrinol. 1999; 113: 302-311Crossref PubMed Scopus (36) Google Scholar, 4.Yazawa T. Yamamoto T. Abé S.I. Endocrinology. 2000; 141: 2027-2032Crossref PubMed Scopus (35) Google Scholar). To screen for genes in the spermatogonia whose expressions might be altered after prolactin treatment, we performed cDNA microarray analyses. Microarrays carrying 5321 independent clones isolated from testes were prepared and hybridized with cDNA synthesized using total RNA extracted from the testes 48 h after newts were injected with prolactin or the vehicle. Expressions of many genes changed in response to prolactin. Among the genes screened, several were down-regulated, and we investigated one clone. Decreased expression of the clone was confirmed by RT-PCR analysis (Fig. 1A, left panels). But after FSH injection the mRNA expression remained almost unchanged compared with the control (Fig. 1A, right panels). Nucleotide sequencing revealed that the clone contained an ∼1.5-kbp cDNA with an open reading frame (477 bp) potentially encoding 159 amino acid residues. The deduced amino acid sequence indicated that the amino-terminal region contained two consensus RNA-binding motifs (ribonucleoprotein (RNP)-1 and RNP-2); the carboxyl-terminal region contained a glycine-rich domain that includes an RG4 domain consisting of four tandems of the arginine-glycine-glycine (RGG) motif. There was 47–76% identity with members of the glycine-rich RNA-binding protein family isolated so far as follows: mammalian RBM3 (RNA-binding motif protein 3) (10.Derry J.M. Kerns J.A. Francke U. Hum. Mol. Genet. 1995; 4: 2307-2311Crossref PubMed Scopus (102) Google Scholar, 11.Danno S. Itoh K. Matsuda T. Fujita J. Am. J. Pathol. 2000; 156: 1685-1692Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar), axolotl RNA-binding protein (RBP) (12.Bhatia R. Dube D.K. Gaur A. Robertson D.R. Lemanski S.L. McLean M.D. Lemanski L.F. Cell Tissue Res. 1999; 297: 283-290Crossref PubMed Scopus (13) Google Scholar), and mammalian and Xenopus cold-inducible RNA-binding protein (CIRP) (13.Nishiyama H. Higashitsuji H. Yokoi H. Itoh K. Danno S. Matsuda T. Fujita J. Gene. 1997; 204: 115-120Crossref PubMed Scopus (142) Google Scholar, 14.Uochi T. Asashima M. Gene. 1998; 211: 245-250Crossref PubMed Scopus (39) Google Scholar, 15.Matsumoto K. Aoki K. Dohmae N. Takio K. Tsujimoto M. Nucleic Acids Res. 2000; 28: 4689-4697Crossref PubMed Scopus (48) Google Scholar) (Fig. 1B). Our clone showed the highest homology to Xenopus CIRP1 and CIRP2, and we named it newt RNA-binding protein (nRBP). As microarray and RT-PCR analyses demonstrated a significant reduction of nRBP mRNA following prolactin treatment (Fig. 1A), we next examined the expression of its protein. Testes were collected at 6, 12, 24, and 48 h following injection of prolactin into newts, and extracts were prepared for Western blot analyses. An antibody that we raised against the whole recombinant protein of nRBP recognized a 19-kDa protein, which had almost the same mobility on SDS-PAGE as the HA-tagged protein (20 kDa) overexpressed in HeLa cells transfected with an expression construct of the isolated cDNA (Fig. 2A, t" @default.
• W2092312800 created "2016-06-24" @default.
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• W2092312800 date "2009-08-01" @default.
• W2092312800 modified "2023-09-27" @default.
• W2092312800 title "Reduced Expression of an RNA-binding Protein by Prolactin Leads to Translational Silencing of Programmed Cell Death Protein 4 and Apoptosis in Newt Spermatogonia" @default.
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