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J. Biol. Chem., Vol. 281, Issue 11, 7498-7514, March 17, 2006
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From the Department of Biological Science and Technology, Faculty of Industrial Science and Technology, Tokyo University of Science, Yamazaki, Noda-shi, Chiba 270-8510, Japan
Received for publication, October 5, 2005 , and in revised form, December 12, 2005.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, a splice variant of mouse PRP19
corresponding to the yeast PRP19 protein, can function as a neuron-astroglial switch during the retinoic acid-primed neural differentiation of P19 cells. The -variant possesses an additional 19 amino acid residues inframe in the N-terminal region of the -variant. The forced expression of the -variant RNA caused the down-regulation of oct-3/4 and nanog mRNA expression during the 12-48 h of the late-early stages of neural differentiation and was sufficient to convert P19 cells into neurons (but not glial cells) when the cells were cultured in aggregated form without retinoic acid. In contrast, the forced expression of the -variant RNA suppressed neuronal differentiation and conversely stimulated astroglial cell differentiation in retinoic acid-primed P19 cells. Based on yeast two-hybrid screening, cyclophilin A was identified as a specific binding partner of the -variant. Luciferase reporter assay mediated by the oct-3/4 promoter revealed that cyclophilin A could act as a transcriptional activator and that its activity was suppressed by the -variant, suggesting that cyclophilin A takes part in the induction of oct-3/4 gene expression, which might lead to neuroectodermal otx2 expression within 12 h of the immediate-early stages of retinoic acid-primed neural differentiation. These results show that the -variant gene plays a pivotal role in neural differentiation and that the -variant participates in neuronal/astroglial cell fate decisions. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In Drosophila, neuronal and glial fates are determined by the gcm (glial cell missing) gene encoding a transcription factor as a binary switch (1). In a gcm mutant, glial cells are converted to neurons, whereas ectopic expression of gcm in neurons causes neuron-glial transformation. Two mammalian gcm homologs, Gcm1 and Gcm2, have been identified in mice, rats, and humans (2-6); however, the sites of Gcm1 and Gcm2 expression are largely restricted to the placenta (6, 7) and para-thyroid glands (6), respectively. Targeted disruption of the Gcm1 gene in mice results in a severe defect in labyrinth formation in the placenta (8, 9). Introduction of the Gcm1 gene into cultured embryonic brain cells results in the induction of an astrocyte lineage. Nonetheless, cultures from Gcm1-deficient mouse brain do not exhibit a significant reduction in the number of astrocytes, suggesting that mouse Gcm1 exhibits the potential to induce gliogenesis, but may function in the generation of a minor subpopulation of glial cells (10). Furthermore, Gcm2-targeted mice exhibit a selective loss of parathyroid glands, but no abnormalities were reported in the nervous system (11). Thus, in mammals, although there is evidence that neurons and glia share a common progenitor (12), the genes that might control a GCM-like switching mechanism have not yet been identified.
Recent studies have shown that the Sox9 transcription factor (13), the QKI RNA-binding protein (14), and extrinsic cues such as bone morphogenetic protein (BMP)2 and ciliary neurotrophic factor (CNTF) (15) promote the differentiation of neural progenitors into glia. Nevertheless, these limited factors may be insufficient for the regulation of glial cell differentiation because mammalian astrocytes exhibit a large heterogeneity, differing in morphology, distribution, molecular types expressed, function, and cell lineage.
In this context, to clarify the molecular mechanisms in neuronal/astroglial fate decisions in mammals, we applied a subtractive cDNA cloning strategy for the retinoic acid (RA)-induced neural differentiation system of mouse P19 embryonic carcinoma cells as described previously (16). By this approach, we isolated PRP19 (precursor RNA processing-19) and its splice variant PRP19, with an additional 19-amino acid residues in-frame. PRP19 and PRP19 are homologs of yeast Prp19, which has been identified in a screen of temperature-sensitive mutants defective in precursor RNA processing of Saccharomyces cerevisiae and shown to be essential for pre-mRNA splicing (17). Nonetheless, there is no report concerning the participation of PRP19 and PRP19 in neural differentiation so far.
In this study, we show that P19 cells modified to express PRP19 constitutively in large quantities no longer required RA for neuronal differentiation. When these cells were cultured in aggregated form, the mRNA expression of neurogenic basic helix-loop-helix (bHLH) transcription factors, including Mash-1, Ngn-1 (neurogenin-1), and NeuroD, was promoted to sufficient levels for the induction of -tubulin III-positive neurons, but not glial fibrillary acidic protein (GFAP)-positive astroglia, accompanied by the down-regulation of oct-3/4 and nanog mRNA expression. On the other hand, the forced expression of Prp19 RNA suppressed neuronal differentiation through the inhibition of CypA (cyclophilin A), which acts as a transcriptional activator, and inversely promoted glial cell differentiation in RA-primed P19 cells. The down-regulation of Prp19 RNA by the antisense 15-mer oligodeoxynucleotides (ODNs) specific for the insertional sequence of Prp19 stimulated neuronal differentiation and suppressed astroglial cell differentiation in RA-primed P19 cells. These results imply that the regulation of alternative splicing of the mouse Prp19 gene plays an important role in neuronal and astroglial cell fate decisions.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-minimal essential medium containing 10% fetal calf serum and 5 x 10-7 M all-trans-RA (Sigma) for 4 days as described previously (16). The cell aggregates were suspended by mild pipetting and transferred to tissue culture dishes. The cells were cultured in RA-free -minimal essential medium containing 10% fetal calf serum for an additional 4 days to induce -tubulin III-positive neurons and for 7 days to induce GFAP-positive astroglial cells. ICR mice were purchased from Charles River Japan (Kanagawa, Japan). Culture of Mouse Neural Stem (NS) Cells—NS cells from hippocampi of ICR mice at postnatal day 7 were cultured in serum-free Dulbecco's modified Eagle's medium/F-12 nutrient mixture containing 20 ng/ml mouse epidermal growth factor (EGF; Sigma) as described previously (18). To induce neural differentiation, the spheres formed by NS cells were plated onto tissue culture dishes precoated with 20 mg/ml poly-L-lysine and cultured in EGF-free medium containing 20 ng/ml recombinant rat CNTF (R&D Systems, Minneapolis, MN) for 2 weeks.
Subtractive Cloning—Tester poly(A)+ RNA was prepared from aggregated P19 cells treated with 5 x 10-7 M RA for 24 h, and firststrand cDNA was hybridized with excess driver RNA prepared from untreated P19 cells, and then a subtractive cDNA probe was prepared by the addition of the cross-linking agent 2,5-diaziridinyl-1,4-benzoquinone and used to screen 9 x 105 plaques of rat brain 5'-stretch plus a 
gt11 cDNA library (Clontech) as described previously (16). The cDNAs inserted into the positive phage were then amplified by PCR using primers based on the phage DNA sequence: 5'-primer, 5'-GCC ACG ACT CCT GGA GCC CG-3'; and 3'-primer, 5'-CAC CAG ACC AAC TGG TAA TG-3'. The amplified cDNAs were digested with EcoRI and subcloned into the EcoRI site of pGEM-7zf(+) (Promega Corp., Madison, WI).
A rat full-length Prp19 cDNA clone was isolated from the second screening of 4.5 x 105 plaques from a rat brain gt10 cDNA library (Clontech) using the 1.1-kb EcoRI cDNA fragment obtained from the first screening as a probe and then subcloned into the EcoRI site of pGEM-7zf(+). The plasmid was designated as pGEM-Prp19.
Primers Used for PCR Amplification—Total RNAs extracted from the spheres of P19 cells and mouse NS cells were reverse-transcribed, and cDNAs were amplified by PCR as described previously (18). The primer sequences used were as follows: mouse Prp19 and Prp19, 5'-CGC CAT GTC CCT GAT CTG CTC G-3' (5'-primer) and 5'-GAT GCC ACC TGC CGG TAC TTG C-3')(3'-primer); glyceraldehyde-3-phosphate dehydrogenase, 5'-CCA CAG TCC ATG CCA TCA CTG CC-3' (5'-primer) and 5'-GGC AGT GAT GGC ATG GAC TGT GG-3' (3'-primer) (19); ribosomal phosphoprotein, 5'-CAG CTC TGG AGA AAC TGC TG-3' (5'-primer) and 5'-GTG TAC TCA GTC TCC ACA GA-3' (3'-primer) (20); oct-3/4, 5'-CCT GGC TAA GCT TCC AAG GGC-3' (5'-primer) and 5'-GTT CTA GCT CCT TCT GCA GGG C-3' (3'-primer) (21); nanog,5'-CTA TGA TCT TTC CTT CTA GAC ACT G-3' (5'-primer) and 5'-AGC CCA AAG CTT GCG TAA GTC TCA TAT T-3' (3'-primer) (22, 23); Mash-1,5'-CAC AAG TCA GCG GCC AAG CAG-3' (5'-primer) and 5'-GAT CCC TCG TCG GAG GAG TAG-3' (3'-primer) (24); Ngn-1, 5'-CCT TTG GAG ACC TGC ATC TC-3' (5'-primer) and 5'-GAT GTA GTT GTA GGC GAA GC-3' (3'-primer) (25); NeuroD, 5'-GCA TGC ACG GGC TGA ACG C-3' (5'-primer) and 5'-GGG ATG CAC CGG GAA GGA AG-3' (3'-primer) (26); GFAP, 5'-TTT CTC CTT GTC TCG AAT GA-3' (5'-primer) and 5'-GGT TTC ATC TTG GAG CTT CT-3' (3'-primer) (25); otx2,5'-CCG ACT TTG CGC CTC CAA ACA A-3' (5'-primer) and 5'-GGT TGA TGG ACC CTT CTA AGG C-3' (3'-primer) (27); retinoic acid receptor (RAR), 5'-CAT CAC AAC TAC CTG CCA GAC TC-3' (5'-primer) and 5'-GTG CGC TTT GCG AAC CTT CTC AA-3' (3'-primer) (28); RAR, 5'-CGC CTC CAC ACC TAG AGG ATA AG-3' (5'-primer) and 5'-TGG GGA ATG TCT GCA ACA GCT GG-3' (3'-primer) (29); and CypA,5'-CGC GAA TTC CAG CCA TGG TCA ACC CCA C-3' (5'-primer) and 5'-CCA CTC GAG TTA GAG CTG TCC ACA GTC G-3' (3'-primer) (30).
The Prp19 PCR product was subcloned into the EcoRV site of pGEM-5zf(+) and designated as pGEM-Prp19. The CypA PCR product was digested with NsiI and inserted into the EcoRI-XhoI site of pACT2 (Clontech) and named pACT2-CypA.
Real-time PCR Analysis of PRP19 and PRP19 Expression during Neural Differentiation—To generate cDNA from P19 cell culture samples, 5 µg of total RNAs was reverse-transcribed with random primer. Real-time PCR was performed using SYBR Green PCR Master Mix (Eurogentec, Seraine, Belgium) according to the manufacturer's protocol. The specific primers used were as follows: Prp19, 5'-ATC AAC AAC CAG CCT CTC TCA GAG-3' (5'-primer) and 5'-TGC AGC ATG ACT GCA TCC C-3' (3'-primer); and Prp19, 5'-CAG AGG AGC AGC TCA TCG ACA-3' (5'-primer) and 5'-AGG GTA GGC CAC TGT GAG GAC T-3' (3'-primer). Fluorescence was detected at the end of each cycle to monitor the amount of PCR product. The quantities of specific mRNA in the sample were measured according to the corresponding gene-specific standard curve. Amplification of specific transcripts was confirmed by a final melting curve profile and gel electrophoresis. The levels of Prp19 and Prp19 transcripts at each time point were calibrated as compared with those of ribosomal phosphoprotein transcripts as an internal control (31).
Northern Blot Analysis—Aliquots of 20 µg of total RNAs were electrophoresed on 6% formaldehyde-containing 1% agarose gel, transferred to Hybond-N+ nylon membrane (Amersham Biosciences, Buckinghamshire, UK), and hybridized with 32P-labeled PCR-amplified probe as described above. Developed x-ray films were scanned into a Macintosh G4, and the expression levels of these RNAs were quantified using the NIH Image 1.62 ppc program.
Construction of Expression Vectors—pcDNA3-EF1--sense Prp19 and pcDNA3-EF1--antisense Prp19 expression vectors were constructed by the insertion of the Prp19 cDNA fragment into the EcoRV site of pcDNA3-EF1- (16). The pcDNA3-EF1--sense Prp19 expression vector was constructed by the insertion of the ApaI-HindIII fragment of pGEM-Prp19 into the BamHI-HindIII site of pcDNA3-EF1--sense Prp19. pcDNA3-EF1--sense CypA and pcDNA3-EF1--antisense CypA expression vectors were constructed by the insertion of the blunt-ended EcoRI-XhoI CypA fragment of pACT2-CypA into the EcoRV site of pcDNA3-EF1-. These vectors were introduced into P19 cells by lipofection with N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium methyl sulfate (Roche Applied Science, Mannheim, Germany) and cultured in the presence of 400 µg/ml G418 (Wako, Tokyo, Japan) for selection. The stable transfectants of the pcDNA3-EF1--sense Prp19, pcDNA3-EF1--antisense Prp19, pcDNA3-EF1--sense Prp19, pcDNA3-EF1--sense CypA, and pcDNA3-EF1--antisense CypA vectors were designated as -S1, -A1, -S1, CypA-S1, and CypA-A1 cells, respectively.
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and PRP19 were prepared using glutathione S-transferase-fused rat PRP19 and synthetic peptides specific for PRP19 (SPALPQSSQWPTLSQ), respectively. Cells were lysed in SDS sample buffer (62.5 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, and 5% 2-mercaptoethanol). Aliquots of 30 µg of cell lysate were heated at 95 °C for 5 min and subjected to 10% SDS-PAGE. The proteins were transferred to a clear blot membrane (Atto Bioscience, Tokyo). The membranes were blocked in 20 mM Tris-HCl-buffered saline (pH 7.4) and 0.01% Tween 20 containing 1% nonfat dry milk for 1 h at room temperature and incubated overnight at 4 °C with anti-PRP19 antibody (1:500 dilution), anti-PRP19 antibody (1:100), anti-CypA antibody (1:10000; Upstate, Lake Placid, NY), anti--tubulin III antibody (1:400; Sigma), anti-GFAP antibody (1:400; Sigma), anti-neural cell adhesion molecule (N-CAM) antibody (1:200; Sigma), anti-polypyrimidine tract-binding protein-associated splicing factor (PSF) antibody (1:100; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-CDC5L (CDC5-like) antibody (1:2000) (32), or anti-actin antibody (1:500; Sigma). After washing three times with 20 mM Tris-HCl-buffered saline (pH 7.4) and 0.01% Tween 20, the membranes were incubated with adequate secondary antibodies. Anti-mouse IgG conjugated with horseradish peroxidase (1:5000; Sigma), anti-guinea pig IgG conjugated with horseradish peroxidase (1:2000; MP Biomedicals, Irvine, CA), anti-rabbit IgG conjugated with horseradish peroxidase (1:5000; BIOSOURCE), or anti-goat IgG conjugated with horseradish peroxidase (1:2000; Santa Cruz Biotechnology, Inc.) was used as a secondary antibody as indicated. Signals were visualized with the ECL system (Amersham Biosciences).
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-tubulin III (1:400) or GFAP (1:400) under the same conditions described above. After washing with Ca2+/Mg2+-free phosphate-buffered saline, the samples were incubated with biotin-conjugated rabbit anti-mouse IgG + IgA + IgM (Nichirei, Tokyo) as a secondary antibody, followed by incubation with peroxidase-conjugated streptavidin (Nichirei). Visualization was carried out using 3,3'-diaminobenzidine. The samples were counterstained with hematoxylin.
Construction of Enhanced Green Fluorescent Protein (EGFP)-fused PRP19 and PRP19 Expression Vectors—EGFP-fused PRP19 and PRP19 expression vectors were constructed by insertion of the entire coding regions of PRP19 and PRP19, respectively, downstream of the EGFP-coding sequence in-frame into the pEGFP-C1 vector (Clontech) and designated as pEGFP-PRP19 and pEGFP-PRP19, respectively. P19 cells were transfected with the pEGFP-PRP19 and pEGFP-PRP19 expression vectors using Lipofectamine PLUSTM (Invitrogen) and cultured in the presence of 400 µg/ml G418 for selection. To see the subcellular localization of PRP19 and PRP19, the cells were fixed with 4% paraformaldehyde and washed with Ca2+/Mg2+-free phosphate-buffered saline. The nuclei were stained with 1 µg/ml Hoechst 33258. The cells were observed under a fluorescence microscope (Axioplan 2, Carl Zeiss, Oberkochen, Germany).
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RNA by Antisense ODNs—The sequence of antisense ODNs used was 5'-TAGGCCACTGTGAGG-3', which corresponds to nucleotides 282-296 of the specific region of mouse Prp19 cDNA; and the sequence of sense ODNs used for the control experiment was 5'-CCTCACAGTGGCCTA-3'. P19 cells were treated with various concentration of ODNs as described previously (33).
Immunoprecipitation—The hemagglutinin (HA)-fused CypA expression vector was constructed by insertion of the blunt-ended BglII open reading frame fragment of pACT2-CypA into the EcoRV site of pcDNA3-EF1-. P19 cells (3 x 105) were transfected with 2 µg of DNA using Lipofectamine PLUSTM. The cells were harvested 48 h after transfection and lysed with immunoprecipitation buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM MgCl2, 0.1% Nonidet P-40, and 1 mM phenylmethylsulfonyl fluoride). For immunoprecipitations, equal amounts of cell lysate proteins were incubated overnight with protein A-Sepharose beads charged with anti-PRP19 or anti-HA antibody at 4 °C. The beads were washed twice with immunoprecipitation buffer. Immunoprecipitated proteins or cell lysates were mixed with 3x SDS sample buffer and separated by SDS-PAGE. Western blot analysis was performed as described above with the appropriate antibodies.
Gel Filtration of the Spliceosome—P19 cells (1 x 108) were homogenized in lysis buffer (20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EGTA, 1 mM dithiothreitol, 10% glycerol, 0.1% Nonidet P-40, and 0.1 mM phenylmethylsulfonyl fluoride) and centrifuged at 105,000 x g for 1 h. The resulting supernatant was applied to a Sepharose 4B column (bed volume of 38.4 ml; Amersham Biosciences AB, Uppsala, Sweden) and eluted with 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM EGTA, 1 mM dithiothreitol, 10% glycerol, and 0.1 mM phenylmethylsulfonyl fluoride. Fractions of 0.78 ml were collected and analyzed by Western blotting with the appropriate antibodies as described above.
Yeast Two-hybrid Screening—Mouse Prp19-specific region cDNA (amino acids 83-101) was inserted into pAS2-1 (Clontech) in-frame with the Gal4 DNA-binding domain as bait. An RA-treated P19 cell cDNA library was constructed using pACT2. Yeast two-hybrid screening was performed as described previously (34).
Chromatin Immunoprecipitation—P19 cells treated with RA for 0 and 6 h were cross-linked with 1% formaldehyde for 10 min. The nuclear fraction was isolated and sonicated to shear genomic chromatin; clarified by centrifugation; precleared with protein A-Sepharose; and immunoprecipitated with anti-CypA antibody, anti-RAR antibody (Santa Cruz Biotechnology, Inc.), or anti-acetylated histone H3 antibody (Upstate). Following immunoprecipitation and immobilization of immunocomplexes, proteinase K digestion was allowed to proceed for 1 h at 45 °C. DNA was purified by phenol/chloroform extraction. PCR was carried out on the purified DNA using primers to the oct-3/4 promoter region (RAREoct; -412 to +39, 5'-CTA GGA CTC GAG ACG GGT GGG TAA G-3' (5'-primer) and 5'-CAC CCG GAT CCG GGG GCC TGG-3' (3'-primer) (35)).
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-galactosidase expression vector pcDNA3.1/Myc-His/LacZ (Invitrogen) together with a total of 1 µg of pcDNA3-EF1--sense CypA, pcDNA3-EF1--sense Prp19, and vacant pcDNA3-EF1- expression vectors in various combinations using Lipofectamine PLUSTM. After 24 h of transfection, the cells were treated with RA for an additional 24 h and lysed with the lysis buffer from the Promega luciferase assay kit, and luciferase activities were determined according to the manufacturer's instructions using a Luminous CT9000D luminometer (DiaIatron, Tokyo). Reporter gene activities were normalized using -galactosidase activity as an internal control. |
RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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cDNA—To isolate the early response genes that participate in the RA-primed neural differentiation of mouse P19 cells, we employed a subtractive cDNA cloning strategy to enrich genes that are highly expressed in P19 cells after 24 h of RA treatment as described previously (16). Using the subtractive probes obtained, 
9 x 105 plaques from the rat brain gt11 cDNA library were screened, and nine clones whose expression levels were stimulated by >2-fold over the untreated control cells were identified. A homology search of the nine clones in the DDBJ, EBI, and GenBankTM Data Banks showed that five were novel cDNA clones, and the remaining four clones were identified as acyl-CoA synthetase III, cytochrome c oxidase, Trip15/CSN2, and fibroblast growth factor receptor-2. In this study, we focused on one of the five novel clones (30-1-2) because the increment in its expression level was highest of the five novel clones during the RA-primed neural differentiation of P19 cells. To isolate the full-length 30-1-2 cDNA, we screened the rat brain gt10 cDNA library using the 1.1-kb EcoRI cDNA fragment obtained from the first screening as a probe and isolated the 1853-bp full-length cDNA clone. The cDNA encodes 504 amino acid residues, and its molecular mass is 55,238 Da. The cDNA sequence was deposited in DDBJ/GenBankTM/EBI Data Bank under accession number AB020022
[GenBank]
(Fig. 1A). A conserved domain search identified five WD40 repeat domains in the C-terminal region and a modified RING finger domain in the N-terminal region. A protein BLAST search in the GenBankTM Data Bank revealed that the protein is identical or closely related to PRP19 in Mus musculus (100% identity), Homo sapiens (99.4%), and S. cerevisiae (36.0%) (Fig. 1B). Therefore, we designated the full-length 30-1-2 cDNA clone isolated from the rat brain cDNA library as rat Prp19. The deduced amino acid sequences of the rat and mouse PRP19 proteins are completely identical, suggesting that they could function in a similar manner. Thus, we used rat Prp19 cDNA throughout the experiment, even though P19 cells were originally established in a mouse embryo (36).
Detection of the PRP19 Splice Variant PRP19—Because Prp19 mRNA expression was up-regulated in the RA-primed neural differentiation of P19 cells, we analyzed the expression pattern of the Prp19 gene during the differentiation of mouse hippocampus-derived NS cells (Fig. 2). The NS cells formed neurospheres in medium containing 20 ng/ml EGF and actively incorporated bromodeoxyuridine (Fig. 2A, panel a) as described previously (18). When EGF-generated spheres were plated onto poly-L-lysine-coated tissue culture dishes after the removal of EGF and cultured in medium containing 20 ng/ml CNTF for 2 weeks, the NS cells differentiated into -tubulin III-positive neurons (Fig. 2A, panel b) and myelin basic protein-positive oligodendrocytes (panel c). The expression level of Prp19 mRNA during the differentiation of NS cells was analyzed by semiquantitative real-time PCR. In the presence of EGF, the expression level of Prp19 mRNA in NS cells was similar to that in untreated P19 cells, whereas the expression level in CNTF-treated NS cells was 1.5-fold higher than that in NS cells cultured with EGF (Fig. 2B). Furthermore, an additional PCR product termed Prp19 was detected in cDNA derived from CNTF-treated NS cells.
Sequence analysis revealed that this Prp19 cDNA possesses an additional 57 bp encoding 19 amino acid residues in-frame (DDBJ/Gen-BankTM/EBI accession number AB222602
[GenBank]
), located 245 bp downstream of the initiation codon of Prp19, and that its molecular mass is 57,301 Da (Fig. 2C). The chromosomal organization of exons encoding Prp19 was determined by alignment of the Prp19 cDNA in the mouse genome data base. The Prp19 gene was deduced to be composed of 16 exons with lengths ranging from 59 to 174 bp (Fig. 3A). Mouse genome sequence analysis revealed that the inserted sequence of Prp19 was derived from intron 3 by alternative splicing at a 3'-AG splice site and is located in-frame just before exon 4 (Fig. 3B). Both PRP19 and PRP19 possess three domains, including a modified RING finger domain, a nuclear localization signal, and WD40 repeat domains (Fig. 3C). To analyze the intracellular localization of proteins, pEGFP-PRP19 and pEGFP-PRP19 expression vectors were constructed and introduced into P19 cells (Fig. 3D). EGFP-fused PRP19 was detected in the nuclei, whereas EGFP-fused PRP19 was observed in the nuclei and cytoplasm. The same results were obtained by immunocytochemical analysis with anti-PRP19 and anti-PRP19 antibodies (data not shown).
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mRNA during Neural Differentiation—To examine the expression levels of Prp19 and Prp19 mRNAs during the neural differentiation of P19 cells, total RNAs were extracted from P19 cells treated with RA for various times and analyzed by real-time PCR. The primers were designed to amplify the specific fragments of two splice variants. The expression of Prp19 mRNA was induced shortly after the addition of RA. Maximal expression was observed after 12 h of treatment and was estimated to be 3.0-fold of the original level at 0 h (Fig. 4A, upper panel). The maximal expression level of Prp19 was estimated to be 2.5-fold of the expression level of the untreated cells, was observed 1-2 days after RA treatment, and decreased steeply thereafter to the basal level (Fig. 4A, lower panel). The expression levels of PRP19 and PRP19 proteins were also analyzed by Western blotting using specific antibodies (Fig. 4B). Within 3 h of RA treatment, the expression level of PRP19 was augmented, as observed for its mRNA expression level (Fig. 4B, upper panel). In contrast, the expression level of PRP19 increased steeply after 1 day of RA treatment and was estimated to be 4.2-fold of the original level at 0 h (Fig. 4B, lower panel). Thus, these results indicated that Prp19 gene expression was induced prior to Prp19 gene expression.
Participation of PRP19 and PRP19 in Neural Differentiation—To demonstrate the participation of PRP19 and PRP19 in neural differentiation, we established P19 cell lines in which the exogenous sense and antisense Prp19 and sense Prp19 RNAs were constitutively expressed. The stable transfectants of the pcDNA3-EF1--antisense Prp19, pcDNA3-EF1--sense Prp19, and pcDNA3-EF1--sense Prp19 expression vectors were designated -A1, -S1 and -S1, respectively. The expression level of PRP19 in -A1 and -S1 cells was estimated to be 0.6- and 1.5-fold of that in empty vector-introduced V1 cells (Fig. 5A). The expression level of PRP19 in -S1 cells was 1.9-fold higher than that in V1 cells, whereas the expression level of PRP19 in -S1 and -A1 cells was similar to that in V1 cells.
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and PRP19 on the neural differentiation of P19 cells by immunocytochemical analysis (Fig. 5B). -S1 cells could differentiate into -tubulin III-positive neurons after the simple aggregation culture without the addition of RA (Fig. 5, B, panel i; and C). In contrast, V1, -A1, and -S1 cells did not differentiate into neurons in the absence of RA (Fig. 5, B, panels a, e, and m; and C). In the presence of RA, all the transfectants differentiated into neurons (Fig. 5, B, panels b, f, j, and n; and D). Nonetheless, the neuronal differentiation of -S1 cells was significantly stimulated compared with that of V1 cells (Fig. 5D). The neuronal differentiation of -A1 and -S1 cells was decreased conversely.
The effects of PRP19 and PRP19 on GFAP-positive astroglial cell differentiation were also examined. Although the differentiation of all transfectants to GFAP-positive astroglial cells was not observed without the addition of RA (Fig. 5B, panels c, g, k, and o), all transfectants differentiated into astroglial cells in the presence of RA (panels d, h, l, and p). The differentiation of -S1 cells to astroglial cells induced by RA was significantly increased (Fig. 5, B, panel p; and E), whereas the astroglial differentiation of -A1 cells was strikingly suppressed (Fig. 5, B, panel h; and E).
We further analyzed the effects of exogenous expression of PRP19 and PRP19 on the expression of neuronal and astroglial cell marker proteins by Western blotting with anti--tubulin III, anti-N-CAM, and anti-GFAP antibodies (Fig. 6). In aggregated -S1 cells, the expression levels of the neuronal markers -tubulin III and 180-kDa neuron-specific N-CAM were markedly induced without any addition of RA (Fig. 6A). The stimulatory effects of the forced expression of PRP19 on the neuronal markers in RA-primed -S1 cells were also observed. On the other hand, the expression of neuronal markers in -S1 and -A1 cells was reduced in the presence of RA. In all transfectants, the expression of GFAP, a marker of astroglial cells, was not observed without RA (Fig. 6B). In the presence of RA, the expression levels of GFAP in -S1 and -S1 cells were significantly enhanced. Conversely, the expression of GFAP in RA-primed -A1 cells was decreased.
These results were consistent with the data from immunocytochemical analysis. Thus, it seems that PRP19 plays a crucial role in the early stages of neural differentiation of P19 cells, whereas PRP19 acts as a stimulator of astroglial cell differentiation and conversely exhibits an inhibitory effect on neuronal differentiation.
Effect of Prp19-specific Antisense ODNs on Neural Differentiation—To further confirm the function of Prp19 in neural differentiation, we examined the effect of Prp19-specific antisense 15-mer ODNs on the neural differentiation of RA-primed P19 cells. When P19 cells were treated with 5 and 10 µM antisense Prp19 ODNs, the expression level of PRP19 protein was reduced to 40 and 30% of the level in untreated control cells, respectively (Fig. 7A). On the other hand, PRP19 expression levels in antisense ODN-treated cells were not affected. Immunocytochemical analysis revealed that, in the presence of the antisense ODNs, the neuronal differentiation of RA-primed P19 cells was dose-dependently stimulated, whereas astroglial cell differentiation was strikingly suppressed (Fig. 7, B-D). The inhibition observed in the presence of sense ODNs was perhaps due to the nonspecific inhibition of protein synthesis, as observed previously (33).
We also examined the effect of antisense ODNs on the expression levels of neuronal and astroglial cell markers in RA-primed P19 cells by Western blotting. The expression of the neuron-specific markers -tubulin III and 180-kDa neuron-specific N-CAM was increased in antisense ODN-treated cells compared with sense ODN-treated cells (Fig. 7E). In contrast, the expression level of GFAP in antisense ODN-treated cells was down-regulated (Fig. 7F). Thus, these results were consistent with the data from the forced expression of Prp19 RNA as described above.
Effect of PRP19 and PRP19 on Neurogenic Gene Expression—The data described above suggested that the forced expression of Prp19 RNA promoted both neurogenesis and gliogenesis, whereas the forced expression of Prp19 RNA promoted astroglial (but not neuronal) differentiation. To further explore the molecular mechanisms underlying the neural differentiation regulated by PRP19 and PRP19, we investigated the expression patterns of neural differentiation-related genes such as oct-3/4, nanog, Mash-1, and GFAP by Northern blotting. The transcription factor Oct-3/4 maintains the undifferentiated state of P19 cells as well as embryonic stem (ES) cells (37). The down-regulation of oct-3/4 mRNA is required for the onset of neural differentiation (16). On the basis of these facts, we analyzed the expression pattern of oct-3/4 mRNA during the neural differentiation of RA-primed V1, -A1, -S1, and -S1 cells (Fig. 8, A, upper panels; and F). The expression level of oct-3/4 mRNA in V1 cells was reduced to 50% of the original level after 28 h of RA treatment and became undetectable after 30 h. In contrast, the expression level of oct-3/4 mRNA in -S1 cells was lower than that in V1 cells and decreased to <50% of the original level after 20 h of RA treatment. Moreover, in aggregated -S1 cells (but not in aggregated V1 cells), the down-regulation of oct-3/4 mRNA expression occurred within 48 h in the absence of RA (Fig. 8, A, lower panels; and F). The decrease in the expression level of oct-3/4 mRNA was slowed in RA-primed -A1 and -S1 cells, and oct-3/4 mRNA expression was continued by at least 40 h.
Nanog is a recently discovered homeodomain transcription factor and is both sufficient and necessary for the maintenance of pluripotency of mouse ES cells and mouse epiblasts, independent of the leukemia inhibitory factor (LIF)/STAT3 (signal transducer and activator of transcription) signaling pathway (22, 23). We therefore analyzed the expression level of nanog mRNA during neural differentiation. Northern blot analysis revealed that nanog mRNA expression was induced in all aggregated transfectants regardless of treatment with RA, and its high level of expression was maintained until the onset of neural differentiation (Fig. 8B, upper panels). In RA-primed -S1 cells, the enhanced expression was lowered to a negligible level within 20 h. In contrast, in -S1 cells, substantial expression was detected even at 28 h after RA addition and became undetectable at 32 h. In V1 and -A1 cells, the enhanced expression disappeared after 24 h. In -S1 cells, the down-regulation of aggregation culture-induced nanog mRNA expression began after 20 h and reached a negligible level at 48 h without the addition of RA (Fig. 8, B, lower panels; and J). On the other hand, in the absence of RA, the down-regulation of nanog transcription was not observed in aggregated V1 cells. From these results, it seems likely that PRP19 participates in the down-regulation of oct-3/4 and nanog gene expression in the early stages of RA-primed neural differentiation of P19 cells, whereas PRP19 suppresses the down-regulation of the expression of these genes.
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-S1 cells was substantially detected after 28 h, when significant down-regulation of oct-3/4 and nanog mRNA expression was observed (Fig. 8, C, upper panel; and G). In contrast, the induction of Mash-1 mRNA expression was observed after 60 h in RA-primed V1 cells, in which oct-3/4 and nanog gene expression was sustained for a longer time compared with -S1 cells treated with RA. Similar to V1 cells, the induction of Mash-1 mRNA expression in RA-primed -A1 and -S1 cells occurred later than in -S1 cells. The induction of Mash-1 gene expression was observed in aggregated -S1 cells in the absence of RA, corresponding to the down-regulation of oct-3/4 and nanog gene expression (Fig. 8, C, lower panel; and K). The expression of Ngn-1 and NeuroD mRNAs was also transiently induced in all transfectants after 3-5 days of RA treatment (data not shown). However, the period of time required for the maximal expression of both mRNAs was different among these transfectants. Maximal expression of the Ngn-1 and NeuroD genes in -S1 cells was observed after 3 days of RA treatment and occurred faster than in any other transfectant. Thus, the results support the idea that PRP19 participates in the induction of neurogenic gene expression through the down-regulation of oct-3/4 and nanog mRNA expression.
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-S1 cells was already observed after 6 days of RA treatment, and its expression level was much more robust than that of any other transfectant throughout neural differentiation. In RA-primed -A1 cells, the induction of GFAP mRNA expression was detected at 8 days, 1-2 days later than that observed in other cell lines. This delay coincided with a lower level of astroglial differentiation determined by immunocytochemical and Western blot analyses using anti-GFAP antibody as shown in Figs. 5 and 6. Under these conditions, the level of ribosomal phosphoprotein mRNA analyzed as an internal control was not significantly altered, although some bias was observed (Fig. 8E).
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in pre-mRNA Splicing—Pre-mRNA splicing is a prerequisite for the expression of most eukaryotic genes. PRP19, CDC5L, and PSF are major components of the spliceosome (32, 42). To determine whether PRP19 and PRP19 participate in neural differentiation through their pre-mRNA splicing functions, we analyzed the spliceosome prepared from RA-primed P19 cells by immunoprecipitation using anti-PRP19 and anti-PRP19 antibodies. As shown in Fig. 9A, PSF and CDC5L were efficiently precipitated by anti-PRP19 (but not anti-PRP19) antibody, even though their quantitative changes were not obvious after 24 h of RA treatment. Sepharose 4B gel filtration analysis of the spliceosome also revealed the partial coexistence of PRP19 and CDC5L in the higher molecular mass fractions corresponding to 930 kDa (Fig. 9B). After 24 h of RA treatment, the localization of both PRP19 and CDC5L was shifted to the lower molecular mass region, showing the possibility that the qualitative change in the spliceosome occurs during neural differentiation. Yeast two-hybrid analysis using PRP19 as bait revealed that PRP19 could directly bind to CDC5L (amino acids 203-803) via its N-terminal region (amino acids 1-166) (Fig. 9C). Thus, it appears that PRP19 (but not PRP19) takes part in pre-mRNA splicing during neural differentiation as one of its major functions.
Identification of PRP19-specific Binding Proteins—To further analyze the role of PRP19 in the process of neural differentiation, cDNA clones encoding mouse PRP19-binding proteins were searched using a yeast two-hybrid screening system. Yeast strain Y153 (Leu-/Trp-/His-) was cotransfected with a bait vector expressing a fusion protein composed of the yeast Gal4 DNA-binding domain and sequence 83-101 of mouse PRP19 and with an RA-treated P19 cell cDNA library directing the synthesis of fusion proteins composed of cDNA-encoded proteins and the Gal4 transcriptional activation domain (Fig. 10A). Strain Y153 contains the HIS3 and lacZ genes linked to the GAL4 promoter. Upon transfection of 1.3 x 106 Y153 cells, 38 His+ clones were developed, and 10 of the 38 clones were -galactosidase-positive. Sequence analysis of the clones showed that three of the 10 clones were novel cDNA clones, and six were identified as known cDNA clones, such as CypA; prothymosin-; endometrial bleeding-associated factor; proteasome activator subunit-4; and ribosomal proteins S7, S15a, and S26. Among them, CypA is specifically expressed in neurons and participates in the neuronal (but not astroglial) differentiation of P19 cells (44, 45). Therefore, in this study, we focused on the functional analysis of CypA in neural differentiation regulated by PRP19 and PRP19. Yeast two-hybrid analysis showed that the isolated CypA cDNA encoding amino acids 61-163 specifically bound to the N-terminal region (amino acids 83-101) of PRP19, although association with full-length PRP19 was not detected (Fig. 10, A and B).
P19 cells were transfected with the pcDNA3-EF1--HA-CypA plasmid vector to express HA-tagged full-length CypA and cultured for 24 h. The cells were then treated with RA for an additional 24 h and subjected to Western blot analysis. Significant expression of HA-tagged CypA and PRP19 was observed (Fig. 10C). Immunoprecipitation of the extract from the cells with anti-HA antibody revealed that CypA coprecipitated with PRP19. In the absence of RA, coprecipitation was not detected, as in the vacant vector-transfected cells. These results imply that the conformational change in PRP19 after RA treatment contributes to the association with CypA.
Involvement of CypA in Neurogenesis—To confirm the participation of CypA in neuronal differentiation, we constructed pcDNA3-EF1--sense CypA and pcDNA3-EF1--antisense CypA expression vectors and introduced them into P19 cells. The resulting stable transfectants were designated CypA-S1 and CypA-A1 cells, respectively. The expression levels of CypA-S1 and CypA-A1 cells were estimated to be 1.32- and 0.68-fold of that of empty vector-transfected V1 cells, respectively (Fig. 11A). In the absence of RA, CypA-S1 cells differentiated into -tubulin III-positive neurons after the simple aggregation culture, as observed in -S1 cells (Fig. 11, B, panel d; and C). In the presence of RA, the neuronal differentiation of CypA-S1 cells was enhanced, whereas the neurogenesis of CypA-A1 cells was vigorously suppressed compared with V1 cells (Fig. 11, B, panels e and h; and D). Although the effect of sense CypA on astroglial cell differentiation was not evident in the presence of RA, the appearance of GFAP-positive astroglia in CypA-A1 cells was strongly suppressed (Fig. 11 B, panels f and i; and E). Thus, these results are consistent with a previous report except for the suppression of gliogenesis (45), suggesting that CypA participates in the early stages of neural differentiation.
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-globulin (160 kDa), and bovine serum albumin (66 kDa) were used as size markers. C, yeast two-hybrid analysis of the interaction between PRP19