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Genomic organization and alternative transcripts of the human PQBP-1 gene
Gene 259 (2000) 69–73 www.elsevier.com/locate/gene
Genomic organization and alternative transcripts of the human PQBP-1 gene Kazuya Iwamoto, Yu-Tzu Huang, Shintaroh Ueda * Department of Biological Sciences, Graduate School of Science, University of Tokyo, Tokyo, Japan Received 14 April 2000; received in revised form 19 June 2000; accepted 25 August 2000 Received by T. Gojobori
Abstract PQBP-1 has been identified as a protein that binds to huntingtin, androgen receptor and transcription factor Brain-2 through their homopolymeric glutamine repeats. We here report the genomic organization of the human PQBP-1 gene and its multiple alternative transcripts. The coding region of PQBP-1 comprises six exons and five introns, and four types of alternative transcript, designated PQBP-1a to PQBP-1d, were found in addition to the PQBP-1 transcript reported originally. All of the PQBP-1 transcripts retain the WW domain in the N-terminal region, a potent transactivator domain. On the other hand, there is a wide variation in their C-terminal regions. Importantly, PQBP-1a and PQBP-1d lack the domain responsible for the interaction with homopolymeric glutamine repeats and a nuclear localization signal. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Brn-2; Npw38; Polyglutamine; Polyglutamine-binding protein; WW domain
1. Introduction Homopolymeric glutamine repeats have functional features that include activation of transcriptional activity in a length-dependent manner (Gerber et al., 1994) and sex determination (Bowles et al., 1999). Sequence comparison of transcription factors reveals a great variation among species in the position and repeat number of homopolymeric amino acid repeats, including polyglutamine (Sumiyama et al., 1996; Nakachi et al., 1997). In Brain-2, one of the class III POU transcription factors, there is a wide variation of the homopolymeric glutamine repeats among vertebrates. The mammalian Brain-2 genes have long homopolymeric glutamine repeats, which have been well conserved among mammals not only in position but also in repeat number. The reptile Brain-2 also has homopolymeric glutamine repeats, but there is a wide variation in repeat number between mammals and reptiles. In addition to the homopolymeric glutamine repeats common to mammals and Abbreviations: cDNA, DNA complementary to RNA; kb, kilobase(s); PCR, polymerase chain reaction; PQBP-1, poly-glutamine tract-binding protein-1; RT-PCR, reverse transcription-PCR. * Corresponding author. Tel.: +81-3-5841-4486; fax: +81-3-3818-7547. E-mail address: [email protected] (S. Ueda)
reptiles, there are reptile-specific homopolymeric glutamine repeats. In Xenopus, which has two Brain-2 homologues, one homologue ( XLPOU3) lacks homopolymeric glutamine repeats and the other ( XLPOU3B) shows a short run of glutamine repeats in a position different from those in the mammalian orthologues. The zebrafish orthologue (zp-47) shows no homopolymeric glutamine repeats (Baltzinger et al., 1992; Hara et al., 1992; Li et al., 1993; Schreiber et al., 1993; Baltzinger et al., 1996; Spaniol et al., 1996; Sumiyama et al., 1996; Nakachi et al., 1997). A similar situation is observed with other transcription factors. These findings suggest the possibility that differences in position and number of homopolymeric glutamine repeats lead to diversification of protein function during evolution (Sumiyama et al., 1996; Nakachi et al., 1997). In the last decade, proteins containing homopolymeric glutamine repeats have been extensively studied from an etiological viewpoint, because expansion of homopolymeric glutamine repeats causes progressive neurodegenerative disorders, so-called triplet diseases, such as X-linked spinal and bulbar muscular atrophy, Huntington’s disease, dentatorubral–pallidoluysian atrophy, and Machado–Joseph disease (Ashley and Warren, 1995; Paulson and Fischbeck, 1996; Perutz, 1999). Furthermore, huntingtin-associated protein, hun-
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tingtin-interacting protein, glyceraldehyde-3-phosphate dehydrogenase, and leucine-rich acidic nuclear protein have been shown to be homopolymeric glutamine repeat-binding proteins (Li et al., 1995; Burke et al., 1996; Kalchman et al., 1997; Matilla et al., 1997). Recently, a novel homopolymeric glutamine repeatbinding protein, PQBP-1, was identified by a yeast twohybrid system using the homopolymeric glutamine repeats of mouse Brain-2 as bait (Imafuku et al., 1998; Waragai et al., 1998). PQBP-1 was also identified as Npw38 through a computer-assisted search of the WW domains from the HomoProtein cDNA Bank ( Kato et al., 1994; Komuro et al., 1999). We report here the presence of multiple transcripts of the human PQBP-1 gene. Interestingly, two of them lack the polar amino acid-rich domain that is responsible for interaction with the homopolymeric glutamine repeats of Brain-2 and huntingtin. This elicits questions regarding functional diversity among alternative transcripts. We also investigate the genomic sequence and define the exon–intron structure of its coding region.
2. Materials and methods DNAs for human PQBP-1 and its alternative transcripts were obtained by RT-PCR with an Advantage cDNA PCR kit (Clontech) using human fetal brain Marathon-Ready cDNAs (Clontech) as templates. Two primer sets were used: 5∞-TGCTATCAGCTATGCCGCTGCCCGTTGC-3∞ (5∞ End Primer) and 5∞-TTTAACCCAGGGCCAGGGAGGCCGAAGC-3∞ (3∞ End Primer); 5∞-GGAATTCATGCCGCTGCCCGTTGCGCTGCAG-3∞ and 5∞-GGAATTCTCAATCCTGCTGCTTGGTTCGGG-3∞. PCR began at 94°C for 1 min, followed by 25 cycles of 94°C for 30 s and 74°C for 1 min. PCR products were purified by agarose gel electrophoresis, and ligated to the pCR2.1 vector using a TOPO TA-cloning kit (Invitrogen). We also used human adult brain cerebral cortex cDNAs (QUICK-clone cDNAs, Clontech) as RT-PCR templates. These cDNA templates are both synthesized from mRNAs that have been purified at least three times on oligo-dT columns. 5∞-ACTTTTCTTCAGCATCTGGGGACACAAG-3∞ and 5∞-GGATGGGGATGGGTGATGTGGCGGACAC-3∞ were used to detect particular alternative transcripts, PQBP-1a and PQBP-1b, respectively, in combination with the 5∞ End Primer. A genomic DNA fragment containing the entire coding region of human PQBP-1 was obtained with EXTaq polymerase ( TaKaRa) using the 5∞ and 3∞ End Primers. The PCR cycle consisted of 30 cycles at 94°C for 30 s and 72°C for 3 min 30 s. The amplified product was cloned into a plasmid vector, pCR2.1, using a TOPO TA-cloning kit as in the case of RT-PCR cloning. Sequencing templates were obtained using a GPS genome Priming System (New
England BioLab.), and nucleotide sequence determination was performed on an ABI 373S sequencer using a Dye Terminater Sequencing kit with AmpliTaq FS (PE Applied Biosystems). The nucleotide sequence data reported in this paper will appear in the DDBJ/EMBL/GenBank international nucleotide sequence database with the following accession numbers: AB041832–AB041836.
3. Results and discussion We obtained the entire coding region of the PQBP-1 gene by RT-PCR amplification using human fetal brain cDNAs as templates. In addition to a band corresponding to the expected size of the PQBP-1 cDNA reported previously, some discrete bands were observed (Fig. 1). These bands were obtained reproducibly using another PCR primer set. The nucleotide sequences of these PCR products showed that they were transcript variants of the PQBP-1; we therefore designated them as PQBP-1a to PQBP-1d. Fig. 2A shows their deduced amino acid sequences, together with the sequence of PQBP-1 ( Waragai et al., 1998; Komuro et al., 1999). We next addressed whether these transcript variants were generated by alternative splicing. We obtained PQBP-1 genomic DNA as a single 4.6 kb band by PCR amplification. Sequence comparison of the genomic PQBP-1 gene with its transcripts revealed that the coding region of PQBP-1 comprises six exons and five introns ( Fig. 3A), showing that these transcript variants were generated by alternative splicing. All the splicing accep-
Fig. 1. Alternative transcripts of human PQBP-1. Expression of PQBP-1 alternative mRNAs was analyzed by RT-PCR using human fetal brain cDNAs ( lane 1) and human adult brain cerebral cortex cDNAs ( lane 2) as templates.
K. Iwamoto et al. / Gene 259 (2000) 69–73
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Fig. 2. Structure of alternative PQBP-1 products. (A) Predicted amino acid sequences. (B) Schematic representation of their domain structures.
tor and donor sites had an AG/GT consensus sequence (Breathnach and Chambon, 1981) (Table 1). The PQBP-1 product is characterized by several domains: acidic region, WW domain, polar amino acid-rich domain, nuclear localization signal (NLS), and C -domain. The WW domain of the PQBP-1 product, 2 which binds to RNA and works as a potent transactivator ( Komuro et al., 1999), is located over two exons, exons 2 and 3. The polar amino acid-rich domain responsible for interaction with homopolymeric glutamine repeats ( Waragai et al., 1998) and the NLS are located in the same exon, exon 4. Seven Alu sequences were found in the PQBP-1 genomic sequence; six in intron 1 and one in intron 2 (data not shown). Fig. 2B shows a schematic representation of the PQBP-1 products. All of them retain the N-terminal acidic region and the WW domain, but have different C-terminal structures. PQBP-1a is generated by alterna-
tive use of two distinct splice acceptor sites in intron 3, which leads to a 14-nucleotide addition in PQBP-1a (see Fig. 3C ). This insertion event causes a reading frame shift that results in PQBP-1a lacking a polar amino acid-rich domain, NLS, and C -domain (Fig. 2). 2 Judging from the ratio of cloned DNAs regardless of cloning preference, the ratio of PQBP-1 to PQBP-1a transcripts was presumed to be approximately 10 to 1. PQBP-1b and PQBP-1c were generated by not fully splicing the introns, but their amino acid sequences remain unchanged due to the existence of a translational stop codon within unspliced intron 4; they had the polar amino acid-rich domain and NLS, but their C-terminals lacked the C -domain. PQBP-1d is generated by splicing 2 out of exon 4. This in-frame alternative splicing causes a lack of the polar amino acid-rich domain and NLS in PQBP-1d, which retains the C -domain. We also 2 obtained all these alternative transcripts of PQBP-1 by
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Fig. 3. Genomic organization of the human PQBP-1 gene and its transcripts. (A) Genomic organization of the entire coding region of the PQBP-1 gene. Exons and introns are indicated by open boxes and solid lines, respectively. (B) Alternative PQBP-1 transcripts. The shaded box represents the exon generated by alternative use of the splice acceptor site shown in (C ). Arrowheads indicate the putative translational stop codon. (C ) Alternative use of the splice acceptor site in intron 3. Upper-case letters indicate coding sequences and italics indicate the additional 14 nucleotides in the alternative transcript PQBP-1a. Table 1 Exon–intron boundaries of the human PQBP-1 gene. Exon and intron sequences are shown in upper- and lower-case letters, respectively cDNA
Exon size (bp)
Splice donor
Intron size (bp)
Splice acceptor
1–67 68–179 180–292 293–577 578–641 642–798
>67 112 113 285 64 >157
CTGGAGCCTG/gtgagacagc ACCCTTCCTG/gtgagcctgg AGTAATGCAG/gtgagttggc AGCAAGAAGG/gtaagctggg ACGCCCCCCG/gtaagtgaca
2609 628 190 214 132
gttctaccag/AACCAGAGGA ggccccatag/CGGGCTCCCT gtgtccccag/ATGCTGAAGA acttccacag/CAGTAAGCCG tcaccggcag/GGGCACGTGG
RT-PCR using human adult cerebral cDNAs as templates and confirmed the presence of PQBP-1a and PQBP-1b using specific primer sets (data not shown). We thus identified four types of PQBP-1 alternative transcripts, but might not exclude the possibility of the existence of rarer transcripts (for example, PQBP-1b with an additional 14 nucleotides). It is known that human PQBP-1 suppresses the ability of mouse Brain-2 to transactivate its target gene through interaction between the polar amino acid-rich region of PQBP-1 and the homopolymeric glutamine repeats of Brain-2 ( Waragai et al., 1998). However, Xenopus and zebrafish have no or fewer homopolymeric glutamine repeats in their Brain-2, compared with mammalian and reptile Brain-2 (Sumiyama et al., 1996; Nakachi et al., 1997). This raises the question of whether PQBP-1 is able to interact with Brain-2 in these lower vertebrates. Alternative splicing produces PQBP-1 transcripts lacking a polar amino acid-rich region, for example PQBP-1a and PQBP-1d. These alternative products would not be expected to interact with the cofactors of PQBP-1. Further studies on PQBP-1 transcripts and their inter-
action with cofactors in lower vertebrates may provide information about functional diversity during evolution and lead to the establishment of animal models of triplet diseases.
Acknowledgement This study was supported by grants from the Ministry of Education, Science, Sports, and Culture of Japan.
References Ashley Jr., C.T., Warren, S.T., 1995. Trinucleotide repeat expansion and human disease. Annu. Rev. Genet. 29, 703–728. Baltzinger, M., Payen, E., Remy, P., 1992. Nucleotide sequence of XLPOU3 cDNA, a member of the POU-domain gene family expressed in Xenopus laevis embryos. Nucleic Acids Res. 20, 1993 Baltzinger, M., Relaix, F., Remy, P., 1996. Transcription of XLPOU3, a brain specific gene, during Xenopus laevis early embryogenesis. Mech. Dev. 58, 103–114. Bowles, J., Cooper, L., Berkman, J., Koopman, P., 1999. Sry requires
K. Iwamoto et al. / Gene 259 (2000) 69–73 a CAG repeat domain for male sex determination in Mus musculus. Nat. Genet. 22, 405–408. Breathnach, R., Chambon, P., 1981. Organization and expression of eucaryotic split genes coding for proteins. Annu. Rev. Biochem. 50, 349–383. Burke, J.R., Enghild, J.J., Martin, M.E., Jou, Y.S., Myers, R.M., Roses, A.D., Vance, J.M., Strittmatter, W.J., 1996. Huntingtin and DRPLA proteins selectively interact with the enzyme GAPDH. Nat. Med. 2, 347–350. Gerber, H.P., Seipel, K., Georgiev, O., Hofferer, M., Hug, M., Rusconi, S., Schaffner, W., 1994. Transcriptional activation modulated by homopolymeric glutamine and proline stretches. Science 263, 808–811. Hara, Y., Rovescalli, A.C., Kim, Y., Nirenberg, M., 1992. Structure and evolution of four POU domain genes expressed in mouse brain. Proc. Natl. Acad. Sci. USA 89, 3280–3284. Imafuku, I., Waragai, M., Takeuchi, S., Kanazawa, I., Kawabata, M., Mouradian, M.M., Okazawa, H., 1998. Polar amino acid-rich sequences bind to polyglutamine tracts. Biochem. Biophys. Res. Commun. 253, 16–20. Kalchman, M.A., Koide, H.B., McCutcheon, K., Graham, R.K., Nichol, K., Nishiyama, K., Kazemi-Esfarjani, P., Lynn, F.C., Wellington, C., Metzler, M., Goldberg, Y.P., Kanazawa, I., Gietz, R.D., Hayden, M.R., 1997. HIP1, a human homologue of S. cerevisiae Sla2p, interacts with membrane-associated huntingtin in the brain. Nat. Genet. 16, 44–53. Kato, S., Sekine, S., Oh, S.W., Kim, N.S., Umezawa, Y., Abe, N., Yokoyama-Kobayashi, M., Aoki, T., 1994. Construction of a human full-length cDNA bank. Gene 150, 243–250. Komuro, A., Saeki, M., Kato, S., 1999. Npw38, a novel nuclear protein possessing a WW domain capable of activating basal transcription. Nucleic Acids Res. 27, 1957–1965. Li, P., He, X., Gerrero, M.R., Mok, M., Aggarwal, A., Rosenfeld,
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M.G., 1993. Spacing and orientation of bipartite DNA-binding motifs as potential functional determinants for POU domain factors. Genes Dev. 7, 2483–2496. Li, X.J., Li, S.H., Sharp, A.H., Nucifora Jr., F.C., Schilling, G., Lanahan, A., Worley, P., Snyder, S.H., Ross, C.A., 1995. A huntingtinassociated protein enriched in brain with implications for pathology. Nature 378, 398–402. Matilla, A., Koshy, B.T., Cummings, C.J., Isobe, T., Orr, H.T., Zoghbi, H.Y., 1997. The cerebellar leucine-rich acidic nuclear protein interacts with ataxin-1. Nature 389, 974–978. Nakachi, Y., Hayakawa, T., Oota, H., Sumiyama, K., Wang, L., Ueda, S., 1997. Nucleotide compositional constraints on genomes generate alanine-, glycine-, and proline-rich structures in transcription factors. Mol. Biol. Evol. 14, 1042–1049. Paulson, H.L., Fischbeck, K.H., 1996. Trinucleotide repeats in neurogenetic disorders. Annu. Rev. Neurosci. 19, 79–107. Perutz, M.F., 1999. Glutamine repeats and neurodegenerative diseases: molecular aspects. Trends Biochem. Sci. 24, 58–63. Schreiber, E., Tobler, A., Malipiero, U., Schaffner, W., Fontana, A., 1993. cDNA cloning of human N-Oct3, a nervous-system specific POU domain transcription factor binding to the octamer DNA motif. Nucleic Acids Res. 21, 253–258. Spaniol, P., Bornmann, C., Hauptmann, G., Gerster, T., 1996. Class III POU genes of zebrafish are predominantly expressed in the central nervous system. Nucleic Acids Res. 24, 4874–4881. Sumiyama, K., Washio-Watanabe, K., Saitou, N., Hayakawa, T., Ueda, S., 1996. Class III POU genes: generation of homopolymeric amino acid repeats under GC pressure in mammals. J. Mol. Evol. 43, 170–178. Waragai, M., Lammers, C.H., Takeuchi, S., Imafuku, I., Udagawa, Y., Kanazawa, I., Kawabata, M., Mouradian, M.M., Okazawa, H., 1998. PQBP-1, a novel polyglutamine tract-binding protein, inhibits transcription activation by Brn-2 and affects cell survival. Human Mol. Genet. 8, 977–987.
Genomic organization and alternative transcripts of the human PQBP-1 gene Kazuya Iwamoto, Yu-Tzu Huang, Shintaroh Ueda * Department of Biological Sciences, Graduate School of Science, University of Tokyo, Tokyo, Japan Received 14 April 2000; received in revised form 19 June 2000; accepted 25 August 2000 Received by T. Gojobori
Abstract PQBP-1 has been identified as a protein that binds to huntingtin, androgen receptor and transcription factor Brain-2 through their homopolymeric glutamine repeats. We here report the genomic organization of the human PQBP-1 gene and its multiple alternative transcripts. The coding region of PQBP-1 comprises six exons and five introns, and four types of alternative transcript, designated PQBP-1a to PQBP-1d, were found in addition to the PQBP-1 transcript reported originally. All of the PQBP-1 transcripts retain the WW domain in the N-terminal region, a potent transactivator domain. On the other hand, there is a wide variation in their C-terminal regions. Importantly, PQBP-1a and PQBP-1d lack the domain responsible for the interaction with homopolymeric glutamine repeats and a nuclear localization signal. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Brn-2; Npw38; Polyglutamine; Polyglutamine-binding protein; WW domain
1. Introduction Homopolymeric glutamine repeats have functional features that include activation of transcriptional activity in a length-dependent manner (Gerber et al., 1994) and sex determination (Bowles et al., 1999). Sequence comparison of transcription factors reveals a great variation among species in the position and repeat number of homopolymeric amino acid repeats, including polyglutamine (Sumiyama et al., 1996; Nakachi et al., 1997). In Brain-2, one of the class III POU transcription factors, there is a wide variation of the homopolymeric glutamine repeats among vertebrates. The mammalian Brain-2 genes have long homopolymeric glutamine repeats, which have been well conserved among mammals not only in position but also in repeat number. The reptile Brain-2 also has homopolymeric glutamine repeats, but there is a wide variation in repeat number between mammals and reptiles. In addition to the homopolymeric glutamine repeats common to mammals and Abbreviations: cDNA, DNA complementary to RNA; kb, kilobase(s); PCR, polymerase chain reaction; PQBP-1, poly-glutamine tract-binding protein-1; RT-PCR, reverse transcription-PCR. * Corresponding author. Tel.: +81-3-5841-4486; fax: +81-3-3818-7547. E-mail address: [email protected] (S. Ueda)
reptiles, there are reptile-specific homopolymeric glutamine repeats. In Xenopus, which has two Brain-2 homologues, one homologue ( XLPOU3) lacks homopolymeric glutamine repeats and the other ( XLPOU3B) shows a short run of glutamine repeats in a position different from those in the mammalian orthologues. The zebrafish orthologue (zp-47) shows no homopolymeric glutamine repeats (Baltzinger et al., 1992; Hara et al., 1992; Li et al., 1993; Schreiber et al., 1993; Baltzinger et al., 1996; Spaniol et al., 1996; Sumiyama et al., 1996; Nakachi et al., 1997). A similar situation is observed with other transcription factors. These findings suggest the possibility that differences in position and number of homopolymeric glutamine repeats lead to diversification of protein function during evolution (Sumiyama et al., 1996; Nakachi et al., 1997). In the last decade, proteins containing homopolymeric glutamine repeats have been extensively studied from an etiological viewpoint, because expansion of homopolymeric glutamine repeats causes progressive neurodegenerative disorders, so-called triplet diseases, such as X-linked spinal and bulbar muscular atrophy, Huntington’s disease, dentatorubral–pallidoluysian atrophy, and Machado–Joseph disease (Ashley and Warren, 1995; Paulson and Fischbeck, 1996; Perutz, 1999). Furthermore, huntingtin-associated protein, hun-
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tingtin-interacting protein, glyceraldehyde-3-phosphate dehydrogenase, and leucine-rich acidic nuclear protein have been shown to be homopolymeric glutamine repeat-binding proteins (Li et al., 1995; Burke et al., 1996; Kalchman et al., 1997; Matilla et al., 1997). Recently, a novel homopolymeric glutamine repeatbinding protein, PQBP-1, was identified by a yeast twohybrid system using the homopolymeric glutamine repeats of mouse Brain-2 as bait (Imafuku et al., 1998; Waragai et al., 1998). PQBP-1 was also identified as Npw38 through a computer-assisted search of the WW domains from the HomoProtein cDNA Bank ( Kato et al., 1994; Komuro et al., 1999). We report here the presence of multiple transcripts of the human PQBP-1 gene. Interestingly, two of them lack the polar amino acid-rich domain that is responsible for interaction with the homopolymeric glutamine repeats of Brain-2 and huntingtin. This elicits questions regarding functional diversity among alternative transcripts. We also investigate the genomic sequence and define the exon–intron structure of its coding region.
2. Materials and methods DNAs for human PQBP-1 and its alternative transcripts were obtained by RT-PCR with an Advantage cDNA PCR kit (Clontech) using human fetal brain Marathon-Ready cDNAs (Clontech) as templates. Two primer sets were used: 5∞-TGCTATCAGCTATGCCGCTGCCCGTTGC-3∞ (5∞ End Primer) and 5∞-TTTAACCCAGGGCCAGGGAGGCCGAAGC-3∞ (3∞ End Primer); 5∞-GGAATTCATGCCGCTGCCCGTTGCGCTGCAG-3∞ and 5∞-GGAATTCTCAATCCTGCTGCTTGGTTCGGG-3∞. PCR began at 94°C for 1 min, followed by 25 cycles of 94°C for 30 s and 74°C for 1 min. PCR products were purified by agarose gel electrophoresis, and ligated to the pCR2.1 vector using a TOPO TA-cloning kit (Invitrogen). We also used human adult brain cerebral cortex cDNAs (QUICK-clone cDNAs, Clontech) as RT-PCR templates. These cDNA templates are both synthesized from mRNAs that have been purified at least three times on oligo-dT columns. 5∞-ACTTTTCTTCAGCATCTGGGGACACAAG-3∞ and 5∞-GGATGGGGATGGGTGATGTGGCGGACAC-3∞ were used to detect particular alternative transcripts, PQBP-1a and PQBP-1b, respectively, in combination with the 5∞ End Primer. A genomic DNA fragment containing the entire coding region of human PQBP-1 was obtained with EXTaq polymerase ( TaKaRa) using the 5∞ and 3∞ End Primers. The PCR cycle consisted of 30 cycles at 94°C for 30 s and 72°C for 3 min 30 s. The amplified product was cloned into a plasmid vector, pCR2.1, using a TOPO TA-cloning kit as in the case of RT-PCR cloning. Sequencing templates were obtained using a GPS genome Priming System (New
England BioLab.), and nucleotide sequence determination was performed on an ABI 373S sequencer using a Dye Terminater Sequencing kit with AmpliTaq FS (PE Applied Biosystems). The nucleotide sequence data reported in this paper will appear in the DDBJ/EMBL/GenBank international nucleotide sequence database with the following accession numbers: AB041832–AB041836.
3. Results and discussion We obtained the entire coding region of the PQBP-1 gene by RT-PCR amplification using human fetal brain cDNAs as templates. In addition to a band corresponding to the expected size of the PQBP-1 cDNA reported previously, some discrete bands were observed (Fig. 1). These bands were obtained reproducibly using another PCR primer set. The nucleotide sequences of these PCR products showed that they were transcript variants of the PQBP-1; we therefore designated them as PQBP-1a to PQBP-1d. Fig. 2A shows their deduced amino acid sequences, together with the sequence of PQBP-1 ( Waragai et al., 1998; Komuro et al., 1999). We next addressed whether these transcript variants were generated by alternative splicing. We obtained PQBP-1 genomic DNA as a single 4.6 kb band by PCR amplification. Sequence comparison of the genomic PQBP-1 gene with its transcripts revealed that the coding region of PQBP-1 comprises six exons and five introns ( Fig. 3A), showing that these transcript variants were generated by alternative splicing. All the splicing accep-
Fig. 1. Alternative transcripts of human PQBP-1. Expression of PQBP-1 alternative mRNAs was analyzed by RT-PCR using human fetal brain cDNAs ( lane 1) and human adult brain cerebral cortex cDNAs ( lane 2) as templates.
K. Iwamoto et al. / Gene 259 (2000) 69–73
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Fig. 2. Structure of alternative PQBP-1 products. (A) Predicted amino acid sequences. (B) Schematic representation of their domain structures.
tor and donor sites had an AG/GT consensus sequence (Breathnach and Chambon, 1981) (Table 1). The PQBP-1 product is characterized by several domains: acidic region, WW domain, polar amino acid-rich domain, nuclear localization signal (NLS), and C -domain. The WW domain of the PQBP-1 product, 2 which binds to RNA and works as a potent transactivator ( Komuro et al., 1999), is located over two exons, exons 2 and 3. The polar amino acid-rich domain responsible for interaction with homopolymeric glutamine repeats ( Waragai et al., 1998) and the NLS are located in the same exon, exon 4. Seven Alu sequences were found in the PQBP-1 genomic sequence; six in intron 1 and one in intron 2 (data not shown). Fig. 2B shows a schematic representation of the PQBP-1 products. All of them retain the N-terminal acidic region and the WW domain, but have different C-terminal structures. PQBP-1a is generated by alterna-
tive use of two distinct splice acceptor sites in intron 3, which leads to a 14-nucleotide addition in PQBP-1a (see Fig. 3C ). This insertion event causes a reading frame shift that results in PQBP-1a lacking a polar amino acid-rich domain, NLS, and C -domain (Fig. 2). 2 Judging from the ratio of cloned DNAs regardless of cloning preference, the ratio of PQBP-1 to PQBP-1a transcripts was presumed to be approximately 10 to 1. PQBP-1b and PQBP-1c were generated by not fully splicing the introns, but their amino acid sequences remain unchanged due to the existence of a translational stop codon within unspliced intron 4; they had the polar amino acid-rich domain and NLS, but their C-terminals lacked the C -domain. PQBP-1d is generated by splicing 2 out of exon 4. This in-frame alternative splicing causes a lack of the polar amino acid-rich domain and NLS in PQBP-1d, which retains the C -domain. We also 2 obtained all these alternative transcripts of PQBP-1 by
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Fig. 3. Genomic organization of the human PQBP-1 gene and its transcripts. (A) Genomic organization of the entire coding region of the PQBP-1 gene. Exons and introns are indicated by open boxes and solid lines, respectively. (B) Alternative PQBP-1 transcripts. The shaded box represents the exon generated by alternative use of the splice acceptor site shown in (C ). Arrowheads indicate the putative translational stop codon. (C ) Alternative use of the splice acceptor site in intron 3. Upper-case letters indicate coding sequences and italics indicate the additional 14 nucleotides in the alternative transcript PQBP-1a. Table 1 Exon–intron boundaries of the human PQBP-1 gene. Exon and intron sequences are shown in upper- and lower-case letters, respectively cDNA
Exon size (bp)
Splice donor
Intron size (bp)
Splice acceptor
1–67 68–179 180–292 293–577 578–641 642–798
>67 112 113 285 64 >157
CTGGAGCCTG/gtgagacagc ACCCTTCCTG/gtgagcctgg AGTAATGCAG/gtgagttggc AGCAAGAAGG/gtaagctggg ACGCCCCCCG/gtaagtgaca
2609 628 190 214 132
gttctaccag/AACCAGAGGA ggccccatag/CGGGCTCCCT gtgtccccag/ATGCTGAAGA acttccacag/CAGTAAGCCG tcaccggcag/GGGCACGTGG
RT-PCR using human adult cerebral cDNAs as templates and confirmed the presence of PQBP-1a and PQBP-1b using specific primer sets (data not shown). We thus identified four types of PQBP-1 alternative transcripts, but might not exclude the possibility of the existence of rarer transcripts (for example, PQBP-1b with an additional 14 nucleotides). It is known that human PQBP-1 suppresses the ability of mouse Brain-2 to transactivate its target gene through interaction between the polar amino acid-rich region of PQBP-1 and the homopolymeric glutamine repeats of Brain-2 ( Waragai et al., 1998). However, Xenopus and zebrafish have no or fewer homopolymeric glutamine repeats in their Brain-2, compared with mammalian and reptile Brain-2 (Sumiyama et al., 1996; Nakachi et al., 1997). This raises the question of whether PQBP-1 is able to interact with Brain-2 in these lower vertebrates. Alternative splicing produces PQBP-1 transcripts lacking a polar amino acid-rich region, for example PQBP-1a and PQBP-1d. These alternative products would not be expected to interact with the cofactors of PQBP-1. Further studies on PQBP-1 transcripts and their inter-
action with cofactors in lower vertebrates may provide information about functional diversity during evolution and lead to the establishment of animal models of triplet diseases.
Acknowledgement This study was supported by grants from the Ministry of Education, Science, Sports, and Culture of Japan.
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