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The Human Thrombospondin 3 Gene: Analysis of Transcription Initiation and an Alternatively Spliced Transcript
Molecular Cell Biology Research Communications 2, 47–52 (1999) Article ID mcbr.1999.0148, available online at http://www.idealibrary.com on
The Human Thrombospondin 3 Gene: Analysis of Transcription Initiation and an Alternatively Spliced Transcript Kenneth W. Adolph* ,1 and Paul Bornstein† *Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, Minnesota 55455; and †Department of Biochemistry, University of Washington, Seattle, Washington 98195
Received July 15, 1999
The involvement of the thrombospondins in diverse and complex processes is due to the modular structures of the polypeptides encoded by the thrombospondin gene family. TSP1 (6) and TSP2 (7–12) have similar structures with amino- and carboxy-terminal domains, a procollagen homology region, and type I (TSP or properdin), type II (EGF-like), and type III (Ca 21binding) repeats. TSP3 (13–16), TSP4 (17), and TSP5/ COMP (18, 19) lack the procollagen homology region and type I repeats. Binding sites for cell surface receptors and protein-protein interactions along the modular structures of the TSP polypeptides account for the extensive range of their molecular and cellular interactions. Differences in the functions of TSP family members are suggested by their distinctive developmental and tissue-specific patterns of expression. In situ hybridization of TSP1, TSP2, and TSP3 cDNA probes to mouse embryo sections showed that each mRNA has a temporally and spatially independent distribution during mouse embryonic development (20). Immunohistochemical analysis of TSP2 protein in mouse tissues revealed its presence in dermal and other connective tissue-forming cells, and in areas of chondrogenesis, osteogenesis, and vasculogenesis (21, 22). That the TSP genes have distinctive patterns of expression is further suggested by analysis of their promoter elements. Comparison of the sequences of the promoter/59 flanking regions of the human TSP2 and TSP1 genes showed no significant sequence identity (23). The human TSP3 promoter, which has no homology with the TSP1 or TSP2 promoters, does have a special feature: its proximity to the gene for metaxin (24, 25), a peripheral mitochondrial membrane protein (26). The TSP3 and metaxin (MTX) genes are divergently transcribed and have a common promoter/ intergenic region of 1.4 kb. A far upstream enhancer of the mouse thrombospondin 3 gene (Thbs3) is located within intron 6 of the metaxin gene (27).
Thrombospondin 3 (TSP3) is a member of a family of modular, extracellular proteins that have been implicated in a diverse number of important biological processes. To contribute to an understanding of the precise roles of human TSP3, aspects of TSP3 gene transcription have been investigated. The TSP3 gene (THBS3) shares a promoter/intergenic region of 1.4 kb with the divergently transcribed metaxin gene, and the existence of TSP3 transcription initiation sites in the TSP3/metaxin intergenic region was investigated by a PCR procedure. Transcripts were detected which initiate in the intergenic region, up to several hundred bases upstream from the major transcription start site. An alternatively spliced transcript of TSP3 was also detected by the PCR procedure. This includes a new exon, exon A*, which replaces exon A. Exon A* is located in the TSP3/metaxin intergenic region, 1 kb 5* of exon A. In addition, transcripts of metaxin were found with extended 5* ends; these overlap the 5* end of the TSP3 alternative transcript. The complexities of TSP3 transcription initiation revealed by this study could contribute to the tissue-specific expression and diverse functions of TSP3. © 1999 Academic Press
The thrombospondin proteins have been implicated in a number of fundamental biological processes: blood coagulation, embryonic development, tissue differentiation, tumor growth and metastasis, angiogenesis, nerve development, wound healing, and inflammation (1–5). This is brought about by participation of the thrombospondins in cellular processes including cell proliferation, adhesion, and migration, and reflects the molecular functions of these proteins in binding to extracellular matrix (ECM) proteins and cell surface receptors, and to cytokines and proteases. 1
Corresponding author. Fax: 612-625-2163. E-mail: ka@brain. biochem.umn.edu. 47
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FIG. 1. Diagram representing the approach to investigate TSP3 transcription initiation. The genomic region containing the TSP3 (THBS3) and metaxin (MTX) exons and introns and the common promoter/intergenic region is shown. The arrows above the genomic region indicate that the genes are divergently transcribed. For TSP3, PCR used each of a series of primers C1, C2, C3 . . . placed increasingly 59 into the TSP3/metaxin intergenic region. The base numbers indicate the 59 end of each primer in relation to the translation start site in exon A. Primers C6 and C7 were located 1116 bp and 1169 bp upstream of the ATG. The amplification reactions were carried out with nested, antisense primers A and B in exon 12, and used a human cDNA mixture as template.
The experiments reported here were aimed at examining aspects of the regulation of TSP3 gene expression. Initiation of TSP3 transcription in the common TSP3/metaxin intergenic region and the existence of alternatively spliced transcripts of TSP3 were studied.
primer Y, 59-CCTCTGGAATGCATGAGAGTAG-39. These oligonucleotides were obtained from Integrated DNA Technologies, Inc. Expand High Fidelity polymerase mixture (Boehringer Mannheim) was used as the thermostable DNA polymerase. PCR conditions, using a Perkin Elmer GeneAmp PCR System 2400, were typically 94°, 45 sec; 65°, 45 sec; 72°, 2 min; 30 cycles. More stringent conditions were employed in some experiments: 94°, 45 sec; 70°, 2 min; 30 cycles.
MATERIALS AND METHODS Identification of alternatively spliced transcripts and transcripts with extended 59 ends. A PCR procedure was used to identify alternatively spliced transcripts of TSP3, and transcripts of TSP3 and metaxin that initiate 59 of the major transcripts. Primers were designed from the sequences of TSP3 cDNA (16), the TSP3/ metaxin intergenic region (25), and metaxin cDNA (25). For TSP3, nested, antisense primers (primers A and B) were placed in exon 12, about 1 kb 39 of the translation initiation site in the cDNA. PCR was carried out with each of a series of primers (C1, C2, C3 . . .), the first being located in the TSP3/metaxin intergenic region 159 bases 59 of the translation initiation site, and the second, third, etc. placed further and further 59. A similar procedure was performed for metaxin, with primers X and Y located in exon 8 and primers Z1, Z2, Z3 . . . in the intergenic region. The template in the PCR reactions was a panel of cDNAs generated using poly A 1 RNA from human tissues (heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas; Clontech) or an equal mixture of these eight cDNAs. Primer A, for first round amplification of the TSP3 gene, had the sequence 59-TAGACCTCAGGGTCTCCAGGAT-39; Primer B, for second round amplification, was 59-AGCTGCCTCCGGAATGGCTCAA-39. The sequences for metaxin were: primer X, 59-TGTCCGGCTGTACCAACAACGA-39;
DNA sequencing and analysis. DNA species corresponding to alternatively spliced transcripts and transcripts extending 59 into the intergenic region were purified for sequencing from preparative agarose gels: DNA bands were cut from 2% agarose/TAE gels and isolated using the QIAEX II Gel Extraction Kit (Qiagen). The DNA samples were directly sequenced with an Applied Biosystems Model 373 sequencer. The
FIG. 2. Detection of an alternatively spliced transcript of TSP3. The agarose gel includes the TSP3 cDNA products generated by second-round PCR using primer B in exon 12 with primer C1 (lane 1), C2 (lane 2), C3 (lane 3), C4 (lane 4), and C5 (lane 5) in the TSP3/metaxin intergenic region. The M lanes contain molecular weight markers: lambda DNA/HindIII (left) and phiX174 DNA/ HaeIII (right). 48
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FIG. 3. (A) Nucleotide sequence of the 59 end of the cDNA corresponding to the TSP3 alternative transcript. Exon A9 and exon B are included. The positions of intron A9 and intron B are indicated by triangles. Transcription factor binding motifs are underlined. (B) Diagram illustrating the pattern of alternative splicing of the TSP3 gene. The organization of the genomic region containing exon A9, exon A, and exon B is included.
sequencing reactions used the Amersham Thermo Sequenase Dye Terminator Cycle Sequencing Kit or the Applied Biosystems Dye Terminator Cycle Sequencing Kit with AmpliTaq DNA polymerase FS. Nucleic acid sequences were analyzed with the GENEPRO software package, Version 5.0 (Riverside Scientific Enterprises, Bainbridge Island, WA). The SIGNAL SCAN program was used to identify potential transcription factor binding sites (28).
through C5 were located in the TSP3/metaxin intergenic region and their 59 ends were, respectively, 159, 280, 492, 714, and 1096 bp upstream from the TSP3 translation start site. The major transcription start site, determined previously (16), is 21 bases upstream from the translation start site. Figure 2 shows the results of PCR experiments using each of these primers with nested primers A and B in exon 12. A human cDNA mixture was used as template. For primers C1 to C4, the sizes of the PCR bands on agarose gels are consistent with these species representing mRNAs that have transcription initiation sites in the TSP3/metaxin intergenic region, further 59 than previously identified (16). This was confirmed by sequencing the ends of the purified bands using primers C1 . . . C4 as the sequencing primers. PCR experiments were also carried out with each of C1 . . . C4 and a pair of nested primers in exon 22 (primers D and E)
RESULTS AND DISCUSSION Existence of an Alternatively Spliced Transcript of the TSP3 Gene and Transcripts Initiating in the TSP3/Metaxin Intergenic Region A diagram illustrating the approach to identify TSP3 alternatively spliced transcripts and transcripts with extended 59 ends is shown in Fig. 1. Primers C1 49
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to test whether the exon/intron structures of these species are the same as the normal, full-length cDNA (16). The sizes of the bands observed suggest that these species have the normal exon/intron structure and differ only in the lengths of their 59 ends. Primers C1 to C4 could be hybridizing to a single, larger transcript. Alternatively, the PCR products could represent transcripts with heterogeneous transcription initiation sites. Primers located further 59 than C4, up to 300 bases 59 of C4, failed to produce a band like those for C1 . . . C4 in Fig. 2. Thus, the transcription initiation sites of these transcripts with longer 59 ends extend well into, but not through, the TSP3/metaxin intergenic region. It was, however, possible to amplify a major band using a primer, C5, placed more than 1 kb 59 of the TSP3 translation start site. Unexpectedly, the band was substantially smaller than the size expected for a transcript extending 1 kb through the intergenic region (Fig. 2). Sequencing the purified DNA fragment revealed that it represents an alternatively spliced transcript. The transcript is composed of a new exon, exon A9, that replaces exon A of the major transcript (16). The 59 end of exon A9 was established by PCR using a series of primers (C6, C7, C8 . . .) increasingly 59 of C5 up to exon 1 of the metaxin gene (Fig. 1), with nested primers A and B in exon 12. The most 59 of these to produce a band was C8, and the sequence of the end of this DNA fragment is included in Fig. 3A. Exon A9 is 209 bp in length, and its 59 end is 137 bp from the translation start site of metaxin. The sequence of the 59 splice junction of intron A9 in the shared promoter region is caGuGTctgg (capital letters indicate conserved bases in splice consensus sequences). The 39 splice junction is the same as that for intron A of the major TSP3 transcript; its sequence is gaccAG. The size of intron A9 is 2498 bp, compared to 1390 bp for intron A. A diagram showing the genomic relationship of alternative exon A9 to exon A of the major transcript and to exon B is presented in Fig. 3B. A TGA stop codon, in-frame with the TSP3 reading frame, is encountered in exon A9 46 bases from its 39 end. The first in-frame ATG, which follows the stop codon and might possibly initiate translation of a truncated TSP3 protein, is present in exon B, 35 bases from its 59 end. However, the ATG sequence in exon B does not contain a good Kozak consensus sequence (29) and there does not appear to be a downstream signal peptide sequence if this translation initiation site is used. A different downstream ATG, in-frame or out-of-frame, might therefore be used as the translation start site of a truncated TSP3 molecule or a different protein. In this regard, there is now increasing evidence for intracellular forms of proteins that are normally secreted, e.g. lysyl oxidase (30). Even if a secreted protein or an intracellular protein is not produced, the alternative TSP3 transcript may still influence expression of the
FIG. 4. Tissue distribution of the TSP3 alternatively spliced transcript. The agarose gel shows the major cDNA band of 1078 bp produced by second-round PCR using primer B in exon 12 and primer C5 located 1 kb upstream of the TSP3 translation start site. The human tissue sources of the cDNAs used as template in the PCR reactions were: lane 1, heart; lane 2, brain; lane 3, placenta; lane 4, lung; lane 5, liver; lane 6, skeletal muscle; lane 7, kidney; lane 8, pancreas. The M lanes contain phiX174 DNA/HaeIII molecular weight markers.
metaxin gene because of the overlap of the 59 ends of the alternative transcript and the longer metaxin transcripts (see below). The search for new exons of TSP3 and new alternatively spliced transcripts was continued by placing additional primers in exon 1, intron 1, exon 2, and intron 2 of metaxin. The primers were equally spaced about 90 –100 bases apart, and PCR was carried out with nested primers A and B in TSP3 exon 12. However, no bands that represented TSP3 transcripts were detected on agarose gels. The experiments described above were all performed using a mixture of human cDNAs (heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas) as template in PCR reactions. To examine the tissue specificity of the TSP3 alternatively spliced transcript, the reactions were carried out using each of the cDNAs individually. The results are shown in Fig. 4 and demonstrate that the alternatively spliced transcript (the major band of 1078 bp) is widely distributed among human tissues. This gives confidence to the conclusion that the alternative transcript is a significant aspect of TSP3 gene transcription. Existence of Metaxin Transcripts Initiating in the TSP3/Metaxin Intergenic Region and Overlap with the TSP3 Alternatively Spliced Transcript To detect human metaxin mRNAs initiating 59 of the major transcript (25), PCR experiments were carried out with a pair of nested primers (X and Y) in exon 8 and a series of primers (Z1, Z2, Z3 . . .) located progressively 59 into the TSP3/metaxin intergenic region. The amplification reactions included a human cDNA mixture as template. Strong bands were observed on agarose gels up to and including primer Z3, located 227 bp 59 of the metaxin translation start site. The sizes of the bands suggested that these species were normal, full50
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FIG. 5. (A) Nucleotide sequence of the cDNA corresponding to the metaxin transcript with the longest detected 59 end. The sequence through exon 1 is shown. Potential transcription factor binding sites are underlined, and the predicted amino acid sequence is included below the nucleotide sequence. (B) Overlap of the 59 ends of the TSP3 alternative transcript and the longest metaxin transcript. The figure shows the alignment of the inverse of the cDNA corresponding to the longest metaxin transcript with the cDNA corresponding to the TSP3 alternative transcript. The metaxin cDNA is at the top and begins with the inverted translation start site (cat), and the TSP3 cDNA (in capital letters) is at the bottom and extends through exon A9.
length metaxin mRNAs with 59 ends initiating in the promoter/intergenic region. Sequencing confirmed this interpretation, as shown for primer Z3 in Fig. 5A. The metaxin transcript revealed with primer Z3 was the longest observed, since primers placed further 59 did not produce bands by PCR. In fact, no other significant bands were detected using primers located through the intergenic region up to the TSP3 translation start site. This would indicate that alternatively spliced transcripts of metaxin, with new exons in the TSP3/ metaxin intergenic region, do not exist. Alignment of the sequences of the longest transcript of metaxin and the longest alternatively spliced transcript of TSP3 showed that the 59 ends overlap (Fig. 5B). This complementarity of the 59 ends of the diver-
gently transcribed mRNAs was found for the sequences determined with primer Z3 for metaxin and primer C8 for TSP3. The overlap of 90 bases seen in Fig. 5B implies that the transcriptional regulatory elements also overlap. Such a finding may have implications for the tissue-specific levels of expression of the two genes. ACKNOWLEDGMENTS This research was supported by a Grant-in-Aid from the University of Minnesota and by National Institutes of Health Grant DE08229.
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3. Bornstein, P., and Sage, E. H. (1994) Methods Enzymol. 245, 62– 85. 4. Bornstein, P. (1995) J. Cell Biol. 130, 503–506. 5. Adams, J. C., Tucker, R. P., and Lawler, J. (1995) The Thrombospondin Gene Family, R. G. Landis, Austin, Texas. 6. Lawler, J., and Hynes, R. O. (1986) J. Cell Biol. 103, 1635–1648. 7. Bornstein, P., O’Rourke, K., Wikstrom, K., Wolf, F. W., Katz, R., Li, P., and Dixit, V. M. (1991) J. Biol. Chem. 266, 12821–12824. 8. Bornstein, P., Devarayalu, S., Li, P., Disteche, C. M., and Framson, P. (1991) Proc. Natl. Acad. Sci. USA 88, 8636 – 8640. 9. LaBell, T. L., Milewicz, D. J. M., Disteche, C. M., and Byers, P. H. (1992) Genomics 12, 421– 429. 10. Laherty, C. D., O’Rourke, K., Wolf, F. W., Katz, R., Seldin, M. F., and Dixit, V. M. (1992) J. Biol. Chem. 267, 3274 –3281. 11. LaBell, T. L., and Byers, P. H. (1993) Genomics 17, 225–229. 12. Shingu, T., and Bornstein, P. (1993) Genomics 16, 78 – 84. 13. Vos, H. L., Devarayalu, S., de Vries, Y., and Bornstein, P. (1992) J. Biol. Chem. 267, 12192–12196. 14. Bornstein, P., Devarayalu, S., Edelhoff, S., and Disteche, C. M. (1993) Genomics 15, 607– 613. 15. Qabar, A. N., Lin, Z., Wolf, F. W., O’Shea, K. S., Lawler, J., and Dixit, V. M. (1994) J. Biol. Chem. 269, 1262–1269. 16. Adolph, K. W., Long, G. L., Winfield, S., Ginns, E. I., and Bornstein, P. (1995) Genomics 27, 329 –336. 17. Lawler, J., Duquette, M., Whittaker, C. A., Adams, J. C., McHenry, K., and DeSimone, D. W. (1993) J. Cell Biol. 120, 1059 –1067.
18. Oldberg, A., Antonsson, P., Lindblom, K., and Heinegard, D. (1992) J. Biol. Chem. 267, 22346 –22350. 19. Newton, G., Weremowicz, S., Morton, C. C., Copeland, N. G., Gilbert, D. J., Jenkins, N. A., and Lawler, J. (1994) Genomics 24, 435– 439. 20. Iruela-Arispe, M. L., Liska, D. J., Sage, E. H., and Bornstein, P. (1993) Dev. Dyn. 197, 40 –56. 21. Kyriakides, T. R., Zhu, Y.-H., Yang, Z., and Bornstein, P. (1998) J. Histochem. Cytochem. 46, 1007–1015. 22. Tooney, P. A., Sakai, T., Sakai, K., Aeschlimann, D., and Mosher, D. F. (1998) Matrix Biol. 17, 131–143. 23. Adolph, K. W., Liska, D. J., and Bornstein, P. (1997) Gene 193, 5–11. 24. Bornstein, P., McKinney, C. E., LaMarca, M. E., Winfield, S., Shingu, T., Devarayalu, S., Vos, H. L., and Ginns, E. I. (1995) Proc. Natl. Acad. Sci. USA 92, 4547– 4551. 25. Long, G. L., Winfield, S., Adolph, K. W., Ginns, E. I., and Bornstein, P. (1996) Genomics 33, 177–184. 26. Armstrong, L. C., Komiya, T., Bergman, B. E., Mihara, K., and Bornstein, P. (1997) J. Biol. Chem. 272, 6510 – 6518. 27. Collins, M., Rojnuckarin, P., Zhu, Y.-H., and Bornstein, P. (1998) J. Biol. Chem. 273, 21816 –21824. 28. Prestridge, D. S. (1996) CABIOS 12, 157–160. 29. Kozak, M. S. (1987) Nucleic Acids Res. 15, 8125– 8148. 30. Li, W. D., Nellaiappan, K., Strassmaier, T., Graham, L., Thomas, K. M., and Kagan, H. M. (1997) Proc. Natl. Acad. Sci. USA 94, 12817–12822.
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The Human Thrombospondin 3 Gene: Analysis of Transcription Initiation and an Alternatively Spliced Transcript Kenneth W. Adolph* ,1 and Paul Bornstein† *Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, Minnesota 55455; and †Department of Biochemistry, University of Washington, Seattle, Washington 98195
Received July 15, 1999
The involvement of the thrombospondins in diverse and complex processes is due to the modular structures of the polypeptides encoded by the thrombospondin gene family. TSP1 (6) and TSP2 (7–12) have similar structures with amino- and carboxy-terminal domains, a procollagen homology region, and type I (TSP or properdin), type II (EGF-like), and type III (Ca 21binding) repeats. TSP3 (13–16), TSP4 (17), and TSP5/ COMP (18, 19) lack the procollagen homology region and type I repeats. Binding sites for cell surface receptors and protein-protein interactions along the modular structures of the TSP polypeptides account for the extensive range of their molecular and cellular interactions. Differences in the functions of TSP family members are suggested by their distinctive developmental and tissue-specific patterns of expression. In situ hybridization of TSP1, TSP2, and TSP3 cDNA probes to mouse embryo sections showed that each mRNA has a temporally and spatially independent distribution during mouse embryonic development (20). Immunohistochemical analysis of TSP2 protein in mouse tissues revealed its presence in dermal and other connective tissue-forming cells, and in areas of chondrogenesis, osteogenesis, and vasculogenesis (21, 22). That the TSP genes have distinctive patterns of expression is further suggested by analysis of their promoter elements. Comparison of the sequences of the promoter/59 flanking regions of the human TSP2 and TSP1 genes showed no significant sequence identity (23). The human TSP3 promoter, which has no homology with the TSP1 or TSP2 promoters, does have a special feature: its proximity to the gene for metaxin (24, 25), a peripheral mitochondrial membrane protein (26). The TSP3 and metaxin (MTX) genes are divergently transcribed and have a common promoter/ intergenic region of 1.4 kb. A far upstream enhancer of the mouse thrombospondin 3 gene (Thbs3) is located within intron 6 of the metaxin gene (27).
Thrombospondin 3 (TSP3) is a member of a family of modular, extracellular proteins that have been implicated in a diverse number of important biological processes. To contribute to an understanding of the precise roles of human TSP3, aspects of TSP3 gene transcription have been investigated. The TSP3 gene (THBS3) shares a promoter/intergenic region of 1.4 kb with the divergently transcribed metaxin gene, and the existence of TSP3 transcription initiation sites in the TSP3/metaxin intergenic region was investigated by a PCR procedure. Transcripts were detected which initiate in the intergenic region, up to several hundred bases upstream from the major transcription start site. An alternatively spliced transcript of TSP3 was also detected by the PCR procedure. This includes a new exon, exon A*, which replaces exon A. Exon A* is located in the TSP3/metaxin intergenic region, 1 kb 5* of exon A. In addition, transcripts of metaxin were found with extended 5* ends; these overlap the 5* end of the TSP3 alternative transcript. The complexities of TSP3 transcription initiation revealed by this study could contribute to the tissue-specific expression and diverse functions of TSP3. © 1999 Academic Press
The thrombospondin proteins have been implicated in a number of fundamental biological processes: blood coagulation, embryonic development, tissue differentiation, tumor growth and metastasis, angiogenesis, nerve development, wound healing, and inflammation (1–5). This is brought about by participation of the thrombospondins in cellular processes including cell proliferation, adhesion, and migration, and reflects the molecular functions of these proteins in binding to extracellular matrix (ECM) proteins and cell surface receptors, and to cytokines and proteases. 1
Corresponding author. Fax: 612-625-2163. E-mail: ka@brain. biochem.umn.edu. 47
1522-4724/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
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FIG. 1. Diagram representing the approach to investigate TSP3 transcription initiation. The genomic region containing the TSP3 (THBS3) and metaxin (MTX) exons and introns and the common promoter/intergenic region is shown. The arrows above the genomic region indicate that the genes are divergently transcribed. For TSP3, PCR used each of a series of primers C1, C2, C3 . . . placed increasingly 59 into the TSP3/metaxin intergenic region. The base numbers indicate the 59 end of each primer in relation to the translation start site in exon A. Primers C6 and C7 were located 1116 bp and 1169 bp upstream of the ATG. The amplification reactions were carried out with nested, antisense primers A and B in exon 12, and used a human cDNA mixture as template.
The experiments reported here were aimed at examining aspects of the regulation of TSP3 gene expression. Initiation of TSP3 transcription in the common TSP3/metaxin intergenic region and the existence of alternatively spliced transcripts of TSP3 were studied.
primer Y, 59-CCTCTGGAATGCATGAGAGTAG-39. These oligonucleotides were obtained from Integrated DNA Technologies, Inc. Expand High Fidelity polymerase mixture (Boehringer Mannheim) was used as the thermostable DNA polymerase. PCR conditions, using a Perkin Elmer GeneAmp PCR System 2400, were typically 94°, 45 sec; 65°, 45 sec; 72°, 2 min; 30 cycles. More stringent conditions were employed in some experiments: 94°, 45 sec; 70°, 2 min; 30 cycles.
MATERIALS AND METHODS Identification of alternatively spliced transcripts and transcripts with extended 59 ends. A PCR procedure was used to identify alternatively spliced transcripts of TSP3, and transcripts of TSP3 and metaxin that initiate 59 of the major transcripts. Primers were designed from the sequences of TSP3 cDNA (16), the TSP3/ metaxin intergenic region (25), and metaxin cDNA (25). For TSP3, nested, antisense primers (primers A and B) were placed in exon 12, about 1 kb 39 of the translation initiation site in the cDNA. PCR was carried out with each of a series of primers (C1, C2, C3 . . .), the first being located in the TSP3/metaxin intergenic region 159 bases 59 of the translation initiation site, and the second, third, etc. placed further and further 59. A similar procedure was performed for metaxin, with primers X and Y located in exon 8 and primers Z1, Z2, Z3 . . . in the intergenic region. The template in the PCR reactions was a panel of cDNAs generated using poly A 1 RNA from human tissues (heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas; Clontech) or an equal mixture of these eight cDNAs. Primer A, for first round amplification of the TSP3 gene, had the sequence 59-TAGACCTCAGGGTCTCCAGGAT-39; Primer B, for second round amplification, was 59-AGCTGCCTCCGGAATGGCTCAA-39. The sequences for metaxin were: primer X, 59-TGTCCGGCTGTACCAACAACGA-39;
DNA sequencing and analysis. DNA species corresponding to alternatively spliced transcripts and transcripts extending 59 into the intergenic region were purified for sequencing from preparative agarose gels: DNA bands were cut from 2% agarose/TAE gels and isolated using the QIAEX II Gel Extraction Kit (Qiagen). The DNA samples were directly sequenced with an Applied Biosystems Model 373 sequencer. The
FIG. 2. Detection of an alternatively spliced transcript of TSP3. The agarose gel includes the TSP3 cDNA products generated by second-round PCR using primer B in exon 12 with primer C1 (lane 1), C2 (lane 2), C3 (lane 3), C4 (lane 4), and C5 (lane 5) in the TSP3/metaxin intergenic region. The M lanes contain molecular weight markers: lambda DNA/HindIII (left) and phiX174 DNA/ HaeIII (right). 48
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FIG. 3. (A) Nucleotide sequence of the 59 end of the cDNA corresponding to the TSP3 alternative transcript. Exon A9 and exon B are included. The positions of intron A9 and intron B are indicated by triangles. Transcription factor binding motifs are underlined. (B) Diagram illustrating the pattern of alternative splicing of the TSP3 gene. The organization of the genomic region containing exon A9, exon A, and exon B is included.
sequencing reactions used the Amersham Thermo Sequenase Dye Terminator Cycle Sequencing Kit or the Applied Biosystems Dye Terminator Cycle Sequencing Kit with AmpliTaq DNA polymerase FS. Nucleic acid sequences were analyzed with the GENEPRO software package, Version 5.0 (Riverside Scientific Enterprises, Bainbridge Island, WA). The SIGNAL SCAN program was used to identify potential transcription factor binding sites (28).
through C5 were located in the TSP3/metaxin intergenic region and their 59 ends were, respectively, 159, 280, 492, 714, and 1096 bp upstream from the TSP3 translation start site. The major transcription start site, determined previously (16), is 21 bases upstream from the translation start site. Figure 2 shows the results of PCR experiments using each of these primers with nested primers A and B in exon 12. A human cDNA mixture was used as template. For primers C1 to C4, the sizes of the PCR bands on agarose gels are consistent with these species representing mRNAs that have transcription initiation sites in the TSP3/metaxin intergenic region, further 59 than previously identified (16). This was confirmed by sequencing the ends of the purified bands using primers C1 . . . C4 as the sequencing primers. PCR experiments were also carried out with each of C1 . . . C4 and a pair of nested primers in exon 22 (primers D and E)
RESULTS AND DISCUSSION Existence of an Alternatively Spliced Transcript of the TSP3 Gene and Transcripts Initiating in the TSP3/Metaxin Intergenic Region A diagram illustrating the approach to identify TSP3 alternatively spliced transcripts and transcripts with extended 59 ends is shown in Fig. 1. Primers C1 49
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to test whether the exon/intron structures of these species are the same as the normal, full-length cDNA (16). The sizes of the bands observed suggest that these species have the normal exon/intron structure and differ only in the lengths of their 59 ends. Primers C1 to C4 could be hybridizing to a single, larger transcript. Alternatively, the PCR products could represent transcripts with heterogeneous transcription initiation sites. Primers located further 59 than C4, up to 300 bases 59 of C4, failed to produce a band like those for C1 . . . C4 in Fig. 2. Thus, the transcription initiation sites of these transcripts with longer 59 ends extend well into, but not through, the TSP3/metaxin intergenic region. It was, however, possible to amplify a major band using a primer, C5, placed more than 1 kb 59 of the TSP3 translation start site. Unexpectedly, the band was substantially smaller than the size expected for a transcript extending 1 kb through the intergenic region (Fig. 2). Sequencing the purified DNA fragment revealed that it represents an alternatively spliced transcript. The transcript is composed of a new exon, exon A9, that replaces exon A of the major transcript (16). The 59 end of exon A9 was established by PCR using a series of primers (C6, C7, C8 . . .) increasingly 59 of C5 up to exon 1 of the metaxin gene (Fig. 1), with nested primers A and B in exon 12. The most 59 of these to produce a band was C8, and the sequence of the end of this DNA fragment is included in Fig. 3A. Exon A9 is 209 bp in length, and its 59 end is 137 bp from the translation start site of metaxin. The sequence of the 59 splice junction of intron A9 in the shared promoter region is caGuGTctgg (capital letters indicate conserved bases in splice consensus sequences). The 39 splice junction is the same as that for intron A of the major TSP3 transcript; its sequence is gaccAG. The size of intron A9 is 2498 bp, compared to 1390 bp for intron A. A diagram showing the genomic relationship of alternative exon A9 to exon A of the major transcript and to exon B is presented in Fig. 3B. A TGA stop codon, in-frame with the TSP3 reading frame, is encountered in exon A9 46 bases from its 39 end. The first in-frame ATG, which follows the stop codon and might possibly initiate translation of a truncated TSP3 protein, is present in exon B, 35 bases from its 59 end. However, the ATG sequence in exon B does not contain a good Kozak consensus sequence (29) and there does not appear to be a downstream signal peptide sequence if this translation initiation site is used. A different downstream ATG, in-frame or out-of-frame, might therefore be used as the translation start site of a truncated TSP3 molecule or a different protein. In this regard, there is now increasing evidence for intracellular forms of proteins that are normally secreted, e.g. lysyl oxidase (30). Even if a secreted protein or an intracellular protein is not produced, the alternative TSP3 transcript may still influence expression of the
FIG. 4. Tissue distribution of the TSP3 alternatively spliced transcript. The agarose gel shows the major cDNA band of 1078 bp produced by second-round PCR using primer B in exon 12 and primer C5 located 1 kb upstream of the TSP3 translation start site. The human tissue sources of the cDNAs used as template in the PCR reactions were: lane 1, heart; lane 2, brain; lane 3, placenta; lane 4, lung; lane 5, liver; lane 6, skeletal muscle; lane 7, kidney; lane 8, pancreas. The M lanes contain phiX174 DNA/HaeIII molecular weight markers.
metaxin gene because of the overlap of the 59 ends of the alternative transcript and the longer metaxin transcripts (see below). The search for new exons of TSP3 and new alternatively spliced transcripts was continued by placing additional primers in exon 1, intron 1, exon 2, and intron 2 of metaxin. The primers were equally spaced about 90 –100 bases apart, and PCR was carried out with nested primers A and B in TSP3 exon 12. However, no bands that represented TSP3 transcripts were detected on agarose gels. The experiments described above were all performed using a mixture of human cDNAs (heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas) as template in PCR reactions. To examine the tissue specificity of the TSP3 alternatively spliced transcript, the reactions were carried out using each of the cDNAs individually. The results are shown in Fig. 4 and demonstrate that the alternatively spliced transcript (the major band of 1078 bp) is widely distributed among human tissues. This gives confidence to the conclusion that the alternative transcript is a significant aspect of TSP3 gene transcription. Existence of Metaxin Transcripts Initiating in the TSP3/Metaxin Intergenic Region and Overlap with the TSP3 Alternatively Spliced Transcript To detect human metaxin mRNAs initiating 59 of the major transcript (25), PCR experiments were carried out with a pair of nested primers (X and Y) in exon 8 and a series of primers (Z1, Z2, Z3 . . .) located progressively 59 into the TSP3/metaxin intergenic region. The amplification reactions included a human cDNA mixture as template. Strong bands were observed on agarose gels up to and including primer Z3, located 227 bp 59 of the metaxin translation start site. The sizes of the bands suggested that these species were normal, full50
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FIG. 5. (A) Nucleotide sequence of the cDNA corresponding to the metaxin transcript with the longest detected 59 end. The sequence through exon 1 is shown. Potential transcription factor binding sites are underlined, and the predicted amino acid sequence is included below the nucleotide sequence. (B) Overlap of the 59 ends of the TSP3 alternative transcript and the longest metaxin transcript. The figure shows the alignment of the inverse of the cDNA corresponding to the longest metaxin transcript with the cDNA corresponding to the TSP3 alternative transcript. The metaxin cDNA is at the top and begins with the inverted translation start site (cat), and the TSP3 cDNA (in capital letters) is at the bottom and extends through exon A9.
length metaxin mRNAs with 59 ends initiating in the promoter/intergenic region. Sequencing confirmed this interpretation, as shown for primer Z3 in Fig. 5A. The metaxin transcript revealed with primer Z3 was the longest observed, since primers placed further 59 did not produce bands by PCR. In fact, no other significant bands were detected using primers located through the intergenic region up to the TSP3 translation start site. This would indicate that alternatively spliced transcripts of metaxin, with new exons in the TSP3/ metaxin intergenic region, do not exist. Alignment of the sequences of the longest transcript of metaxin and the longest alternatively spliced transcript of TSP3 showed that the 59 ends overlap (Fig. 5B). This complementarity of the 59 ends of the diver-
gently transcribed mRNAs was found for the sequences determined with primer Z3 for metaxin and primer C8 for TSP3. The overlap of 90 bases seen in Fig. 5B implies that the transcriptional regulatory elements also overlap. Such a finding may have implications for the tissue-specific levels of expression of the two genes. ACKNOWLEDGMENTS This research was supported by a Grant-in-Aid from the University of Minnesota and by National Institutes of Health Grant DE08229.
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