©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Human Tenascin-R
COMPLETE PRIMARY STRUCTURE, PRE-mRNA ALTERNATIVE SPLICING AND GENE LOCALIZATION ON CHROMOSOME 1q23-q24 (*)

(Received for publication, December 8, 1995)

Barbara Carnemolla (§) Alessandra Leprini Laura Borsi Germano Querzé Stefania Urbini Luciano Zardi

From the Laboratory of Cell Biology, Istituto Nazionale per la Ricerca sul Cancro, Largo Rosanna Benzi, 10, 16132 Genoa, Italy

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have established the primary structure of human tenascin-R (TN-R), a component of the extracellular matrix of the central nervous system, by sequencing cDNA clones which cover its complete coding region. The deduced amino acid sequence of human TN-R (1358 amino acids) showed a homology to chicken and rat TN-R of 75 and 93%, respectively. By reverse transcriptase-polymerase chain reaction we have studied the existence of TN-R isoforms generated by pre-mRNA alternative splicing in various human astrocytomas and meningiomas. Our findings demonstrate the existence of a human isoform in which one fibronectin-like repeat is omitted. Northern blot analysis of the poly(A)-rich RNA from different tissues showed two mRNAs having sizes of about 10 and 11 kilobases. Using DNA from a panel of human-hamster and human-mouse somatic cell hybrids and by fluorescence in situ hybridization, we have assigned the gene for human TN-R to the region 1q23-q24. The mouse mutation loop-tail (Lp), which has been proposed as a model for human neural tube defects, maps to region of mouse chromosome 1 syntenic with human 1q23-q24.


INTRODUCTION

Tenascin-R (TN-R), (^1)also known as janusin, J1-160/180, or restriction (Kruse et al., 1985; Faissner et al., 1988; Pesheva et al., 1989; Nöremberg et al., 1992; Fuss et al., 1993) is a member of the TN family (Erickson, 1993, 1994; Chiquet-Ehrismann, 1995). While other members of the TN family have been found in many different tissues and organs (Crossin et al., 1986; Natali et al., 1991; Crossin, 1994; Matsumoto et al., 1994), TN-R has been detected mainly in the central nervous system. TN-R is localized around motor neurons and on motor axons in the spinal cord (Rathjen et al., 1991). TN-R is also localized in the cerebellum, hippocampus, and olfactory bulb. Immunofluorescence and in situ hybridization studies indicate that TN-R is associated with the surface of neurons, myelinating oligodendrocytes, and type-2 astrocytes. TN-R is also produced by cells of chick retinal tissues. In the chicken brain, TN-R is detectable at embryonic days 6-16, but is barely detectable in the adult. This time-restricted distribution has suggested an involvement of TN-R in central nervous system development (Pesheva et al., 1989; Rathjen et al., 1991; Bartsch et al., 1993; Fuss et al., 1993; Wintergerst et al., 1993).

We began studies on TN-R to investigate its functions in the human central nervous system and to possibly associate disorders with its altered expression in humans. As a first step we have determined its primary structure, isoforms generated by its alternative splicing and its gene locus. The information and reagents described here may represent the basis for studies on human TN-R functions in normal and pathological conditions.


MATERIALS AND METHODS

Isolation and Sequencing of Human Tenascin-R cDNA and Genomic Clones, Reverse Transcriptase-Polymerase Chain Reaction (PCR) and Northern Blot Analysis

Using RNA from a human astrocytoma and oligonucleotides from two very conserved regions of rat TN-R, established by comparison with the cDNA sequence of chicken TN-R (BC-172fw: agcctgtacaagctccgtataggaggctac (3903-3932 of the rat cDNA sequence), BC-173rev: ttctccatcccctttgtagaaatgaagatg (4143-4172 of the rat cDNA sequence), BC-184fw: ttcaagacgatcacagagaccaccgtgga (1422-1450 of the rat cDNA sequence), BC-185rev: ccttccggctgcagcctccccttagccagt (1801-1830 of rat cDNA sequence) (Nöremberg et al., 1992; Fuss et al., 1993)), we have obtained by reverse transcriptase-PCR two DNA fragments of human TN-R (established by comparison of their size and sequence with those of rat TN-R). The two P labeled cDNA probes were used to screen an adult human brain (substantia nigra) ZAP II-cDNA library (Stratagene, La Jolla, CA). Eight clones, covering the complete human TN-R coding sequence (EMBL accession no. Z67996), were isolated from the library and transformed into pBluescript plasmids by in vivo excision according to manufacturer's instructions (Stratagene). Both strands of each clone were sequenced using either T3, T7, or synthetic oligonucleotide primers and Sequenase 2.0 DNA sequencing kit (U. S. Biochemical Corp.). Protein and nucleotide sequences were analyzed and compared using Microgenie (Beckman Instruments Inc., Fullerton, CA).

To isolate the genomic clones genR6 and genR2, a human genomic FIXII phage library (Stratagene) from human placenta was screened with two P-labeled cDNA probes from base 420 to 690 and from base 3832 to 4101 of the human TN-R sequence, respectively.

Reverse transcriptase-PCR was carried out using rTth DNA polymerase (Perkin-Elmer), according to the manufacturer's instructions.

Poly(A)-rich RNA preparations from human meningiomas and astrocytomas and Northern blots were carried out according to Borsi et al.(1992), while ``human multiple tissue Northern blots,'' containing 2 µg/lane of poly(A)-rich RNA from different non-fetal human tissues were obtained from Clontech Laboratories (Palo Alto, CA). Southern blots were performed according to Sambrook et al. (1989). All oligonucleotides were synthesized by TIB-MOLBIOL (Genoa, Italy).

Chromosomal and Subchromosomal Localization

For chromosomal localization, genomic DNA from a panel of Chinese hamster-human and mouse-human hybrid cell lines (BIOS Laboratories, New Haven, CT), previously digested using the restriction enzyme TaqI and blotted onto nylon filters, was hybridized with a P-labeled probe corresponding to the human TN-R cDNA clone R1 (see Fig. 2). Human TN-R fragments were easily distinguished from mouse and hamster TN-R fragments for their different pattern of TaqI digestion. Approximately 100 ng of R1 probe (see Fig. 2) was P-labeled to high specific activity using the BIOS Tag-It kit (BIOS Laboratories). Following purification of the labeled probe on Pharmacia ``Nick'' columns, hybridization was carried out overnight at 65 °C in a minimal volume of BIOS Speed-Hyb solution. Final washes were made at 65 °C in 0.25 times SSC, 0.2% SDS. For autoradiography the blots were exposed with intensifying screens at -70 °C.


Figure 2: Model of the domain structure of a human TN-R subunit. The EGF-L and FN-L repeats, as well as the fibrinogen-like sequence, are indicated. The FN-L repeat A, whose expression is regulated by the alternative splicing of the pre-mRNA, is shaded. The potential N-linked glycosylation sites are indicated by small dashes. Also indicated are the overlapping cDNA clones (the coding region is given in bold) covering the complete coding region of human TN-R which has been sequenced to deduce the TN-R primary structure. Also reported in the figure is the Southern blot analysis, using the P-labeled cDNA probe R6, of the reverse transcriptase-PCR products of 715 and 445 base pairs obtained using the BC-281 and BC-291 primers indicated in the figure.



Subchromosomal localization was carried out through fluorescence in situ hybridization using the genomic TN-R clone genR2, about 15 kilobases long, which includes all the exons of the fibrinogen-like sequence and a 3` untranscribed sequence.

Purified DNA from phage genomic clone genR2 was labeled with digoxigenin-dUTP by nick translation. Labeled probe was combined with sheared human DNA and hybridized to normal metaphase chromosomes derived from phytohemagglutinin-stimulated peripheral blood lymphocytes in a solution containing 50% formamide, 10% dextran sulfate, and 2 times SSC. Specific hybridization signals were detected by incubating the hybridized slides with fluoresceinated antidigoxigenin antibodies followed by counter-staining with propidium iodide.


RESULTS AND DISCUSSION

The deduced amino acid sequence of human TN-R is reported in Fig. 1. The first 23 amino acids correspond to a typical signal peptide (Kreil, 1981; Von Heijne, 1986). The domain structure of a human TN-R subunit is depicted in Fig. 2and shows, as for both rat and chicken TN-R, 4.5 epidermal growth factor-like (EGF-L), 9 fibronectin-like (FN-L) repeats, and a fibrinogen-like sequence.


Figure 1: Human TN-R sequence arranged in groups of homologous repeats. Potential N-linked glycosylation sites are underlined. All cysteine (C) residues are highlighted as are the conserved tryptofan (W), leucin (L), and threonine (T) residues used to align the FN-L type III repeats. Numbers on the right indicate the residue number; numbers on the left indicate the FN-L repeat number. The FN-L repeat A undergoes alternative splicing. (The EMBL accession number of the sequence is Z67996.)



The larger human TN-R reading frame is 4074 bases, corresponding to 1358 amino acids, while the chicken and rat TN-R are 1356 and 1353 amino acids, respectively. The percentage of identity between the deduced amino acid and nucleotide sequences of the protein coding region of the human TN-R are 93 and 87%, respectively, when compared with rat TN-R, and 75% for both amino acid and nucleotide sequences when compared with chicken TN-R. Comparative analysis of similarities of different regions of human TN-R with human TN-C and chicken and rat TN-R is reported in Table 1.



Two regions of alternative splicing of the TN-R pre-mRNA have been described: in chicken an isoform lacking a 45-amino acid sequence close to the N-terminal has been identified (Nöremberg et al., 1992), while in rat the FN-L repeat ``A'' may be omitted in a small percentage of molecules, as demonstrated by reverse transcriptase-PCR (Fuss et al., 1993). In order to study the two described splicing regions in human TN-R, we have analyzed RNA from five human astrocytomas and five meningiomas, using reverse transcriptase-PCR with primers localized within the FN-III 4 and 6 (BC-281, bases 2050-2069 and BC-291, bases 2734-2765 of the human TN-R sequence (EMBL accession number Z67996)) and in the regions flanking the splicing area in the N-terminal knob (BC-323, bases 271-290 and BC-324, bases 551-560 of the human TN-R sequence (EMBL accession number Z67996)). In the N-terminal knob we observed, through reverse transcriptase-PCR, a single band product corresponding to the large form. The cloning and sequencing of the genomic clone genR6 containing the 5` region of the TN-R gene demonstrated that the human splicing region homologous to that observed in the chicken was within an exon which included almost all the N-terminal knob (data not shown). However, while this data does not exclude the possibility that alternative splicing occurs in this region of the human TN-R transcript, the results from reverse transcriptase-PCR experiments demonstrated that the small isoform, if expressed in human, represents a very minor form. On the contrary we demonstrated in RNA from both astrocytomas and meningiomas, again by reverse transcriptase-PCR and by Southern blot of the obtained products, the presence of a small percentage of TN-R mRNA molecules in which the FN III repeat A was omitted. In this case as well, however, the mRNA lacking the A repeat represents less than 10% of the total TN-R mRNA, as judged by the relative amounts of the amplification products (Fig. 2).

Using the cDNA of the clone R1 (see Fig. 2) as probe, we have analyzed, by Northern blot, poly(A)-rich RNA from different normal adult tissues. In all tissues, with the exception of the brain, the TN-R mRNA was undetectable (Fig. 3A). Furthermore, we have analyzed, by high resolution Northern blot, poly(A)-rich RNA from human astrocytomas and meningiomas. In both cases we observed two bands of 11 and 10 kilobases, each representing about 50% of total TN-R mRNA (Fig. 3B). Considering that the larger reading frame for TN-R is composed of about 4000 bases and that the alternative splicing region may account for less than 500 bases, this observation suggests that there are nearly 6 kilobases, which are not translated and that the difference between the two TN-R mRNAs is not solely explained by the alternative splicing region(s) thus far observed. The finding of a long untranslated sequence in the mRNA of human TN-R is in keeping with the observation of a chicken TN-R mRNA of about 8.5 kilobases. Furthermore, Fuss et al.(1993) showed in rat TN-R a 3`-untranslated sequence of about 2500 bases and did not find the potential polyadenylation sites, suggesting an even longer 3`-untranslated region. The differences in size observed between the two human TN-R mRNAs (Fig. 3B) might be explained by the utilization of different polyadenylation sites; this hypothesis coincides with what we have observed analyzing the reported 3`-untranslated region of rat TN-R (Fuss et al., 1993), in which we found a potential AATAAA polyadenylation site between bases 5522 and 5528. The complete sequence of the 3` region of the TN-R transcript will clarify this hypothesis.


Figure 3: A, Northern blot analysis of poly(A)-rich RNA from different human tissues, using the cDNA probe R1 (see Fig. 2). Among the tissues tested, TN-R mRNA was detected only in the brain extract. B, Northern blot analysis of RNA extracted from two brain neoplasias (astrocytoma and meningioma). Two TN-R mRNAs of about 10 and 11 kilobases were detected.



By Southern blot analysis of genomic DNA from human-hamster and human-mouse somatic cell hybrids containing different human chromosomes previously digested using TaqI, we localized the TN-R gene on human chromosome 1 (Table 2). Furthermore, by fluorescence in situ hybridization using a human TN-R genomic clone genR2 (of about 14 kilobases, which includes all the exons of the fibrinogen-like sequence and a 3`-untranslated region), we have sublocalized the human TN-R gene within the chromosome. The initial experiments led to the specific labeling of the long arm of a group A chromosome. Further experiments were conducted in which a probe from the P58 locus, which is known to localize to 1p36, was cohybridized with genR2. These experiments resulted in the specific labeling of 1p36 and the long arm of chromosome 1. Measurements of 10 specifically hybridized chromosomes 1 indicated that genR2 was localized at a position which is 27% of the distance from the heterochromatic-euchromatic boundary to the telomere of chromosome arm 1q, an area that corresponds to the interface between bands 1q23 and 1q24. A total of 80 metaphase cells were analyzed, with 46 exhibiting specific labeling.



The TN-R gene locus is thus in close proximity to the LIM homeobox transcription factor 1, to several proto oncogenes and to chromosomal break points associated with various neoplastic diseases (Carrol et al., 1984; Williams et al., 1984; Chaganti et al., 1986; Nomura et al., 1989; Seldin and Kruh, 1989; Kamps et al., 1990; Nourse et al., 1990; German et al., 1994; Klemsz et al., 1994; Miranda et al., 1994). The region of synteny between this part of human chromosome 1 and mouse chromosome 1 has been well characterized, and it has been demonstrated that there is conservation of both gene order and intergenic distance (Kingsmore et al., 1989; Oakey et al., 1992). The semi-dominant mouse mutation loop-tail (Lp), which has been proposed as a model for a subset of human neural tube defects, also maps to this region of chromosome 1 (Mullick et al., 1995; Stanier et al., 1995). Lp homozygotes die in utero or shortly after birth following failure of the neural tube to close. Stanier et al.(1995) have suggested that the human homolog of Lp is likely to reside at human 1q21-23. Localization of the murine TN-R gene is in progress.


FOOTNOTES

*
This work was supported in part by funds from the Associazione Italiana per la Ricerca sul Cancro (AIRC) and the Consiglio Nazionale delle Ricerche (CNR), ``Progetto finalizzato: applicazioni cliniche della ricerca oncologica.'' The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Laboratory of Cell Biology, Istituto Nazionale per la Ricerca sul Cancro, Largo Rosanna Benzi, 10, 16132 Genoa, Italy. Tel.: 39-10-3534901 (or 352855); Fax: 39-10-352855; lzardi{at}cisi.unige.it.

(^1)
The abbreviations used are: TN-R, tenascin-R; PCR, polymerase chain reaction; FN-L, fibronectin-like.


ACKNOWLEDGEMENTS

We thank Thomas Wiley for manuscript revision. We are grateful to Drs. Roberto Gherzi, Francisco Baralle, Mario Sessarego, and Cristina Cuoco for helpful discussions. We thank Dr. David Hughes for helpful discussions and for the analysis of syntenic regions of mouse genome.

Note Added in Proof-Sequence on genomic clones has indicated that the residue 167 (see Fig. 1) is not E, but G as in every other tenascin molecule. The spliced type III repeat that we called ``A'' (see Fig. 2) is called ``R'' by other authors, in TN-R from other species.


REFERENCES

  1. Bartsch, U., Pesheva, P., Raff, M., and Schachner, M. (1993) Glia 9, 57-69 [Medline] [Order article via Infotrieve]
  2. Borsi, L., Carnemolla, B., Nicolò, G., Spina, B., Tanara, G., and Zardi, L. (1992) Int. J. Cancer 52, 688-692 [Medline] [Order article via Infotrieve]
  3. Carrol, A. J., Crist, W. M., Parmley, R. T., Roper, M., Cooper, M. D., and Finley, W. H. (1984) Blood 63, 761-724
  4. Chaganti, R. S. K., Balazs, I., Jhanwar, S. C., Murty, V. V. V. S., Koduru, P. R. K., Grzeschik, K.-H, and Stavnezer, E. (1986) Cytogenet. Cell Genet. 43, 181-186 [Medline] [Order article via Infotrieve]
  5. Chiquet-Ehrismann, R. (1995) Experientia (Basel) 51, 9-10
  6. Crossin, K. L. (1994) Persp. Dev. Neurobiol. 2, 21-32
  7. Crossin, K. L., Hoffman, S., Grumet, M., Thiery, J. P., and Edelman, G. M. (1986) J. Cell Biol. 102, 1917-1930 [Abstract]
  8. Erickson, H. P. (1993) Curr. Opin. Cell Biol. 5, 869-876 [Medline] [Order article via Infotrieve]
  9. Erickson, H. P. (1994) Persp. Dev. Neurobiol. 2, 9-19
  10. Faissner, A., Kruse, J., Chiquet-Ehrismann, R., and Mackie, E. (1988) Differentiation 37, 104-114 [Medline] [Order article via Infotrieve]
  11. Fuss, B., Wintergerst, E.-S., Bartsch, U., and Schachner, M. (1993) J. Cell Biol. 120, 1237-1249 [Abstract]
  12. German, M. S., Wang, J., Fernald, A. A., Espinosa, R., III, Le Beau, M. M., and Bell, G. I. (1994) Genomics 24, 403-404 [CrossRef][Medline] [Order article via Infotrieve]
  13. Kamps, M. P., Murre, C., Sun, X., and Baltimore, D. (1990) Cell 60, 547-555 [Medline] [Order article via Infotrieve]
  14. Kingsmore, S. F., Watson, M. L., Howard, T. A., and Seldin, M. F. (1989) EMBO J. 8, 4073-4080 [Abstract]
  15. Klemsz, M., Hromas, R., Raskind, W., Bruno, E., and Hoffman, R. (1994) Genomics 20, 291-294 [CrossRef][Medline] [Order article via Infotrieve]
  16. Kreil, G. (1981) Annu. Rev. Biochem. 50, 317-348 [Medline] [Order article via Infotrieve]
  17. Kruse, J., Keilhauer, G., Faissner, A., Timpl, R., and Schachner, M. (1985) Nature 316, 146-148 [Medline] [Order article via Infotrieve]
  18. Matsumoto, K., Saga, Y., Ikemura, T., Sakakura, T., and Chiquet-Ehrismann, R. (1994) J. Cell Biol. 125, 483-493 [Abstract]
  19. Miranda, C., Minoletti, F., Greco, A., Sozzi, G., and Pierotti, M. A. (1994) Genomics 23, 714-715 [CrossRef][Medline] [Order article via Infotrieve]
  20. Mullick, A., Trasler, D., and Gros, P. (1995) Genomics 26, 479-488 [CrossRef][Medline] [Order article via Infotrieve]
  21. Natali, P. G., Nicotra, M. R., Bigotti, A., Botti, C., Castellani, P., Risso, A. M., and Zardi, L. (1991) Int. J. Cancer 47, 811-816 [Medline] [Order article via Infotrieve]
  22. Nomura, N., Sasamoto, S., Ishii, S., Date, T., Matsui, M., and Ishizaki, R. (1989) Nucleic Acids Res. 17, 5489-5500 [Abstract]
  23. Nörenberg, U., Wille, H., Wolff, J. M., Frank, R., and Rathjen, F. G. (1992) Neuron 8, 849-863 [Medline] [Order article via Infotrieve]
  24. Nourse, J., Mellentin, J. D., Galili, N., Wilkinson, J., Stanbridge, E., Smith, S. D., and Cleary, M. L. (1990) Cell 6O, 535-545
  25. Oakey, R. J., Watson, M. L., and Seldin, M. F. (1992) Hum. Mol. Genet. 1, 613-620 [Abstract]
  26. Pesheva, P., Spiess, E., and Schachner, M. (1989) J. Cell Biol. 109, 1765-1778 [Abstract]
  27. Rathjen, F. G., Wolff, J. M., and Chiquet-Ehrismann, R. (1991) Development (Camb.) 113, 151-164
  28. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  29. Seldin, M. F., and Kruh, G. D. (1989) Genomics 4, 221-223 [Medline] [Order article via Infotrieve]
  30. Stanier, P., Henson, J. N., Moore, G. E., and Copp, A. J. (1995) Genomics 26, 473-478 [CrossRef][Medline] [Order article via Infotrieve]
  31. Von Heijne, G. (1986) Nucleic Acids Res. 14, 4683-4690 [Abstract]
  32. Williams, D. L., Look, A. T., Melvin, S. L., Roberson, P. K., Dahl, G., Flake, T., and Strass, S. (1984) Cell 36, 101-109 [Medline] [Order article via Infotrieve]
  33. Wintergerst, E. S., Fuss, B., and Bartsch, U. (1993) Eur. J. Neurosci. 5, 299-310 [Medline] [Order article via Infotrieve]

©1996 by The American Society for Biochemistry and Molecular Biology, Inc.