Title:

Splice choice antagonists as therapeutic agents

Document Type and Number:

United States Patent 7402434

Abstract:

The invention relates to methods and reagents for influencing alternative RNA splicing in living cells. More particularly, the invention relates to novel means for influencing RNA splice choice in living cells using polynucleotide-based reagents that compete for binding sites in nucleotide binding proteins, and novel methods for using these reagents as therapeutics.

Inventors:

Newman, Stuart A. (23 Iroquois Rd., Pleasantville, NY, US)
Bronstein, Natalie B. (78 Morningside Dr., Patterson, NY, US)

Plaque It!

CROSS REFERENCE TO RELATED PATENT APPLICATIONS

This application is related to and claims priority on U.S. provisional patent application Ser. No. 60/202,657 filed May 8, 2000.

Claims:

We claim:

1. A method of modifying an activity of at least one human or avian hnRNP A protein within at least one cell, which comprises the steps of: introducing into the cell a plurality of RNA polynucleotide sequences comprising FGFR2 exon 8 sequences capable of binding to the hnRNP A protein; and interacting the RNA polynucleotide sequences with the hnRNP A protein within the cell, wherein the RNA polynucleotide sequences compete with at least one endogenous RNA sequence for interacting with the hnRNP A protein.

2. The method of claim 1, wherein the polynucleotide sequences are introduced into the cell by electroporation.

3. The method of claim 1, wherein the polynucleotide sequences are introduced into the cell by applying the polynucleotide sequences to a surface of the cell.

4. The method of claim 3, wherein the polynucleotide sequences are packaged in at least one liposome.

5. The method of claim 3, wherein the polynucleotide sequences are applied to a surface of the cell along with a detergent.

6. The method of claim 1, wherein the cell is at least one tissue culture cell.

7. The method of claim 1, wherein the cell is at least one avian cell.

8. The method of claim 1, wherein the cell is at least one non-human tissue culture cell.

9. The method of claim 1, wherein the polynucleotide sequences further comprise at least one isolated and purified RNA molecule.

10. The method of claim 1, wherein the polynucleotide sequences further comprise at least one synthetic RNA molecule.

11. The method of claim 1, wherein the polynucleotide sequences further comprise at least one synthetic RNA analog.

12. The method of claim 1, wherein the polynucleotide sequences are single-stranded.

13. The method of claim 1, wherein the step of interacting the polynucleotide sequences to the hnRNP A protein further comprises regulating the activity of the hnRNP A protein.

14. The method of claim 1, further comprising the step of determining an effect on RNA processing by monitoring at least one resulting phenotypic characteristic selected from the group consisting of abnormal skin, cell, tissue, organ, vertebral body, neural tissue, skeletal, and limb development.

15. A method of modifying an activity of at least one human or avian hnRNP A1 protein within at least one cell comprising the steps of: introducing into the cell a plurality of RNA polynucleotide sequences comprising FGFR2 exon 8 sequences capable of binding to the hnRNP A1 protein; and interacting the RNA polynucleotide sequences with the hnRNP A1 protein within the cell, wherein the RNA polynucleotide sequences compete with at least one endogenous RNA sequence for interacting with the hnRNP A1 protein.

16. The method of claim 15, further comprising the step of determining an effect on RNA processing by monitoring at least one resulting phenotypic characteristic selected from the group consisting of abnormal skin, cell, tissue, organ, vertebral body, neural tissue, skeletal, and limb development.

17. The method of claim 1, wherein the hnRNP A protein is selected from the group consisting of hnRNP A1 protein, hnRNP A1^Bprotein, and hnRNP A2 protein.

18. A method of modifying an activity of at least one human or avian hnRNP A protein within at least one cell, which comprises the steps of: introducing into the cell a plurality of RNA polynucleotide sequences comprising at least one intronic splicing silencer, wherein the polynucleotide sequences are isolated and purified RNA molecules, synthetic RNA molecules, or synthetic RNA analogs; interacting the RNA polynucleotide sequences with the hnRNP A protein within the cell, wherein the RNA polynucleotide sequences compete with at least one endogenous RNA sequence for interacting with the hnRNP A protein; and altering the splicing of the at least one endogenous RNA sequence.

19. The method of claim 18, wherein the hnRNP A protein is selected from the group consisting of hnRNP A1 protein, hnRNP A1^Bprotein, and hnRNP A2 protein.

20. A method of modifying an activity of at least one human or avian hnRNP A protein within at least one cell, comprising the steps of: introducing into the cell a plurality of RNA polynucleotide sequences comprising at least one intronic splicing enhancer; and interacting the RNA polynucleotide sequences with the hnRNP A protein within the cell, wherein the RNA polynucleotide sequences compete with at least one endogenous RNA sequence for interacting with the hnRNP A protein.

21. The method of claim 20, wherein the hnRNP A protein is selected from the group consisting of hnRNP A1 protein, hnRNP A1^Bprotein, and hnRNP A2 protein.

22. A method of modifying an activity of at least one human or avian hnRNP A protein within at least one cell, which comprises the steps of: introducing into the cell a plurality of RNA polynucleotide sequences comprising at least one exonic splicing silencer, wherein the polynucleotide sequences are isolated and purified RNA molecules, synthetic RNA molecules, or synthetic RNA analogs; interacting the RNA polynucleotide sequences with the hnRNP A protein within the cell, wherein the RNA polynucleotide sequences compete with at least one endogenous RNA sequence for interacting with the hnRNP A protein; and altering the splicing of the at least one endogenous RNA sequence.

23. The method of claim 22, wherein the hnRNP A protein is selected from the group consisting of hnRNP A1 protein, hnRNP A1^Bprotein, and hnRNP A2 protein.

24. A method of modifying an activity of at least one human or avian hnRNP A protein within at least one cell, which comprises the steps of: introducing into the cell a plurality of RNA polynucleotide sequences comprising at least one exonic splicing enhancer, wherein the polynucleotide sequences are isolated and purified RNA molecules, synthetic RNA molecules, or synthetic RNA analogs; interacting the RNA polynucleotide sequences with the hnRNP A protein within the cell, wherein the RNA polynucleotide sequences compete with at least one endogenous RNA sequence for interacting with the hnRNP A protein; and altering the splicing of the at least one endogenous RNA sequence.

25. The method of claim 24, wherein the hnRNP A protein is selected from the group consisting of hnRNP A1 protein, hnRNP A1^Bprotein, and hnRNP A2 protein.

Description:

GOVERNMENT SUPPORT

This work was supported by a grant from the National Institutes of Health (1 R03 HD37194: Spatiotemporal regulation of fibronectin).

FIELD OF THE INVENTION

REFERENCES CITED

Adam, S. A., Nakagawa, T., Swanson, M. S., Woodruff, T. K., and Dreyfuss, G. (1986). mRNA polyadenylate-binding protein: gene isolation and sequencing and identification of a ribonucleoprotein consensus sequence. Mol Cell Biol 6, 2932-43.
Aitchison, J. D., Blobel, G., and Rout, M. P. (1996). Kap104p: a karyopherin involved in the nuclear transport of messenger RNA binding proteins. Science 274, 624-7.
Akamatsu, W., Okano, H. J., Osumi, N., Inoue, T., Nakamura, S., Sakakibara, S., Miura, M., Matsuo, N., Darnell, R. B., and Okano, H. (1999). Mammalian ELAV-like neuronal RNA-binding proteins HuB and HuC promote neuronal development in both the central and the peripheral nervous systems. Proc Natl Acad Sci U S A 96, 9885-90.
Alvarez, I. S., Araujo, M., and Nieto, M. A. (1998). Neural induction in whole chick embryo cultures by FGF. Dev Biol 199, 42-54.
Amendt, B. A., Hesslein, D., Chang, L. J., and Stoltzfus, C. M. (1994). Presence of negative and positive cis-acting RNA splicing elements within and flanking the first tat coding exon of human immunodeficiency virus type 1. Mol Cell Biol 14, 3960-70.
Amendt, B. A., Si, Z. H., and Stoltzfus, C. M. (1995). Presence of exon splicing silencers within human immunodeficiency virus type 1 tat exon 2 and tat-rev exon 3: evidence for inhibition mediated by cellular factors [published erratum appears in Mol Cell Biol 1995 Nov;15(11):6480]. Mol Cell Biol 15, 4606-15.
Amrein, H., Gorman, M., and Nothiger, R. (1988). The sex-determining gene tra-2 of Drosophila encodes a putative RNA binding protein [published erratum appears in Cell 1989 Jul 28;58(2):following 419]. Cell 55, 1025-35.
Anderson, P. J., Hall, C. M., Evans, R. D., Hayward, R. D., and Jones, B. M. (1999). The feet in Apert's syndrome. J Pediatr Orthop 19, 504-7.
Arman, E., Haffner-Krausz, R., Chen, Y., Heath, J. K., and Lonai, P. (1998). Targeted disruption of fibroblast growth factor (FGF) receptor 2 suggests a role for FGF signaling in pregastrulation mammalian development. Proc Natl Acad Sci U S A 95, 5082-7.
Arman, E., Haffner-Krausz, R., Gorivodsky, M., and Lonai, P. (1999). Fgfr2 is required for limb outgrowth and lung-branching morphogenesis. Proc Natl Acad Sci U S A 96, 11895-9.
Arrigo, S. J., and Chen, I. S. (1991). Rev is necessary for translation but not cytoplasmic accumulation of HIV-1 vif, vpr, and env/vpu 2 RNAs. Genes Dev 5, 808-19.
Arts, G. J., Fornerod, M., Mattaj, I. W. (1998). Identification of a nuclear export receptor for tRNA. Current Biology 8, 305-314.
Aviezer, D., Safran, M., and Yayon, A. (1999). Heparin differentially regulates the interaction of fibroblast growth factor-4 with FGF receptors 1 and 2. Biochem Biophys Res Commun 263, 621-6.
Baba-Aissa, F., Van den Bosch, L., Wuytack, F., Raeymaekers, L., and Casteels, R. (1998). Regulation of the sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) 2 gene transcript in neuronal cells. Brain Res Mol Brain Res 55, 92-100.
Balinsky, B. I. (1970). “An Introduction to Embryology.” W. B. Saunders, Philadelphia.
Bandziulis, R. J., Swanson, M. S., and Dreyfuss, G. (1989). RNA-binding proteins as developmental regulators. Genes Dev 3, 431-7.
Bell, L. R., Horabin, J. I., Schedl, P., and Cline, T. W. (1991). Positive autoregulation of sex-lethal by alternative splicing maintains the female determined state in Drosophila. Cell 65, 229-39.
Beyer, A. L., Christensen, M. E., Walker, B. W., and LeStourgeon, W. M. (1977). Identification and characterization of the packaging proteins of core 40S hnRNP particles. Cell 11, 127-38.
Biamonti, G., Buvoli, M., Bassi, M. T., Morandi, C., Cobianchi, F., and Riva, S. (1989). Isolation of an active gene encoding human hnRNP protein A1. Evidence for alternative splicing. J Mol Biol 207, 491-503.
Bischoff, F. R., and Gorlich, D. (1997). RanBP1 is crucial for the release of RanGTP from importin beta-related nuclear transport factors. FEBS Lett 419, 249-54.
Blanchette, M., and Chabot, B. (1997). A highly stable duplex structure sequesters the 5′ splice site region of hnRNP A1 alternative exon 7B. Rna 3, 405-19.
Boche, I., and Fanning, E. (1997). Nucleocytoplasmic recycling of the nuclear localization signal receptor alpha subunit in vivo is dependent on a nuclear export signal, energy, and RCC1. J Cell Biol 139, 313-25.
Bonifaci, N., Moroianu, J., Radu, A., and Blobel, G. (1997). Karyopherin beta2 mediates nuclear import of a mRNA binding protein. Proc Natl Acad Sci U S A 94, 5055-60.
Borja, A. Z. M., Meijers, C., and Zeller, L. (1993). Expresssion of alternatively spliced bRGR first coding exons and antisense mRNAs during chicken embryogenesis. Developmental Biology 157, 110-118.
Brand, B., Christ, B., and Jacob, H. J. (1985). An experimental analysis of the developmental capacities of distal parts of avian leg buds. Am. J. Anat. 173, 321-340.
Broad, T. E., and Ham, R. G. (1983). Growth and adipose differentiation of sheep preadipocyte fibroblasts in serum-free medium. Eur J Biochem 135, 33-9.
Burd, C. G., and Dreyfuss, G. (1994). RNA binding specificity of hnRNP A1: significance of hnRNP A1 high-affinity binding sites in pre-mRNA splicing. Embo J 13, 1197-204.
Burd, C. G., Swanson, M. S., Gorlach, M., and Dreyfuss, G. (1989). Primary structures of the heterogeneous nuclear ribonucleoprotein A2, B1, and C2 proteins: a diversity of RNA binding proteins is generated by small peptide inserts. Proc Natl Acad Sci U S A 86, 9788-92.
Burgess, W. H., and Maciag, T. (1989). The heparin-binding (fibroblast) growth factor family of proteins. Annu Rev Biochem 58, 575-606.
Burke, D., Wilkes, D., Blundell, T. L., and Malcolm, S. (1998). Fibroblast growth factor receptors: lessons from the genes. Trends Biochem Sci 23, 59-62.
Burrus, L. W., and Olwin, B. B. (1989). Isolation of a receptor for acidic and basic fibroblast growth factor from embryonic chick. J Biol Chem 264, 18647-53.
Buvoli, M., Cobianchi, F., Bestagno, M. G., Mangiarotti, A., Bassi, M. T., Biamonti, G., and Riva, S. (1990). Alternative splicing in the human gene for the core protein A1 generates another hnRNP protein. Embo J 9, 1229-35.
Buvoli, M., Cobianchi, F., and Riva, S. (1992). Interaction of hnRNP A1 with snRNPs and pre-mRNAs: evidence for a possible role of A1 RNA annealing activity in the first steps of spliceosome assembly. Nucleic Acids Res 20, 5017-25.
Caceres, J. F., Misteli, T., Screaton, G. R., Spector, D. L., and Krainer, A. R. (1997). Role of the modular domains of SR proteins in subnuclear localization and alternative splicing specificity. J Cell Biol 138, 225-38.
Caceres, J. F., Screaton, G. R., and Krainer, A. R. (1998). A specific subset of SR proteins shuttles continuously between the nucleus and the cytoplasm. Genes Dev 12, 55-66.
Caceres, J. F., Stamm, S., Helfman, D. M., and Krainer, A. R. (1994). Regulation of alternative splicing in vivo by overexpression of antagonistic splicing factors. Science 265, 1706-9.
Calvio, C., Neubauer, G., Mann, M., and Lamond, A. I. (1995). Identification of hnRNP P2 as TLS/FUS using electrospray mass spectrometry. Rna 1, 724-33.
Caputi, M., Casari, G., Guenzi, S., Tagliabue, R., Sidoli, A., Melo, C. A., and Baralle, F. E. (1994). A novel bipartite splicing enhancer modulates the differential processing of the human fibronectin EDA exon. Nucleic Acids Res 22, 1018-22.
Caputi, M., Mayeda, A., Krainer, A. R., and Zahler, A. M. (1999). hnRNP A/B proteins are required for inhibition of HIV-1 pre-mRNA splicing. Embo J 18, 4060-7.
Chabot, B. (1996). Directing alternative splicing: cast and scenarios. Trends Genet 12, 472-8.
Chabot, B., Blanchette, M., Lapierre, I., and La Branche, H. (1997). An intron element modulating 5′ splice site selection in the hnRNP A1 pre-mRNA interacts with hnRNP A1. Mol Cell Biol 17, 1776-86.
Champion-Arnaud, P., Ronsin, C., Gilbert, E., Gesnel, M. C., Houssaint, E., and Breathnach, R. (1991). Multiple mRNAs code for proteins related to the BEK fibroblast growth factor receptor. Oncogene 6, 979-87.
Chan, C. T., and Thorogood, P. (1999). Pleiotropic features of syndromic craniosynostoses correlate with differential expression of fibroblast growth factor receptors 1 and 2 during human craniofacial development. Pediatr Res 45, 46-53.
Chung, S. Y., and Wooley, J. (1986). Set of novel, conserved proteins fold pre-messenger RNA into ribonucleosomes. Proteins 1, 195-210.
Cobianchi, F., Calvio, C., Stoppini, M., Buvoli, M., and Riva, S. (1993). Phosphorylation of human hnRNP protein A1 abrogates in vitro strand annealing activity. Nucleic Acids Res 21, 949-55.
Cobianchi, F., Karpel, R. L., Williams, K. R., Notario, V., and Wilson, S. H. (1988). Mammalian heterogeneous nuclear ribonucleoprotein complex protein A1. Large-scale overproduction in Escherichia coli and cooperative binding to single-stranded nucleic acids. J Biol Chem 263, 1063-71.
Cobianchi, S. F., Biamonti, G., Bassi, M. T., Buvoli, M., and Riva, S. (1990). In “The Eukaryotic Nucleus: Structure and Function” (P. a. W. Strauss, S., Ed.), Vol. Two, pp. 561, Caldwell, N.J.
Cochrane, A. W., Jones, K. S., Beidas, S., Dillon, P. J., Skalka, A. M., and Rosen, C. A. (1991). Identification and characterization of intragenic sequences which repress human immunodeficiency virus structural gene expression. J Virol 65, 5305-13.
Cohn, M. J., Izpisua-Belmonte, J. C., Abud, H., Heath, J. K., and Tickle, C. (1995). Fibroblast growth factors induce additional limb development from the flank of chick embryos. Cell 80, 739-746.
Crossley, P. H., Martinez, S., and Martin, G. R. (1996). Midbrain development induced by FGF8 in the chick embryo. Nature 380, 66-8.
Crozat, A., Aman, P., Mandahl, N., and Ron, D. (1993). Fusion of CHOP to a novel RNA-binding protein in human myxoid liposarcoma. Nature 363, 640-4.
Dahlberg, J. E., and Lund, E. (1998). Functions of the GTPase Ran in RNA export from the nucleus. Curr Opin Cell Biol 10, 400-8.
De Moerlooze, L., Spencer-Dene, B., Revest, J., Hajihosseini, M., Rosewell, I., and Dickson, C. (2000). An important role for the IIIb isoform of fibroblast growth factor receptor 2 (FGFR2) in mesenchymal-epithelial signalling during mouse organogenesis. Development 127, 483-92.
Dejgaard, K., Leffers, H., Rasmussen, H. H., Madsen, P., Kruse, T. A., Gesser, B., Nielsen, H., and Celis, J. E. (1994). Identification, molecular cloning, expression and chromosome mapping of a family of transformation upregulated hnRNP-K proteins derived by alternative splicing. J Mol Biol 236, 33-48.
Del Gatto, F., and Breathnach, R. (1995). A Crouzon syndrome synonymous mutation activates a 5′ splice site within the IIIc exon of the FGFR2 gene. Genomics 27, 558-559.
Del Gatto, F., Gesnel, M. C., and Breathnach, R. (1996). The exon sequence TAGG can inhibit splicing. Nucleic Acids Reseach 24, 2017-2021.
Del Gatto, F., and Breathnach, R. (1995). Exon and intron sequences, respectively, repress and activate splicing of a fibroblast growth factor receptor 2 alternative exon. Mol Cell Biol 15, 4825-34.
Del Gatto-Konczak, F., Olive, M., Gesnel, M. C., and Breathnach, R. (1999). hnRNP A1 recruited to an exon in vivo can function as an exon splicing silencer. Mol Cell Biol 19, 251-60.
Dempsey, L. A., Sun, H., Hanakahi, L. A., and Maizels, N. (1999). G4 DNA binding by LR1 and its subunits, nucleolin and hnRNP D, A role for G-G pairing in immunoglobulin switch recombination. J Biol Chem 274, 1066-71.
Dessau, W., Von Der Mark, H., Von Der Mark, K., and Fischer, S. (1980). Changes in the patterns of collagens and fibronectin during limb-bud chondrogenesis. J. Embryol. Exp. Morphol. 57, 51-60.
Dolnick, B. J. (1993). Cloning and characterization of natural occurring natural antisense RNA to human thymidylate synthase RNA. Nucleic Acids Research 21, 1747-1752.
Dono, R., and Zeller, R. (1994). Cell-type-specific nuclear translocation of fibroblast growth factor-2 isoforms during chicken kidney and limb morphogenesis. Dev Biol 163, 316-30.
Downie, S. A. (1994) Cellular and molecular differences between fore and hind limb precartilage mesenchyme: relation to skeletal pattern formation. In “Cell Biology and Anatomy”, pp. 51. New York Medical College, Valhalla, N.Y.
Downie, S. A., and Newman, S. A. (1994). Morphogenetic differences between fore and hind limb precartilage mesenchyme: relation to mechanisms of skeletal pattern formation. Dev. Biol. 162, 195-208.
Downie, S. A., and Newman, S. A. (1995). Different roles for fibronectin in the generation of fore and hind limb precartilage condensations. Developmental Biology 172, 519-530.
Doye, V., and Hurt, E. (1997). From nucleoporins to nuclear pore complexes. Curr Opin Cell Biol 9, 401-11.
Dreyfuss, G. (1986). Structure and function of nuclear and cytoplasmic ribonucleoprotein particles. Annu Rev Cell Biol 2, 459-98.
Dreyfuss, G., Matunis, M. J., Pinol-Roma, S., and Burd, C. G. (1993). hnRNP proteins and the biogenesis of mRNA. Annu. Rev. Biochem. 62, 289-321.
Dreyfuss, G., Swanson, M. S., and Pinol-Roma, S. (1988). Heterogeneous nuclear ribonucleoprotein particles and the pathway of mRNA formation. Trends Biochem Sci 13, 86-91.
Eckenstein, F. P. (1994). Fibroblast growth factors in the nervous system. J Neurobiol 25, 1467-80.
Eckner, R., Ellmeier, W., and Birnstiel, M. L. (1991). Mature mRNA 3′ end formation stimulates RNA export from the nucleus. Embo J 10, 3513-22.
Elicone, C., Lui, M., Geromanos, S., Erdjument-Bromage, H., and Tempst, P. (1994). Microbore reversed-phase high-performance liquid chromatographic purification of peptides for combined chemical sequencing-laser-desorption mass spectrometric analysis. J Chromatogr A 676, 121-37.
Ellerbrok, H., Serpente, N., Pancino, G., Vanhee, C., D'Auriol, L., Sitbon, M., and Vaquero, C. (1993). Sequences in the rev-responsive element responsible for premature translational arrest in the human-immunodeficiency-virus-type-1 envelope. Eur J Biochem 216, 459-67.
Eperon, I. C., Ireland, D. C., Smith, R. A., Mayeda, A., and Krainer, A. R. (1993). Pathways for selection of 5′ splice sites by U1 snRNPs and SF2/ASF. Embo J 12, 3607-17.
Fallon, J. F., Lopez, A., Ros, M. A., Savage, M. P., Olwin, B. B., and Simandl, B. K. (1994). FGF-2: apical ectodermal ridge growth signal for chick limb development. Science. 264, 104-107.
Faura, M., Renau-Piqueras, J., Bachs, O., and Bosser, R. (1995). Differential distribution of heterogeneous nuclear ribonucleoproteins in rat tissues. Biochem Biophys Res Commun 217, 554-60.
Fischer, U., Huber, J., Boelens, W. C., Maftaj, I. W., and Luhrmann, R. (1995). The HIV-1 Rev activation domain is a nuclear export signal that accesses an export pathway used by specific cellular RNAs. Cell 82, 475-83.
Fogerty, F. J., and Mosher, D. F. (1990). Mechanisms for organization of fibronectin matrix. Cell Differ Dev 32, 439-50.
Fornerod, M., Ohno, M., Yoshida, M., and Mattaj, I. W. (1997). CRM1 is an export receptor for leucine-rich nuclear export signals [see comments]. Cell 90, 1051-60.
Frenz, D. A., Akiyama, S. K., Paulsen, D. F., and Newman, S. A. (1989a). Latex beads as probes of cell surface-extracellular matrix interactions during chondrogenesis: evidence for a role for amino-terminal heparin-binding domain of fibronectin. Dev. Biol. 136, 87-96.
Frenz, D. A., Jaikaria, N. S., and Newman, S. A. (1989b). The mechanism of precartilage mesenchymal condensation: a major role for interaction of the cell surface with the amino-terminal heparin-binding domain of fibronectin. Dev. Biol. 136, 97-103.
Fridell, R. A., Truant, R., Thorne, L., Benson, R. E., and Cullen, B. R. (1997). Nuclear import of hnRNP A1 is mediated by a novel cellular cofactor related to karyopherin-beta. J Cell Sci 110, 1325-31.
Fu, X. D. (1995). Thesuperfamily of arginine/serine-rich splicing factors. RNA 1, 663-680.
Fukuda, M., Asano, S., Nakamura, T., Adachi, M., Yoshida, M., Yanagida, M., and Nishida, E. (1997). CRM1 is responsible for intracellular transport mediated by the nuclear export signal. Nature 390, 308-11.
Gallego, M. E., Sirand-Pugnet, P., Durosay, P., Clouet d'Orval, B., d'Aubenton-Carafa, Y., Brody, E., Expert-Bezancon, A., and Marie, J. (1996). Tissue-specific splicing of two mutually exclusive exons of the chicken beta-tropomyosin pre-mRNA: positive and negative regulations. Biochimie 78, 457-65.
Gardner, R. L. (1983). International Review of Experimental Pathology 24, 63-133.
Ge, H., and Manley, J. L. (1990). A protein factor, ASF, controls cell-specific alternative splicing of SV40 early pre-mRNA in vitro. Cell 62, 25-34.
Gilbert, S. F. (1997). “Developmental biology.” Sinauer Associates, Sunderland, Mass.
Glant, T. T., Hadhazy, C. S., Mikecz, K., and Sipos, A. (1985). Appearance and persistence of fibronectin in cartilage. Specific interaction of fibronectin with collage type II. Histochemistry 82, 149-158.
Goldfarb, D. S. (1997). Nuclear transport: proliferating pathways. Curr Biol 7, R13-6.
Gorlich, D. (1997). Nuclear protein import. Curr Opin Cell Biol 9, 412-9.
Gorlich, D., and Mattaj, I. W. (1996). Nucleocytoplasmic transport. Science 271, 1513-8.
Gould, S. E., Upholt, W. B., and Kosher, R. A. (1995). Characterization of chicken syndecan-3 as a heparan sulfate proteoglycan and its expression during embryogenesis. Dev. Biol. 168, 438-451.
Graham, I. R., Hamshere, M., and Eperon, I. C. (1992). Alternative splicing of a human alpha-tropomyosin muscle-specific exon: identification of determining sequences. Mol Cell Biol 12, 3872-82.
Greeve, J., Lellek, H., Rautenberg, P., and Greten, H. (1998). Inhibition of the apolipoprotein B mRNA editing enzyme-complex by hnRNP C1 protein and 40S hnRNP complexes. Biol Chem 379, 1063-73.
Grothe, C., and Meisinger, C. (1995). Fibroblast growth factor (FGF)-2 sense and antisense mRNA and FGF receptor type 1 mRNA are present in the embryonic and adult rat nervous system: specific detection by nuclease protection assay. Neurosci Lett 197, 175-8.
Haines, D. S., Strauss, K. I., and Gillespie, D. H. (1991). Cellular response to double-stranded RNA. J Cell Biochem 46, 9-20.
Hajihosseini, M. K., and Dickson, C. (1999). A subset of fibroblast growth factors (Fgfs) promote survival, but Fgf-8b specifically promotes astroglial differentiation of rat cortical precursor cells. Mol Cell Neurosci 14, 468-85.
Hall, B. K. (1983). Cartilage: Development, Differentiation, and Growth, Vol. Two. Academic Press, New York.
Hall, B. K., and Miyake, T. (1992). The membranous skeleton: the role of cell condensations in vertebrate skeletogenesis. Anat Embryol. Berl. 186, 107-124.
Hamburger, V., and Hamilton, H. L. (1951). A series of normal stages in the development of the chick embryo. J. Morphol. 88, 49-92.
Hamm, J., and Mattaj, I. W. (1990). Monomethylated cap structures facilitate RNA export from the nucleus. Cell 63, 109-18.
Han, J. K. (1997). Expression of the fibroblast growth factor-2 gene during chick development. Mol Cells 7, 208-13.
Hassfeld, W., Chan, E. K. L., Mathison, D. A., Portman, D., Dreyfuss, G., Steiner, G., and Tan, E. M. (1998). Molecular definition of heterogeneous nuclear ribonucleoprotein R (hnRNP R) using autoimmune antibody: immunological relationship with hnRNP P. Nucleic Acids Res 26, 439-45.
Herold, A., Truant, R., Wiegand, H., and Cullen, B. R. (1998). Determination of the functional domain organization of the importin alpha nuclear import factor. J Cell Biol 143, 309-18.
Higuchi, M., Single, F. N., Kohler, M., Sommer, B., Sprengel, R., and Seeburg, P. H.
(1993). RNA editing of AMPA receptor subunit GluR-B: a base-paired intron-exon structure determines position and efficiency. Cell 75, 1361-70.
Hildebrandt, M., and Nellen, W. (1992). Differential antisense transcription from the Dictyostelium EB4 gene locus: implications on antisense-mediated regulation of mRNA stability. Cell 69, 197-204.
Honore, B., Vorum, H., and Baandrup, U. (1999). hnRNPs H, H′ and F behave differently with respect to posttranslational cleavage and subcellular localization. FEBS Lett 456, 274-80.
Hopper, A. K., Traglia, H. M., and Dunst, R. W. (1990). The yeast RNA1 gene product necessary for RNA processing is located in the cytosol and apparently excluded from the nucleus. J Cell Biol 111, 309-21.
Hu, M. C., Qiu, W. R., Wang, Y. P., Hill, D., Ring, B. D., Scully, S., Bolon, B., DeRose, M., Luethy, R., Simonet, W. S., Arakawa, T., and Danilenko, D. M. (1998). FGF-18, a novel member of the fibroblast growth factor family, stimulates hepatic and intestinal proliferation. Mol Cell Biol 18, 6063-74.
Huang, Y., and Carmichael, G. C. (1996). Role of polyadenylation in nucleocytoplasmic transport of mRNA. Mol Cell Biol 16, 1534-42.
Hynes, R. O. (1990). “Fibronectins.” Springer-Verlag, New York.
Idriss, H., Kumar, A., Casas-Finet, J. R., Guo, H., Damuni, Z., and Wilson, S. H. (1994). Regulation of in vitro nucleic acid strand annealing activity of heterogeneous nuclear ribonucleoprotein protein A1 by reversible phosphorylation. Biochemistry 33, 11382-90.
Ignotz, R. A., Endo, T., and Massague, J. (1987). Regulation of fibronectin and type I collagen mRNA levels by transforming growth factor-beta. J. Biol. Chem. 262, 6643-6646.
Inoue, K., Hoshijima, K., Sakamoto, H., and Shimura, Y. (1990). Binding of the Drosophila sex-lethal gene product to the alternative splice site of transformer primary transcript. Nature 344, 461-3.
Izaurralde, E., Jarmolowski, A., Beisel, C., Mattaj, I. W., Dreyfuss, G., and Fischer, U. (1997a). A role for the M9 transport signal of hnRNP A1 in mRNA nuclear export. J Cell Biol 137, 27-35.
Izaurralde, E., Kutay, U., von Kobbe, C., Mattaj, I. W., and Gorlich, D. (1997b). The asymmetric distribution of the constituents of the Ran system is essential for transport into and out of the nucleus. Embo J 16, 6535-47.
Izaurralde, E., McGuigan, C., and Mattaj, I. W. (1995). Nuclear localization of a cap-binding protein complex. Cold Spring Harb Symp Quant Biol 60, 669-75.
Jang, J. H., Wang, F., and Kan, M. (1997). Heparan sulfate is required for interaction and activation of the epithelial cell fibroblast growth factor receptor-2111b with stromal-derived fibroblast growth factor-7. In Vitro Cell Dev Biol Anim 33, 819-24.
Jarmolowski, A., Boelens, W. C., Izaurralde, E., and Mattaj, I. W. (1994). Nuclear export of different classes of RNA is mediated by specific factors. J Cell Biol 124, 627-35.
Johnson, D. E., and Williams, L. T. (1993). Structural and functional diversity in the FGF receptor multigene family. Adv Cancer Res 60, 1-41.
Joseph-Silverstein, J., Consigli, S. A., Lyser, K. M., and Ver Pault, C. (1989). Journal of Cell Biology, 2459-2466.
Jung, J., Zheng, M., Goldfarb, M., and Zaret, K. S. (1999). Initiation of mammalian liver development from endoderm by fibroblast growth factors. Science 284, 1998-2003.
Kaffman, A., Rank, N. M., O'Neill, E. M., Huang, L. S., and O'Shea, E. K. (1998). The receptor Msn5 exports the phosphorylated transcription factor Pho4 out of the nucleus. Nature 396, 482-6.
Kalderon, D., Roberts, B. L., Richardson, W. D., and Smith, A. E. (1984). A short amino acid sequence able to specify nuclear location. Cell 39, 499-509.
Kamma, H., Horiguchi, H., Wan, L., Matsui, M., Fujiwara, M., Fujimoto, M., Yazawa, T., and Dreyfuss, G. (1999). Molecular characterization of the hnRNP A2/B1 proteins: tissue-specific expression and novel isoforms. Exp Cell Res 246, 399-411.
Kamma, H., Portman, D. S., and Dreyfuss, G. (1995). Cell type-specific expression of hnRNP proteins. Exp Cell Res 221, 187-96.
Kardon, G. (1998). Muscle and tendon morphogenesis in the avian hind limb. Development 125, 4019-32.
Kasashima, K., Terashima, K., Yamamoto, K., Sakashita, E., and Sakamoto, H. (1999). Cytoplasmic localization is required for the mammalian ELAV-like protein HuD to induce neuronal differentiation. Genes Cells 4, 667-683.
Kaufman, M. H. a. B., J. B. L. (1999). “The Anatomical Basis of Mouse DevelopmentThe Anatomical Basis of Mouse Development.” Academic Press, New Yok.
Kay, B. K., Sawhney, R. K., and Wilson, S. H. (1990). Potential for two isoforms of the A1 ribonucleoprotein in Xenopus laevis. Proc Natl Acad Sci U S A 87, 1367-71.
Kettunen, P., Karavanova, I., and Thesleff, I. (1998). Responsiveness of developing dental tissues to fibroblast growth factors: expression of splicing alternatives of FGFR1, -2, -3, and of FGFR4; and stimulation of cell proliferation by FGF-2, -4, -8, and -9. Dev Genet 22, 374-85.
Kiledjian, M., Burd, C. G., Portman, D. S., and Dreyfuss, G. (1994). Structure and Function of hnRNP Proteins. In “Frontiers in Molecular Biology” (K. a. M. Nagai, I. W., Ed.), pp. 127-149. Oxford: IRL Press.
Kiledjian, M., and Dreyfuss, G. (1992). Primary structure and binding activity of the hnRNP U protein: binding RNA through RGG box. Embo J 11, 2655-64.
Kimelman, D., and Kirschner, M. (1987). Synergistic induction of mesoderm by FGF and TGF-beta and the identification of an mRNA coding for FGF in the early Xenopus embryo. Cell 51, 869-77.
Kimelman, D., and Kirschner, M. W. (1989). An antisense mRNA directs the covalent modification of the transcript encoding fibroblast growth factor in Xenopus oocytes. Cell 59, 687-96.
Kishore, R., Bronstein, N., Ismail, Z., Zhang, Q., Taylor, T., and Newman, S. A. (Submitted for publication). Spatiotemporal regulation and in vitro expression of avian hnRNP A1, an mRNA shuttle protein-exon splicing silencer.
Kishore, R., Samuel, M., Khan, M. Y., Hand, J., Frenz, D. A., and Newman, S. A. (1997). Interaction of the NH2-terminal domain of fibronectin with heparin: role of the O-loops of the type I modules. J. Biol. Chem. 272, 17078-17085.
Knee, R., and Murphy, P. R. (1997). Regulation of gene expression by natural antisense RNA transcripts. Neurochem Int 31, 379-92.
Kosher, R. A., Walker, K. H., and Ledger, P. W. (1982). Temporal and spatial distribution of fibronectin during development of the embryonic chick limb bud. Cell. Differ. 11, 217-228.
Krainer, A. R., Conway, G. C., and Kozak, D. (1990). The essential pre-mRNA splicing factor SF2 influences 5′ splice site selection by activating proximal sites. Cell 62, 35-42.
Kulyk, W. M., Rodgers, B. J., Greer, K., and Kosher, R. A. (1989). Promotion of embryonic chick limb cartilage differentiation by transforming growth factor-beta. Dev. Biol. 135, 424-430.
Kumar, A., Williams, K. R., and Szer, W. (1986). Purification and domain structure of core hnRNP proteins A1 and A2 and their relationship to single-stranded DNA-binding proteins. J Biol Chem 261, 11266-73.
Kumar, A., and Wilson, S. H. (1990). Studies of the strand-annealing activity of mammalian hnRNP complex protein A1. Biochemistry. 29, 10717-10722.
Kutay, U., Bischoff, F. R., Kostka, S., Kraft, R., and Gorlich, D. (1997). Export of importin alpha from the nucleus is mediated by a specific nuclear transport factor [see comments]. Cell90, 1061-71.
Kutay, U., Lipowsky, G., Izaurralde, E., Bischoff, F. R., Schwarzmaier, P., Hartmann, E., and Gorlich, D. (1998). Identification of a tRNA-specific nuclear export receptor. Mol Cell 1, 359-69.
Larsen, W. J. (1997). “Human Embryology.” Churchill Livingston, N.Y.
Lazar, M. A., Hodin, R. A., Cardona, G., and Chin, W. W. (1990). Gene expression from the c-erbA alpha/Rev-ErbA alpha genomic locus. Potential regulation of alternative splicing by opposite strand transcription. J Biol Chem 265,12859-63.
Leonard, C. M., Fuld, H. M., Frenz, D. A., Downie, S. A., Massague, J., and Newman, S. A. (1991). Role of transforming growth factor-beta in chondrogenic pattern formation in the embryonic limb: stimulation of mesenchymal condensation and fibronectin gene expression by exogenous TGF-beta and evidence for endogenous TGF-beta-like activity. Dev. Biol. 145, 99-109.
Leonard, C. M., and Newman, S. A. (1987). Nuclear events during early chondrogenesis: phosphorylation of the precartilage 35.5-kDa domain-specific chromatin protein and its regulation by cyclic AMP. Dev. Biol. 120, 92-100.
Lin, X., Buff, E. M., Perrimon, N., and Michelson, A. M. (1999). Heparan sulfate proteoglycans are essential for FGF receptor signaling during Drosophila embryonic development. Development 126, 3715-23.
Linkhart, T. A., Clegg, C. H., and Hauschika, S. D. (1981). Myogenic differentiation in permanent clonal mouse myoblast cell lines: regulation by macromolecular growth factors in the culture medium. Dev Biol 86, 19-30.
Lizarraga, G., Ferrari, D., Kalinowski, M., Ohuchi, H., Noji, S., Kosher, R. A., and Dealy, C. N. (1999). FGFR2 signaling in normal and limbless chick limb buds. Dev Genet 25, 331-8.
Lopez, A. J. (1998). Alternative splicing of pre-mRNA: developmental consequences and mechanisms of regulation. Annu Rev Genet 32, 279-305.
Luhrmann, R. (1990). Functions of U-snRNPs. Mol Biol Rep 14, 183-92.
Lynch, K. W., and Maniatis, T. (1995). Synergistic interactions between two distinct elements of a regulated splicing enhancer. Genes Dev 9, 284-93.
Lynch, K. W., and Maniatis, T. (1996). Assembly of specific SR protein complexes on distinct regulatory elements of the Drosophila doublesex splicing enhancer. Genes Dev 10, 2089-101.
MacCabe, J. A., Blaylock, R. L., Jr., Latimer, J. L., and Pharris, L. J. (1991). Fibroblast growth factor and culture in monolayer rescue mesoderm cells destined to die in the developing avian wing. J Exp Zool 257, 208-13.
Mackem, S., and Mahon, K. A. (1991). Ghox 4.7: A chick homeobox gene expressed primarily in limb buds with limb-type differences in expression. Development 112, 791-806.
Mahajan, R., Delphin, C., Guan, T., Gerace, L., and Melchior, F. (1997). A small ubiquitin-related polypeptide involved in targeting RanGAP1 to nuclear pore complex protein RanBP2. Cell 88, 97-107.
Makarenkova, H., and Patel, K. (1999). Gap junction signalling mediated through connexin-43 is required for chick limb development. Dev Biol 207, 380-92.
Manche, L., Green, S. R., Schmedt, C., and Mathews, M. B. (1992). Interactions between double-stranded RNA regulators and the protein kinase DAI. Mol Cell Biol 12, 5238-48.
Manley, J. L., and Tacke, R. (1996). SR proteins and splicing control. Genes Dev 10, 1569-79.
Martinez, S., Crossley, P. H., Cobos, I., Rubenstein, J. L., and Martin, G. R. (1999). FGF8 induces formation of an ectopic isthmic organizer and isthmocerebellar development via a repressive effect on Otx2 expression. Development 126, 1189-200.
Massague, J. (1987). The TGF-beta family of growth and differentiation factors. Cell. 49, 437-438.
Matunis, E. L., Matunis, M. J., and Dreyfuss, G. (1992). Characterization of the major hnRNP proteins from Drosophila melanogaster. J Cell Biol 116, 257-69.
Matunis, M. J., Coutavas, E., and Blobel, G. (1996). A novel ubiquitin-like modification modulates the partitioning of the Ran-GTPase-activating protein RanGAP1 between the cytosol and the nuclear pore complex. J Cell Biol 135, 1457-70.
Mayeda, A., Helfman, D. M., and Krainer, A. R. (1993). Modulation of exon skipping and inclusion by heterogeneous nuclear ribonucleoprotein A1 and pre-mRNA splicing factor SF2/ASF [published erratum appears in Mol Cell Biol 1993 Jul;13(7):4458]. Mol Cell Biol 13, 2993-3001.
Mayeda, A., and Krainer, A. R. (1992). Regulation of alternative pre-mRNA splicing by hnRNP A1 and splicing factor SF2. Cell 68, 365-75.
Mayeda, A., Munroe, S. H., Caceres, J. F., and Krainer, A. R. (1994). Function of conserved domains of hnRNP A1 and other hnRNP A/B proteins. Embo J 13, 5483-95.
Melcher, T., Maas, S., Higuchi, M., Keller, W., and Seeburg, P. H. (1995). Editing of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor GluR-B pre-mRNA in vitro reveals site-selective adenosine to inosine conversion. J Biol Chem 270, 8566-70.
Melchior, F., and Gerace, L. (1998). Two-way trafficking with Ran. Trends Cell Biol 8, 175-9.
Melchior, F., Paschal, B., Evans, J., and Gerace, L. (1993). Inhibition of nuclear protein import by nonhydrolyzable analogues of GTP and identification of the small GTPase Ran/TC4 as an essential transport factor [published erratum appears in J Cell Biol 1994 Jan; 124(1-2):217]. J Cell Biol 123, 1649-59.
Merino, E., Balbas, P., Puente, J. L., and Bolivar, F. (1994). Antisense overlapping open reading frames in genes from bacteria to humans. Nucleic Acids Res 22, 1903-8.
Merrill, B. M., Stone, K. L., Cobianchi, F., Wilson, S. H., and Williams, K. R. (1988). Phenylalanines that are conserved among several RNA-binding proteins form part of a nucleic acid-binding pocket in the A1 heterogeneous nuclear ribonucleoprotein. J Biol Chem 263, 3307-13.
Michael, W. M., Choi, M., and Dreyfuss, G. (1995). A nuclear export signal in hnRNP A1: a signal-mediated, temperature-dependent nuclear protein export pathway. Cell 83, 415-22.
Michael, W. M., Eder, P. S., and Dreyfuss, G. (1997). The K nuclear shuttling domain: a novel signal for nuclear import and nuclear export in the hnRNP K protein. Embo J 16, 3587-98.
Miki, T., Bottaro, D. P., Fleming, T. P., Smith, C. L., Burgess, W. H., Chan, A. M., and Aaronson, S. A. (1992). Determination of ligand-binding specificity by alternative splicing: two distinct growth factor receptors encoded by a single gene. Proc Natl Acad Sci U S A 89, 246-50.
Min, H., Turck, C. W., Nikolic, J. M., and Black, D. L. (1997). A new regulatory protein, KSRP, mediates exon inclusion through an intronic splicing enhancer. Genes and Development 11, 1023-1036.
Miralles, F., Czernichow, P., and Scharfmann, R. (1999). Pancreatic acinar AR42J cells express functional nerve growth factor receptors. J Endocrinol 160, 433-42.
Miura, T., and Shiota, K. (2000). Extracellular matrix environment influences chondrogenic pattern formation in limb bud micromass culture: Experimental verification of theoretical models. Anat Rec 258, 100-107.
Miyajima, N., Horiuchi, R., Shibuya, Y., Fukushige, S., Matsubara, K., Toyoshima, K., and Yamamoto, T. (1989). Two erbA homologs encoding proteins with different T3 binding capacities are transcribed from opposite DNA strands of the same genetic locus. Cell 57, 31-9.
Montecucco, C., Caporali, R., Cobianchi, F., and Biamonti, G. (1996). Identification of autoantibodies to the I protein of the heterogeneous nuclear ribonucleoprotein complex in patients with systemic sclerosis. Arthritis Rheum 39, 1669-76.
Moore, M. S., and Blobel, G. (1993). The GTP-binding protein Ran/TC4 is required for protein import into the nucleus. Nature 365, 661-3.
Mosher, D. F. (1984). Physiology of fibronectin. Annu Rev Med 35, 561-75.
Munroe, S. H., and Dong, X. F. (1992). Heterogeneous nuclear ribonucleoprotein A1 catalyzes RNA.RNA annealing. Proc Natl Acad Sci U S A 89, 895-9.
Murphy, P. R., and Knee, R. S. (1994). Identification and characterization of an antisense RNA transcript (gfg) from the human basic fibroblast growth factor gene. Mol Endocrinol 8, 852-9.
Nakielny, S., and Dreyfuss, G. (1997a). Import and export of the nuclear protein import receptor transportin by a mechanism independent of GTP hydrolysis. Curr Biol 8, 89-95.
Nakielny, S., and Dreyfuss, G. (1997b). Nuclear export of proteins and RNAs. Curr Opin Cell Biol 9, 420-9.
Nakielny, S., Shaikh, S., Burke, B., and Dreyfuss, G. (1999). Nup153 is an M9-containing mobile nucleoporin with a novel Ran-binding domain. Embo J 18, 1982-95.
Nakielny, S., Siomi, M. C., Siomi, H., Michael, W. M., Pollard, V., and Dreyfuss, G. (1996). Transportin: nuclear transport receptor of a novel nuclear protein import pathway. Exp Cell Res 229, 261-6.
Nellen, W., and Lichtenstein, C. (1993). What makes an mRNA anti-sense-itive? Trends Biochem Sci 18, 419-23.
Neville, M., Stutz, F., Lee, L., Davis, L. I., and Rosbash, M. (1997). The importin-beta family member Crm1p bridges the interaction between Rev and the nuclear pore complex during nuclear export. Curr Biol 7, 767-75.
Newman, S. A. (1977). Lineage and pattern in the developing wing bud. In “Vertebrate Limb and Somite Morphogenesis” (D. A. Ede, J. R. Hinchliffe, and M. Balls, Eds.), pp. 181-197. Cambridge University Press, Cambridge.
Newman, S. A. (1988). Lineage and pattern in the developing vertebrate limb. Trends. Genet. 4, 329-332.
Newman, S. A. (1996). Sticky fingers: Hox genes and cell adhesion in vertebrate limb development. BioEssays 18, 171-174.
Newman, S. A., Frisch, H. L., Perle, M. A., and Tomasek, J. J. (1981a). Limb development: Aspects of differentiation, pattern formation and morphogenesis. In “Morphogenesis and Pattern Formation” (T. G. Connolly, L. L. Brinkley, and B. M. Carlson, Eds.), pp. 163-178. Raven Press, New York.
Newman, S. A., Pautou, M. -P., and Kieny, M. (1981b). The distal boundary of myogenic primordia in chimeric avian limb buds and its relation to an accessible population of cartilage progenitor cells. Dev. Biol. 84, 440-448.
Newman, S. A., and Tomasek, J. J. (1996). Morphogenesis of connective tissues. In “Extracellular Matrices” (W. D. Comper, Ed.), Vol. 2: Molecular Components and Interactions, pp. 335-369. Harwood Academic Publishers, Reading, U.K.
Nigg, E. A. (1997). Nucleocytoplasmic transport: signals, mechanisms and regulation. Nature 386, 779-87.
Niswander, L., and Martin, G. R. (1992). Fgf-4 expression during gastrulation, myogenesis, limb and tooth development in the mouse. Development 114, 755-68.
Niswander, L., Tickle, C., Vogel, A., Booth, I., and Martin, G. R. (1993). FGF-4 replaces the apical ectodermal ridge and directs outgrowth and patterning of the limb. Cell. 75, 579-587.
Noji, S., Koyama, E., Myokai, F., Nohno, T., Ohuchi, H., Nishikawa, K., and Taniguchi, S. (1993). Differential expression of three chick FGF receptor genes, FGFR1, FGFR2 and FGFR3, in limb and feather development. Prog Clin Biol Res, 645-54.
Oberlender, S. A., and Tuan, R. S. (1994). Expression and functional involvement of N-cadherin in embryonic limb chondrogenesis. Development 120, 177-187.
Oberosler, P., and Nellen, W. (1997). Functional activity and developmental regulation of DdRBP1, a RNA binding protein in Dictyostelium discoideum. Biol Chem 378, 1353-60.
Ohbayashi, N., Hoshikawa, M., Kimura, S., Yamasaki, M., Fukui, S., and Itoh, N. (1998). Structure and expression of the mRNA encoding a novel fibroblast growth factor, FGF18. J Biol Chem 273, 18161-4.
Ohtsubo, M., Kai, R., Furuno, N., Sekiguchi, T., Sekiguchi, M., Hayashida, H., Kuma, K., Miyata, T., Fukushige, S., Murotsu, T., and et al. (1987). Isolation and characterization of the active cDNA of the human cell cycle gene (RCC1) involved in the regulation of onset of chromosome condensation. Genes Dev 1, 585-93.
Oldberg, A., and Ruoslahti, E. (1982). Interactions between chondroitin sulfate proteoglycan, fibronectin, and collagen. J Biol Chem 257, 4859-63.
Oldridge, M., Lunt, P. W., Zackai, E. H., McDonald-McGinn, D. M., Muenke, M., Moloney, D. M., Twigg, S. R., Heath, J. K., Howard, T. D., Hoganson, G., Gagnon, D. M., Jabs, E. W., and Wilkie, A. O. (1997). Genotype-phenotype correlation for nucleotide substitutions in the IgII-IgIII linker of FGFR2. Hum Mol Genet 6, 137-43.
Oldridge, M., Wilkie, A. O., Slaney, S. F., Poole, M. D., Pulleyn, L. J., Rutland, P., Hockley, A. D., Wake, M. J., Goldin, J. H., Winter, R. M., and et al. (1995). Mutations in the third immunoglobulin domain of the fibroblast growth factor receptor-2 gene in Crouzon syndrome. Hum Mol Genet 4, 1077-82.
Oldridge, M., Zackai, E. H., McDonald-McGinn, D. M., Iseki, S., Morriss-Kay, G. M., Twigg, S. R., Johnson, D., Wall, S. A., Jiang, W., Theda, C., Jabs, E. W., and Wilkie, A. O. (1999). De novo alu-element insertions in FGFR2 identify a distinct pathological basis for Apert syndrome. Am J Hum Genet 64, 446-61.
Olwin, B. B., Arthur, K., Hannon, K., Hein, P., McFall, A., Riley, B., Szebenyi, G., Zhou, Z., Zuber, M. E., Rapraeger, A. C., and et al. (1994). Role of FGFs in skeletal muscle and limb development. Mol Reprod Dev 39, 90-100; discussion 100-1.
Ornitz, D. M. (2000). FGFs, heparan sulfate and FGFRs: complex interactions essential for development. Bioessays 22, 108-112.
Orr-Urtreger, A., Bedford, M. T., Burakova, T., Arman, E., Zimmer, Y., Yayon, A., Givol, D., and Lonai, P. (1993). Developmental localization of the splicing alternatives of fibroblast growth factor receptor-2 (FGFR2). Dev Biol 158, 475-86.
Orr-Urtreger, A., Givol, D., Yayon, A., Yarden, Y., and Lonai, P. (1991). Developmental expression of two murine fibroblast growth factor receptors, flg and bek. Development 113, 1419-34.
Ossareh-Nasari, B., Bachelerie, F., and Dargemont, C. (1997). Evidence for a role of CRM1 in signal-mediated nuclear protein export. Science 278, 141-144.
Park, W. J., Theda, C., Maestri, N. E., Meyers, G. A., Fryburg, J. S., Dufresne, C., Cohen, M. M., Jr., and Jabs, E. W. (1995). Analysis of phenotypic features and FGFR2 mutations in Apert syndrome. Am J Hum Genet 57, 321-8.
Patstone, G., Pasquale, E. B., and Maher, P. A. (1993). Different members of the fibroblast growth factor receptor family are specific to distinct cell types in the developing chicken embryo. Dev. Biol. 155,107-123.
Paulsen, D. F., and Solursh, M. (1988). Microtiter micromass cultures of limb-bud mesenchymal cells. In. Vitro. Cell. Dev. Biol. 24, 138-147.
Perle, M. A., Leonard, C. M., and Newman, S. A. (1982). Developmentally regulated nonhistone proteins: evidence for deoxyribonucleic acid binding role and localization near deoxyribonuclease I sensitive domains of precartilage cell chromatin. Biochemistry. 21, 2379-2386.
Perle, M. A., and Newman, S. A. (1980). Talpid²mutant of the chicken with perturbed cartilage development has an altered precartilage-specific chromatin protein. Proc. Natl. Acad. Sci. U. S. A, 77, 4828-4830.
Peters, K. G., Werner, S., Chen, G., and Williams, L. T. (1992). Two FGF receptor genes are differentially expressed in epithelial and mesenchymal tissues during limb formation and organogenesis in the mouse. Development 114, 233-43.
Pinol-Roma, S., Choi, Y. D., Matunis, M. J., and Dreyfuss, G. (1988). Immunopurification of heterogeneous nuclear ribonucleoprotein particles reveals an assortment of RNA-binding proteins [published erratum appears in Genes Dev 1988 Apr;2(4):490]. Genes Dev 2, 215-27.
Pinol-Roma, S., and Dreyfuss, G. (1991). Transcription-dependent and transcription-independent nuclear transport of hnRNP proteins. Science 253, 312-4.
Pinol-Roma, S., and Dreyfuss, G. (1992). Shuttling of pre-mRNA binding proteins between nucleus and cytoplasm. Nature 355, 730-2.
Pinol-Roma, S., Swanson, M. S., Gall, J. G., and Dreyfuss, G. (1989). A novel heterogeneous nuclear RNP protein with a unique distribution on nascent transcripts. J Cell Biol 109, 2575-87.
Pollard, V. W., Michael, W. M., Nakielny, S., Siomi, M. C., Wang, F., and Dreyfuss, G. (1996). A novel receptor-mediated nuclear protein import pathway. Cell 86, 985-94.
Pontius, B. W., and Berg, P. (1990). Renaturation of complementary DNA strands mediated by purified mammalian heterogeneous nuclear ribonucleoprotein A1 protein: implications for a mechanism for rapid molecular assembly. Proc Natl Acad Sci U S A 87, 8403-7.
Portman, D. S., and Dreyfuss, G. (1994). RNA annealing activities in HeLa nuclei. Embo J 13, 213-21.
Rabbitts, T. H., Forster, A., Larson, R., and Nathan, P. (1993). Fusion of the dominant negative transcription regulator CHOP with a novel gene FUS by translocation t(12;16) in malignant liposarcoma. Nat Genet 4, 175-80.
Radu, A., Moore, M. S., and Blobel, G. (1995). The peptide repeat domain of nucleoporin Nup98 functions as a docking site in transport across the nuclear pore complex. Cell 81, 215-22.
Rappolee, D. A., Basilico, C., Patel, Y., and Werb, Z. (1994). Expression and function of FGF-4 in peri-implantation development in mouse embryos. Development 120, 2259-69.
Riley, B. B., Savage, M. P., Simandl, B. K., Olwin, B. B., and Fallon, J. F. (1993). Retroviral expression of FGF-2 (bFGF) affects patterning in chick limb bud. Development 118, 95-104.
Robbins, J., Dilworth, S. M., Laskey, R. A., and Dingwall, C. (1991). Two interdependent basic domains in nucleoplasmin nuclear targeting sequence: identification of a class of bipartite nuclear targeting sequence. Cell 64, 615-23.
Robinow, S., and White, K. (1988). The locus elav of Drosophila melanogaster is expressed in neurons at all developmental stages. Dev Biol 126, 294-303.
Roth, M. B., Zahler, A. M., and Stolk, J. A. (1991). A conserved family of nuclear phosphoproteins localized to sites of polymerase II transcription. J Cell Biol 115, 587-96.
Rout, M. P., Blobel, G., and Aitchison, J. D. (1997). A distinct nuclear import pathway used by ribosomal proteins. Cell 89, 715-25.
Rubin, J. S., Osada, H., Finch, P. W., Taylor, W. G., Rudikoff, S., and Aaronson, S. A. (1989). Purification and characterization of a newly identified growth factor specific for epithelial cells. Proc Natl Acad Sci U S A 86, 802-6.
Rush, M. G., Drivas, G., and D'Eustachio, P. (1996). The small nuclear GTPase Ran: how much does it run? Bioessays 18, 103-12.
Saccone, S., Biamonti, G., Maugeri, S., Bassi, M. T., Bunone, G., Riva, S., and Della Valle, G. (1992). Assignment of the human heterogeneous nuclear ribonucleoprotein A1 gene (HNRPA1) to chromosome 12q13.1 by cDNA competitive in situ hybridization. Genomics 12, 171-4.
Sadiq, M., Hildebrandt, M., Maniak, M., and Nellen, W. (1994). Developmental regulation of antisense-mediated gene silencing in Dictyostelium. Antisense Res Dev 4, 263-7.
Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). “Molecular Cloning. A Laboratory Manual.” Cold Spring Harbor Laboratory Press, Cold Spring Harbor.
Sanger, F., Nicklen, S., and Coulson, A. R. (1977). DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. U. S. A. 74, 5463-5467.
Saunders, J. W., Jr. (1948). The proximo-distal sequence of origin of the parts of the chick wing and the role of the ectoderm. J. Exp. Zool. 108, 363-402.
Savage, M. P., and Fallon, J. F. (1995). FGF-2 mRNA and its antisense message are expressed in a developmentally specific manner in the chick limb bud and mesonephros. Dev. Dyn. 202, 343-353.
Savage, M. P., Hart, C. E., Riley, B. B., Sasse, J., Olwin, B. B., and Fallon, J. F. (1993). Distribution of FGF-2 suggests it has a role in chick limb bud growth. Dev. Dyn. 198, 159-170.
Schofield, J. N., and Wolpert, L. (1990). Effect of TGF-beta 1, TGF-beta 2, and bFGF on chick cartilage and muscle cell differentiation. Exp Cell Res 191, 144-8.
Scotet, E., Reichmann, R., Breathnach, R., and Houssaint, E. (1995). Oncoprotein fos activation in epithelial cells induces and epithelio-mensenchymal conversion and changes the receptor encoded by the FGFR-2 mRNA from K-SAM to BEK. Oncology Report 2, 203-207.
Serrero, G., and Khoo, J. C. (1982). An in vitro model to study adipose differentiation in serum-free medium. Anal Biochem 120, 351-9.
Seyedin, S. M., Segarini, P. R., Rosen, D. M., Thompson, A. Y., Bentz, H., and Graycar, J. (1987). Cartilage-inducing factor-B is a unique protein structurally and functionally related to transforming growth factor-beta. J Biol Chem 262, 1946-9.
Shamim, H., Mahmood, R., Logan, C., Doherty, P., Lumsden, A., and Mason, I. (1999). Sequential roles for Fgf4, En1 and Fgf8 in specification and regionalisation of the midbrain. Development 126, 945-59.
Sheikh, H., and Mason, I. (1996). Polarising activity of FGF-8 in the avian midbrain. Int J Dev Biol Suppl, 117S-118S.
Si, Z., Amendt, B. A., and Stolzfus, C. M. (1997). Splicing efficiency of huamn immunodeficiency virus type 1 tat RNA is determined by both a suboptimal 3′ splice site and a 10 nucleotide exon splicing silencer element located with tat exon 2. Nucleic Acids Reseach 25, 861-867.
Simons, R. W. (1993). The control of prokayotic and eukaryotic gene expression by antturally occurring antisense RNA. In “Antisense Research and Applications” (S. T. a. L. Crooke, B., Ed.), pp. 97-124. CRC Press Inc., Boca Raton.
Siomi, H., and Dreyfuss, G. (1995). A nuclear localization domain in the hnRNP A1 protein. J Cell Biol 129, 551-60.
Siomi, H., and Dreyfuss, G. (1997). RNA-binding proteins as regulators of gene expression. Curr Opin Genet Dev 7, 345-53.
Siomi, M. C., Eder, P. S., Kataoka, N., Wan, L., Liu, Q., and Dreyfuss, G. (1997). Transportin-mediated nuclear import of heterogeneous nuclear RNP proteins. J Cell Biol 138, 1181-92.
Siomi, M. C., Fromont, M., Rain, J. C., Wan, L., Wang, F., Legrain, P., and Dreyfuss, G. (1998). Functional conservation of the transportin nuclear import pathway in divergent organisms. Mol Cell Biol 18, 4141-8.
Slack, J. M., Darlington, B. G., Heath, J. K., and Godsave, S. F. (1987). Mesoderm induction in early Xenopus embryos by heparin-binding growth factors. Nature 326, 197-200.
Sommer, B., Kohler, M., Sprengel, R., and Seeburg, P. H. (1991). RNA editing in brain controls a determinant of ion flow in glutamate-gated channels. Cell 67, 11-9.
Stade, K., Ford, C. S., Guthrie, C., and Weis, K. (1997). Exportin 1 (Crm1p) is an essential nuclear export factor. Cell 90, 1041-50.
Steitz, J. A., Black, D. L., Gerke, V., Parker, K. A., Dramer, A., et al. (1988). . In “Structure and function of Major and Minor Small Nuclear Ribonucleoproteins” (M. L. Birnsteil, Ed.), pp. 115-154. SpringerNerlag, Berlin/New York.
Stolt, P., and Zillig, W. (1993). Antisense RNA mediates transcriptional processing in an archaebacterium, indicating a novel kind of RNase activity. Mol Microbiol 7, 875-82.
Storey, K. G., Goriely, A., Sargent, C. M., Brown, J. M., Burns, H. D., Abud, H. M., and Heath, J. K. (1998). Early posterior neural tissue is induced by FGF in the chick embryo. Development 125, 473-84.
Sweet, D. J., and Gerace, L. (1996). A GTPase distinct from Ran is involved in nuclear protein import. J Cell Biol 133, 971-83.
Szebenyi, G., and Fallon, J. F. (1999). Fibroblast growth factors as multifunctional signaling factors. Int Rev Cytol 185, 45-106.
Szebenyi, G., Savage, M. P., Olwin, B. B., and Fallon, J. F. (1995). Changes in the expression of fibroblast growth factor receptors mark distinct stages of chondrogenesis in vitro and during chick limb skeletal patterning. Dev. Dyn. 204, 446-456.
Tempst, P., Link, A. J., Riviere, L. R., Fleming, M., and Elicone, C. (1990). Internal sequence analysis of proteins separated on polyacrylamide gels at the submicrogram level: improved methods, applications and gene cloning strategies. Electrophoresis. 11, 537-553.
Tolnay, M., Lambris, J. D., and Tsokos, G. C. (1997). Transcriptional regulation of the complement receptor 2 gene: role of a heterogeneous nuclear ribonucleoprotein. J Immunol 159, 5492-501.
Tolnay, M., Vereshchagina, L. A., and Tsokos, G. C. (1999). Heterogeneous nuclear ribonucleoprotein DOB is a sequence-specific DNA-binding protein. Biochem J 338, 417-25.
Tomasek, J. J., Mazurkiewicz, J. E., and Newman, S. A. (1982). Nonuniform distribution of fibronectin during avian limb development. Dev. Biol. 90, 118-126.
Truant, R., Fridell, R. A., Benson, R. E., Bogerd, H., and Cullen, B. R. (1998). Identification and functional characterization of a novel nuclear localization signal present in the yeast Nab2 poly(A)+ RNA binding protein. Mol Cell Biol 18,1449-58.
Valcarcel, J., Singh, R., Zamore, P. D., and Green, M. R. (1993). The protein Sex-lethal antagonizes the splicing factor U2AF to regulate alternative splicing of transformer pre-mRNA. Nature 362, 171-5.
Van Obberghen-Schilling, E., Roche, N. S., Flanders, K. C., Sporn, M. B., and Roberts, A. (1988). Transforming growth factor beta-1 positively regulates its own expression in normal and transformed cells. J. Biol. Chem. 263, 7741-7746.
Vanhee-Brossollet, C., Thoreau, H., Serpente, N., D'Auriol, L., Levy, J. P., and Vaquero, C. (1995). A natural antisense RNA derived from the HIV-1 env gene encodes a protein which is recognized by circulating antibodies of HIV+ individuals. Virology 206, 196-202.
Vanhee-Brossollet, C., and Vaquero, C. (1998). Do natural antisense transcripts make sense in eukaryotes? Gene 211, 1-9.
Voelker, R. A., Graves, J., Gibson, W., and Eisenberg, M. (1990). Mobile element insertions causing mutations in the Drosophila suppressor of sable locus occur in DNase I hypersensitive subregions of 5′-transcribed nontranslated sequences. Genetics 126, 1071-82.
Vogel, A., Rodriguez, C., and Izpisua-Belmonte, J. C. (1996). Involvement of FGF-8 in initiation, outgrowth and patterning of the vertebrate limb. Development 122, 1737-1750.
Von Besser, H., Schnabel, P., Wieland, C., Fritz, E., Stanwsky, R., et al. (1990). Chromosoma 100, 37-47.
Wagner, E. G., and Simons, R. W. (1994). Antisense RNA control in bacteria, phages, and plasmids. Annu Rev Microbiol 48, 713-42.
Walicke, P., Cowan, W. M., Ueno, N., Baird, A., and Guillemin, R. (1986). Fibroblast growth factor promotes survival of dissociated hippocampal neurons and enhances neurite extension. Proc Natl Acad Sci U S A 83, 3012-6.
Walshe, J., and Mason, I. (2000). Expression of FGFR1, FGFR2 and FGFR3 during early neural development in the chick embryo. Mech Dev 90, 103-10.
Wang, J., and Manley, J. L. (1997). Regulation of pre-mRNA splicing in metazoa. Curr Opin Genet Dev 7, 205-11.
Wang, J., Xiao, S. H., and Manley, J. L. (1998). Genetic analysis of the SR protein ASF/SF2: interchangeability of RS domains and negative control of splicing. Genes Dev 12, 2222-33.
Watanabe, A., and Ide, H. (1993). Basic FGF maintains some characteristics of the progress zone of chick limb bud in cell culture. Dev Biol 159, 223-31.
Wedeen, C. J., and Figueroa, N. B. (1998). Expression of actin mRNA in embryos of the leech Helobdella triserialis. Int J Dev Biol 42, 581-90.
Weighardt, F., Biamonti, G., and Riva, S. (1995). Nucleo-cytoplasmic distribution of human hnRNP proteins: a search for the targeting domains in hnRNP A1. J Cell Sci 108, 545-55.
Weighardt, F., Biamonti, G., and Riva, S. (1996). The roles of heterogeneous nuclear ribonucleoproteins (hnRNP) in RNA metabolism. Bioessays 18, 747-56.
Weis, K. (1998). Importins and exporting: how to get in and out of the nucleus [published erratum appears in Trends Biochem Sci 1998 Jul;23(7):235]. Trends Biochem Sci 23, 185-9.
Weis, K., Ryder, U., and Lamond, A. I. (1996). The conserved amino-terminal domain of hSRP1 alpha is essential for nuclear protein import. Embo J 15, 1818-25.
Wen, W., Harootunian, A. T., Adams, S. R., Feramisco, J., Tsien, R. Y., Meinkoth, J. L., and Taylor, S. S. (1994). Heat-stable inhibitors of cAMP-dependent protein kinase carry a nuclear export signal. J Biol Chem 269, 32214-20.
Wilke, T. A., Gubbels, S., Schwartz, J., and Richman, J. M. (1997). Expression of fibroblast growth factor receptors (FGFR1, FGFR2, FGFR3) in the developing head and face. Dev Dyn 210, 41-52.
Wilkie, A. O., Slaney, S. F., Oldridge, M., Poole, M. D., Ashworth, G. J., Hockley, A. D., Hayward, R. D., David, D. J., Pulleyn, L. J., Rutland, P., and et al. (1995). Apert syndrome results from localized mutations of FGFR2 and is allelic with Crouzon syndrome [see comments]. Nat Genet 9, 165-72.
Yamada, K. M., and Kennedy, D. W. (1985). Amino acid sequence specificities of an adhesive recognition signal. J Cell Biochem 28, 99-104.
Yang, Q., Rout, M. P., and Akey, C. W. (1998). Three-dimensional architecture of the isolated yeast nuclear pore complex: functional and evolutionary implications. Molecular Cell 1, 223-234.
Yang, X., Bani, M. R., Lu, S. J., Rowan, S., Ben-David, Y., and Chabot, B. (1994). The A1 and A1B proteins of heterogeneous nuclear ribonucleoparticles modulate 5′ splice site selection in vivo. Proc Natl Acad Sci U S A 91, 6924-8.
Yayon, A., Zimmer, Y., Shen, G. H., Avivi, A., Yarden, Y., and Givol, D. (1992). A confined variable region confers ligand specificity on fibroblast growth factor receptors: implications for the origin of the immunoglobulin fold. Embo J 11, 1885-90.
Yoshida, T., Kokura, K., Makino, Y., Ossipow, V., and Tamura, T. A. (1999). Heterogeneous nuclear RNA-ribonucleoprotein F binds to DNA via an oligo(dG)-motif and is associated with RNA polymerase II. Genes Cells 4, 707-719.
Zahler, A. M., Neugebauer, K. M., Lane, W. S., and Roth, M. B. (1993). Distinct functions of SR proteins in alternative pre-mRNA splicing. Science 260, 219-22.
Zhang, Q. (1995). Characterization of the nuclear cAMP signalling pathway during limb cartilage differentiation. In “Cell Biology and Anatomy”, pp. 126. New York Medical College, Valhalla, N.Y.
Zhang, Q., Carr, D. W., Lerea, K. M., Scott, J. D., and Newman, S. A. (1996). Nuclear localization of type II cAMP-dependent protein kinase during limb cartilage differentiation is associated with a novel developmentally-regulated A-kinase anchoring protein. Developmental Biology 176, 51-61.
Zhu, X., Sasse, J., and Lough, J. (1999). Evidence that FGF receptor signaling is necessary for endoderm-regulated development of precardiac mesoderm. Mech Ageing Dev 108, 77-85.
Zieve, G. W., and Sauterer, R. A. (1990). Cell biology of the snRNP particles. Crit Rev Biochem Mol Biol 25, 1-46.

BACKGROUND OF THE INVENTION

The spatial and temporal coordination of gene expression during embryogenesis involves a variety of regulatory mechanisms, of which those acting at the transcriptional level have been most intensively studied (Davidson et al., 1998; Gellon and McGinnis, 1998; Gray and Levine, 1996; Mannervik et al., 1999). Less is known about mechanisms that control differential production and accumulation of specific proteins at various sites in the developing embryo at the post-transcriptional level, causing the RNA transcript to be spliced appropriately, or regulating transport of the spliced mRNA to the cytoplasm. Recent interest in the role of differential splicing in development and the factors and mechanisms by which it is accomplished (Chabot, 1996; Lopez, 1998), and a growing understanding of the determinants of nucleo-cytoplasmic transport of particular mRNAs (Piñol-Roma and Dreyfuss, 1992; Siomi and Dreyfuss, 1997; Weis, 1998), has set the stage for a systematic analysis of how these RNA processing factors contribute to regional and cell type specificity in the embryo.

The ribonucleoprotein hnRNP A1 is of particular interest in this regard, as it functions in both RNA splice site selection and nucleus-to-cytoplasm transport of mRNA. In its capacity as a splicing factor, this protein modulates 5′ splice site selection in a group of gene products, some of which contain a well-characterized RNA sequence determinant (Burd and Dreyfuss, 1994). Among these are the pre-mRNAs of the HIV type 1 tat protein (Del Gatto-Konczak et al., 1999), FGF receptor 2 (FGFR2) (Del Gatto-Konczak et al., 1999), and hnRNP A1 itself (Chabot et al., 1997). In its role in nucleus-to-cytoplasm transport, hnRNP A1 acts as a “shuttle” protein (Piñol-Roma and Dreyfuss, 1992), and is characterized by a novel amino acid motif termed M9, which contains both nuclear localization and nuclear export activities (Michael et al., 1995; Siomi and Dreyfuss, 1995).

The known functions of hnRNP A1 as an RNA shuttle protein and in splice choice selection are exerted in a gene product-specific fashion (Dreyfuss et al., 1993). The tissue-restricted spatiotemporal patterns in the protein's expression reported are therefore likely to be a causal component of the process by which cell types become distinctive from one another during organogenesis. A subclass of primary transcripts (including hnRNP A1 itself, Chabot et al., 1997; Mayeda et al., 1994) is differentially spliced by a process that depends on hnRNP A1. Control of splice choice appears to involve the antagonism of constitutive splicing factors such as SF2/ASF by members of the hnRNP A/B family of proteins (Mayeda and Krainer, 1992, Mayeda et al., 1993, 1994; Del Gatto-Konczak et al., 1999). Once spliced, these RNAs are transported into the cytoplasm by a process that involves hnRNP A1 and transportin 1 (Nakielny and Dreyfuss, 1998, Nakielny et al., 1999). This implies that the regulation of hnRNP A1 levels within living cells during development plays a key role in cell type diversification.

Earlier studies have surveyed the distribution of hnRNP A1 in a limited set of adult cell types (Kamma et al., 1995; Faura et al., 1995) including the developing germ cells of postnatal mice. A study by the inventors and their colleagues documenting the characterization of the sequence of chicken hnRNP A1 and its spatiotemporal and organ-specific expression during embryogenesis is hereby incorporated by reference in its entirety. (Bronstein et al., 2001). hnRNP A1 protein is abundantly expressed in early stage epithelia such as skin, extraembryonic membranes, and neuroectoderm; epithelioid tissues, such as liver; as well as “secondary” epithelia and epithelioid tissues derived from mesenchymes, e.g., heart muscle, skeletal muscle, kidney tubules, sinusoidal vascular endothelium, and precartilage condensations. It is not clear, however whether this pattern represents an authentic expression theme, or simply the prevalence both of epithelioid tissues in the early embryo and of hnRNP A1 expression. (Bronstein et al., 2001).

The expression of hnRNP A1 in differentiating neuroectoderm and dorsal root ganglia broadly coincides with patterns of expression of members of the Hu class of RNA binding protein genes in the chicken (Wakamatsu and Weston, 1997) and it is significant in this regard that the Hu family of proteins results from extensive alternative splicing of Hu gene products during neurogenesis (Okano and Darnell, 1997). However, while the expression of the two RNA binding proteins may be partly overlapping, they are not entirely so: the typical DRG cell nuclei expressing hnRNP A1 are larger than those expressing Hu and vertebral body cartilage expresses hnRNP A1 but not Hu.

The transcription of natural antisense RNA cognate to exonic sequences of the hnRNP A1 gene in many of the same tissues that are producing sense transcript is an unusual phenomenon, but one that is not as rare as previously thought (Dolnick, 1997; Vanhee-Brossollet and Vaquero, 1998). Antisense RNA probably functions as a post-transcriptional inhibitor of gene expression (Knee and Murphy, 1997). This regulatory mechanism may be particularly relevant during development—natural antisense transcripts of several developmentally active growth factors—fibroblast growth factor-2 (Savage and Fallon, 1995), bone morphogenetic protein-2 (Feng et al., 1997), and transforming growth factor-2 (Coker et al., 1998)—have been detected at significant levels, the first two in embryonic tissues. There is one previous report of differential expression of natural antisense RNA expression of a splicing factor gene (Sureau et al., 1997). Moreover, since hnRNP A1 can promote RNA-RNA strand annealing (Cobianchi et al., 1993; Idriss et al., 1994) it is itself a potential component of natural antisense regulatory mechanisms (Oberosler and Nellen, 1997).

The organ- and tissue-specific sense and antisense hnRNP A1 RNA expression patterns seen at different stages are consistent with the idea that antisense expression may be playing a regulatory role during development. For example, in kidney and liver virtually all cells at the early stages express the sense transcript. But whereas the protein product is also broadly distributed in liver the more limited distribution of the protein in kidney may be related to the more localized distribution of antisense RNA during development of this organ.

Because hnRNP A1 helps regulate nuclear-cytoplasmic transport and alternative splicing for well-defined classes of transcripts, its own regulation can provide the basis for post-transcriptional control of the partitioning of organ primordia into distinct gene expression domains. It is therefore significant that the hnRNP A1 gene is widely transcribed throughout the early embryo and its encoded protein is subject to numerous demonstrated and potential autoregulatory effects at the post-transcriptional level: it helps splice its own pre-mRNA, it may transport its own mRNA from the nucleus to the cytoplasm, and it may participate in the regulation of its own synthesis by its gene's antisense transcript. Small changes in the balance of any of these processes, or of other possible but speculative ones, such as unmasking of maternally inherited hnRNP A1, or even transfer of the protein from one cell to another, could thus activate a post-transcriptional cascade leading to the local expression of hnRNP A1, and with it the expression of its target gene products.

The recent recognition that a large proportion of the genes constituting the human genome are alternatively spliced (Ewing and Green, 2000) (a recent estimate indicates that 38% of human mRNAs contain possible alternative splice forms; Bretta et al., 2000) highlights the centrality of the developmental regulation of hnRNP A1 and other nonconstitutive splicing factors in the generation of complexity in vertebrate organisms.

Classes of hnRNP Proteins

In eukaryotes, heterogeneous nuclear RNAs (hnRNAs), which are the products of RNA polymerase II, are extensively processed to produce messenger RNAs (mRNAs). mRNA processing includes capping, splicing, and polyadenylation (Dreyfuss et al., 1993) and involves the association of the hnRNAs with nuclear proteins collectively known as ribonucleoprotein (RNP) complexes (Dreyfuss et al., 1993; Michael et al., 1995). RNPs that directly bind to hnRNAs are classified as the hnRNPs and are involved in the splicing and shuttling of pre-mRNAs. Others are categorized into special classes such as small nuclear ribonuclear proteins (snRNPs) and include the U snRNPs (Bandziulis et al., 1989; Dreyfuss et al., 1993; Dreyfuss et al., 1988; Luhrmann, 1990; Steitz, 1988; Zieve and Sauterer, 1990). The mature transcript produced from the hnRNA-hnRNP-snRNP complex is transported to the cytoplasm by specific hnRNPs where it may associate with yet another set of RNPs involved in translational regulation and mRNA stability (Bandziulis et al., 1989; Dreyfuss et al., 1993; Luhrmann, 1990; Steitz, 1988; Zieve and Sauterer, 1990).

hnRNP proteins are highly conserved throughout the vertebrates, as well as having sequence homologies in the invertebrate Drosophila (Amrein et al., 1988; Robinow and White, 1988) (Bell et at., 1991; Dreyfuss et at., 1993; Inoue et at., 1990; Kay et at., 1990; Roth et al., 1991; Voelker et al., 1990; Von Besser, 1990), and are the most abundant proteins found in the nucleus (Dreyfuss, 1986, Dreyfuss, 1993). In HeLa cells two-dimendional gel electrophoresis has resolved 20 major groups of proteins. These proteins are designated as the heterogeneous nuclear ribonucleoproteins (hnRNPs) A1 (˜34 kDa) to hnRNP U (˜120 kDa), and categorized by structural motifs (Cobianchi, 1990; Dreyfuss et al., 1993; Matunis et al., 1992; Pinol-Roma et at., 1988). Furthermore, sequence analysis has determined that hnRNPs have one or more RNA-binding modules referred to as the RNP motif or RNA Recognition Motif (RRM) in addition to at least one other auxiliary domain (Dreyfuss et al., 1993). The RNP motif contains two consensus sequences, RNP1 and RNP2, within a domain of approximately 90 amino acid residues that are located about 30 amino acids from each other (Dreyfuss et al., 1993; Dreyfuss et al., 1988). The RNP 1 module is an octapeptide, Lys/Arg-Gly-Phe/Tyr-Gly/Ala-Phe-Val-X-Phen/Tyr, SEQ ID NO: 7 in the Sequence Listing (Adam et al., 1986; Dreyfuss et al., 1993), while the RNP2 module is a hexapeptide rich in aromatic and aliphatic amino acids and is less well conserved (Dreyfuss et al., 1993; Dreyfuss et al., 1988). Both of these consensus sequences are directly related to RNA binding (Dreyfuss et al., 1993; Merrill et al., 1988).

Functional and structural categories of human hnRNPs include:

- (i) hn RNP A2/B1 complexes with the snRNPs and plays a role in splicing pre-mRNAs. Though localized in the nucleus of most tissues, A2 is also found in the cytoplasm of the squamous epithelium of the skin and the esophagus, and abundant amounts of A2 are found in the medulla, but not the cortex of the adrenal gland. Both A2 and B1 are found throughout spermatogenesis while A1 expression is repressed in spermatocytes (Kamma et al., 1999).
- (ii) hnRNP C1 is involved in the post-translation base change of cytosine to uracil in the apolipoprotein (apo) B mRNA which codes for the catalytic subunit APOBEC-1, a protein involved in splicesome assembly. C1 may regulate apoB mRNA editing thus restricting the activity of the catalytic subunit (Greeve et al., 1998).
- (iii) hnRNP D is involved in the immunoglobulin heavy chain recombination process by binding to the switching regions in conjunction with a B cell-specific duplex DNA binding factor (Dempsey et al., 1999), while transcriptional regulation of the complement receptor 2 (CR2) is achieved by hnRNP DOB through its binding of both single and double stranded DNA (Tolnay et al., 1997; Tolnay et al., 1999).
- (iv) hnRNP K may play a role in cytosine-rich pre-mRNA metabolism and cell cycle progression. Highly upregulated levels of K have been found in transformed keratinocytes (Dejgaard et al., 1994).
- (v) hnRNP H, H′ are posttranslationally cleaved to produce the C-terminal proteins H(C) and H(C′) both having a molecular weight of 35 kDa with localization primarily in the nucleus. In contrast, hnRNP F varies with its localization depending on the cell type and is predominantly cytoplasmic in some cells which may be important its function (Honore et al., 1999).
- (vi) Autoantibodies of hnRNP A1, A2, B have been found in individuals with connective tissue diseases. In addition to the A/B proteins, hnRNP I has been found in patients with systemic sclerosis (SSc) and in particular, in individuals with pre-SSc or limited SSc. The A/B and I protein complexes may elicit autoimmune responses (Montecucco et al., 1996).
- (vii) The hnRNP L protein, having an unknown function, is found both as a component of the hnRNP complex as well as in discrete nonnucleolar structures of the nucleoplasm in HeLa cells (Pinol-Roma et al., 1989).
- (viii) Finally hnRNP R, an hnRNP P-like protein, was isolated from yet another individual with autoimmune symptoms and may be a component of subcellular particles that are found in autoimmune diseases (Hassfeld et al., 1998). This protein may have some relationship to the gene product of the TLS/FUS gene, an RNA binding protein identical to hnRNP P2, and first identified as a fusion protein in human myxoid liposarcomas (Calvio et al., 1995; Crozat et al., 1993; Hassfeld et al., 1998; Rabbitts et al., 1993).

In addition, the hnRNP classes of RNA-binding proteins have been shown to be developmentally important in many embryonic tissues including the formation and maintenance of the nervous system (Dreyfuss et al., 1993), sex determination in Drosophila melanogaster (Bandziulis et al., 1989; Del Gatto-Konczak et al., 1999; Lynch and Maniatis, 1995; Lynch and Maniatis, 1996), neuronal splice activation (Del Gatto-Konczak et al., 1999; Min, 1997) and maintenance (Dreyfuss, 1993), and epithelial/mesenchymal differentiation (Johnson and Williams, 1993). In Drosophila, the embryonic lethal abnormal visual (ELAV) system proteins are required for correct differentiation and maintenance of neurons. In mammals the ELAV-like neuronal RNA-binding proteins HuB, HuC, and HuD are implicated in neuronal development and differentiation in both the central and peripheral nervous systems (Akamatsu et al., 1999; Kasashima et al., 1999). In other systems such as the human immunodeficiency virus (HIV-1) hnRNPs are involved in regulating exon 2 of the tat splicing gene (Del Gatto-Konczak et al., 1999; Si, 1997).

hnRNP A1

The hnRNP A1 protein contains two RNP consensus motifs, a glycine-rich auxiliary domain at its carboxy-terminus (Burd and Dreyfuss, 1994; Burd et al., 1989; Buvoli et al., 1990; Merrill et al., 1988), as well as an RGG box, also at its carboxy-terminus (Kiledjian and Dreyfuss, 1992). In addition to these motifs, the hnRNP A1 class of proteins contain a nuclear localization signal, within a domain of approximately 38 amino acids at the carboxy-terminal region of the protein (Michael et al., 1995). This motif, referred to as M9, is a novel nuclear localization signal (NLS)/nuclear export signal (NES) and is not homologous to the classical nuclear localization signal (NLS) found, for example in either the large T antigen of the SV40 virus or the bipartite basic NLS of nucleoplasmin (Izaurralde et al., 1997b; Kalderon et al., 1984; Michael et al., 1995; Robbins et al., 1991; Weighardt et al., 1995). The presence of the M9 motif allows hnRNPs to shuttle continuously between the nucleus and the cytoplasm (Dreyfuss et al., 1993). hnRNPs of the A1, A2/B1, D, E, I and K classes have this capability, while those of the C1, C2, and U class are found restricted to the nucleus (Izaurralde et al., 1997b; Michael et al., 1995; Pinol-Roma and Dreyfuss, 1992). Furthermore, hnRNP A1 is found bound to the poly (A)⁺ tail of RNA polymerase II transcripts in both the nucleus and the cytoplasm and data suggest that the hnRNP A1 protein is transported out of the nucleus with the mature message during the export process (Pinol-Roma and Dreyfuss, 1992). FIG. 1a shows the cDNA sequence designated SEQ ID NO:1 and FIG. 1b shows the amino acid sequence of chicken hnRNP A1 (indicated by CHKA1) designated SEQ ID NO:2 compared to the human hnRNP A1 amino acid sequence (indicated by HUMA1) designated SEQ ID NO:3.

FIG. 2 illustrates the structure of the human core hnRNP proteins A1, A1^B, A2 and B1. The RNP-2 and RNP-1 conserved submotifs of RRM1 and RRM2, and the G domains of each protein are shown. hnRNP A1 (SEQ ID NO. 8) and A1^B or hnRNP A2 (SEQ ID NO. 9) and B1 (SEQ ID NO. 10) are identical except for extra amino acid regions indicated by boxes. The sequences of the RNP-1 and RNP-2 submotifs are aligned. The dots in the alignment indicate amino acid identities. All recombinant proteins are in authentic form except for post-translational modifications. The numbers indicate the position of amino acid residues from the initiation codon Met1. Based on published cDNA sequences (Burd, 1989; Buvoli, 1990). After Mayeda et al. (1994).

hnRNP A1 and Splice Choices

In a multi-step process, uracil rich small nuclear ribonuclear proteins (U snRNPs) in association with the core hnRNPs A1, A2, B1, B2, C1, C2, and C3 (classified by increasing molecular weight), bind to the pre-mRNAs in an ordered manner at specific sequences forming the spliceosome (Beyer et al., 1977; Chung and Wooley, 1986; Del Gatto, 1996; Dreyfuss, 1986; Kumar et al., 1986; Mayeda and Krainer, 1992). Alternative splicing allows for the functional and structural diversity of gene products by the addition or deletion of elements as small as a single amino acid (as seen in the Pax-3 and Pax-7 gene products) (Lopez, 1998). Additional means of obtaining protein variants from a single transcript in a cell-specific manner include splice activation and splice repression (Del Gatto-Konczak et al., 1999).

Alternative splicing may involve the use of alternative 5′ or 3′ splice sites, optional exons, exclusive exons, or retained introns (Lopez, 1998). Except for intron retention, splicing patterns are under competitive control of splicing proteins (Lopez, 1998). Splice activation may involve multi-protein complexes on pre-mRNAs. An example of this is seen in the activation of the female specific dsx exon of Drosophila melanogaster by the female specific proteins, tra (transformer), tra-2 and SR (splice regulator proteins rich in arginine/glycine repeats) (Del Gatto-Konczak et al., 1999; Lynch and Maniatis, 1995; Lynch and Maniatis, 1996; Wang et al., 1998). In the mouse, the c-scr exon N1 is activated by the KSRP splicing factor (KH-type splicing regulator) (Min, 1997; Wang and Manley, 1997) which induces the assembly of five other proteins including hnRNP F (a pre-mRNA splicing factor which is associated with the TATA-binding protein, essential for transcription initiation (Del Gatto-Konczak et al., 1999; Min, 1997; Yoshida et al., 1999). This multiprotein complex activates the intronic splicing enhancer that splices the neuronal specific c-scr N1 exon in vitro (Del Gatto-Konczak et al., 1999; Min, 1997).

Splice repression involves protein binding to an intronic 3′ splice site and is seen in the female-specific Sxl protein of Drosophila. This interaction effectively blocks U2 snRNP and U2AF (U2 snRNP auxiliary factor) (Del Gatto, 1996; Del Gatto-Konczak et al., 1999; Lopez, 1998; Valcarcel et al., 1993). Other protein complexes may use exon sequences for splice repression.

Vertebrate genes including the human fibroblast growth factor receptor 2 gene (fgfr2), and the human immunodeficiency virus type 1 (HIV-1) tat gene contain exons that have sequences acting as exonic splice silencers (ESS) (Amendt et al., 1994; Amendt et al., 1995; Baba-Aissa et al., 1998; Caputi et al., 1994; Caputi et al., 1999; Del Gatto, 1995; Del Gatto, 1996; Del Gatto-Konczak et al., 1999; Gallego et al., 1996; Graham et al., 1992; Si, 1997).

The ESS of the human FGFR2 pre-mRNA contains a UAGG sequence in the kgfr exon (keratinocyte growth factor receptor-exon 8)(Del Gatto, 1996; Del Gatto and Breathnach, 1995; Del Gatto-Konczak et al., 1999). This sequence has homology to the high affinity consensus sequence 5′-UAGGGA/U-3′ recognized by hnRNP A1 (Del Gatto-Konczak et al., 1999). In in vitro studies, Del Gatto-Konczak et al. (1999) have demonstrated that hnRNP A1 can modulate splice choices by binding to a 10 mer ESS designated S10 (5′-UAGGGCAGGC-3′, SEQ ID NO: 5 in the Sequence Listing) or to a 6 mer ESS designated S6 (5′-UAGGGC-3′).

In in vitro studies, RNA molecules containing the splicing silencer sequence from the human fibroblast receptor 2 kgfr exon (IIIb) were capable of directing splice choice selection by the recruitment hnRNP A1 (Del Gatto-Konczak et al., 1999). When the following point mutations were introduced into the S6 ESS UCGGGC or UACGGC a two-fold decrease in hnRNP A1 binding was detected (Del Gatto-Konczak et al., 1999). Furthermore, it was determined that the targeting of hnRNP A1 to the ESS domain was through the glycine-rich motif at the C-terminus of the protein. In the human hnRNP A1 protein, the glycine-rich domains are found between residues 189-320: the RGG motif is specifically located at residues 189-247, followed by another glycine-rich motif from residues 239-320 (Del Gatto-Konczak et al., 1999). Silencing of the k-sam (kgfr) exon in these in vitro studies required the entire glycine-rich motif By examining the corresponding sequence in the chicken kgfr exon (IIIb exon 8) of fgfr2 it has been determined that the sequence corresponding to the human ESS is 5′-UAGGGAGGGC-3′, SEQ ID NO: 6 in the Sequence Listing).

Studies involving hnRNP A1 proteins demonstrated that it is capable of promoting RNA molecules to base pair into double stranded structures, therefore influencing pre-mRNA splicing by snRNPs (Burd and Dreyfuss, 1994; Buvoli et al., 1992; Eperon et al., 1993; Kumar and Wilson, 1990; Munroe and Dong, 1992; Pontius and Berg, 1990; Portman and Dreyfuss, 1994). In in vitro assays hnRNP A1, as well as the RNA binding protein splicing factor 2 (ASF/SF2) (a member of the SR nuclear phosphoprotein family) were capable of making splice choices at the 5′ splice site of pre-mRNAs that contain multiple 5′ splice sites and are essential for constitutive splicing (Caceres et al., 1997; Caceres et al., 1998; Del Gatto, 1996; Fu, 1995; Ge and Manley, 1990; Krainer et al., 1990; Manley and Tacke, 1996; Mayeda et al., 1993; Mayeda and Krainer, 1992; Mayeda et al., 1994; Munroe and Dong, 1992; Zahler et al., 1993).

In vitro studies suggest that hnRNP A1 and ASF/SF2 may act antagonistically and that the hnRNP A/B family of splicing proteins regulates the SR family both in vitro and in vivo (Caceres et al., 1998; Caceres et al., 1994; Mayeda and Krainer, 1992; Yang et al., 1994). In in vitro experiments, excess hnRNP A1 favored the distal 5′ splice site, in contrast to excess ASF/SF2 favoring proximal 5′ splice sites in a concentration-dependent manner resulting in alternate splicing patterns of many genes in specific cell types (Del Gatto, 1996; Mayeda et al., 1993; Mayeda and Krainer, 1992; Mayeda et al., 1994; Munroe and Dong, 1992). Burd and Dreyfuss (1994) have shown that the consensus sequence 5′-UAGGGA/U-3′ is a high affinity binding site of hnRNP A1 and that this sequence is similar to the 5′ and 3′ splice sites in vertebrate pre-mRNAs. In addition, the ability of hnRNP A1 to bind to this consensus sequence increased if it was duplicated and separated by two nucleotides, resulting in a dissociation constant of 1×10⁻⁹M. While hnRNP A1 proteins are capable of binding to other pre-mRNA sites, binding affinity varies greatly over a >100 fold range, therefore classifying these proteins as sequence specific RNA binding proteins (Burd and Dreyfuss, 1994).

hnRNP A1 is also involved in self-splicing. The 4.6 kb human hnRNP A1 mRNA containing 10 exons encodes for the 34 kDa hnRNP A1 protein. The pre-mRNA for hnRNP A1 can be differentially spliced to produce the A1 form and A1^Bform (Buvoli et al., 1990). It has been shown that the human hnRNP A1^Bprotein (FIG. 2) with an apparent molecular weight of 38 kDa, corresponds to the protein previous designated as hnRNP B2 (Buvoli et al., 1990). The A1^Bsplice variant which contains an extra exon in the C-terminal region glycine-rich region (156 bp; 52 amino acids) has a higher affinity for ssDNA than the 34 kDa form though its abundance in the cell is only ˜5% that of hnRNP A1 (Buvoli et al., 1990).

More recently, Blanchette and Chabot (1997) have shown that alternative splicing of the hnRNP A1 pre-mRNA yields the A1 and A1^Bforms via 5′ splice selection and exon skipping, and that this process requires conserved elements. Studies have shown that the addition of the alternate exon 7B in the mature mRNA produces the hnRNP A1^Bprotein (Buvoli et al., 1990). Furthermore, Blanchette and Chabot have demonstrated that the conserved intron element (CE1) upstream from exon 7B favors distal 5′ splice site selection. SR proteins, including SF2, which favor the proximal 5′ splice selection site, require U1 snRNP and U2AF when involved in the 5′ splice site stimulation of a 3′ splice site, as seen in the male specific 3′ splice site of tra in Drosophila (Blanchette and Chabot, 1997; Valcarcel et al., 1993). Interestingly, the CE1 element does not interfere with U1 snRNP binding and led to the discovery of an additional element CE610, which is located downstream from exon 7B. CE610 is also involved in distal 5′ splice site selection by secondary structure formation and exon skipping (Blanchette and Chabot, 1997). Since the SR family of splice selection proteins and hnRNP A1 act antagonistically for 5′ splice choices, where the SRs choose the 5′ proximal site and the hnRNPs the 5′ distal site (Weighardt et al., 1996), hnRNP A1 may be involved in modulating its own splicing (Blanchette and Chabot, 1997; Chabot et al., 1997; Del Gatto-Konczak et al., 1999; Mayeda et al, 1994).

Fibroblast Growth Factors (FGFs), Fibroblast Growth Factor Receptors (FGFRs), and FGFR-2 Splice Variants

Fibroblast growth factors (FGFs) are important mitogens in both cell proliferation and differentiation, but in some cases may act as antagonists and inhibit differentiation. Examples of FGF induced differentiation are seen in the stimulation of pre-adipocyte fibroblasts (Broad and Ham, 1983; Johnson and Williams, 1993; Serrero and Khoo, 1982), and hippocampal neurite outgrowth (Johnson and Williams, 1993; Walicke et al., 1986). Developmental roles have been demonstrated in embryonic mesodermal induction in Xenopus (Kimelman and Kirschner, 1987; Slack et al., 1987), and the inhibition of differentiation of myotubes has been shown in skeletal muscle (Linkhart et al., 1981). In addition to acidic FGF (aFGF or FGF1) and basic FGF (bFGF or FGF2), the family of FGFs, including keratinocyte growth factor (KGF) have been shown to stimulate the proliferation of mesenchymal and neuroectodermal cell types (Burgess and Maciag, 1989; Johnson and Williams, 1993). Using immunohistochemical analysis on chick embryo sections, FGF2 has been localized to the heart, myotome, limbs and muscles (Han, 1997; Joseph-Silverstein, 1989) as well as to the notochord, neural tissue, gut cells, and tubules in the mesonephric and metanephric kidneys (Dono and Zeller, 1994; Han, 1997). In addition to the previously mentioned tissues, Han (1997) localized this mitogen to the developing pharyngeal arches, specifically the maxilla and mandible. FGF2 plays an important role in morphogenesis and pattern formation in the vertebrate limb (Han, 1997; Noji et al., 1993; Riley et al., 1993; Savage et al., 1993), as well as in kidney development (Dono and Zeller, 1994; Han, 1997).

Receptors for the 19 known fibroblast growth factors (FGFs) (Hu et al., 1998; Ohbayashi et al., 1998) include the tyrosine kinase fibroblast growth factor receptors (FGFRs) (Johnson and Williams, 1993), the CFR receptor or cytosine rich FGFR, (Burrus and Olwin, 1989) and the heparan sulfate proteoglycans (HSPGs). In chicken, the genes for fgfrs1, 2, and 3 and 4 (fgfr-related kinase or frek) as well as the kgfr (exon IIIb-keratinocyte growth factor receptor) and bek (exon IIIc-bacterial expressed kinase) splice variants for receptors 1 and 2 have been cloned (Szebenyi et al., 1995). The vertebrate FGFRs contain the domains as described by Johnson and Williams, (1993). Modifications of FGFR isoforms are due to alternative splicing of the pre-mRNAs for each gene. In a schematic representation of human FGFR1, the extracellular region of the molecule has the following domains including a signal peptide region at its N-terminus, followed by three immunoglubulin-like (Ig-like) domains with an acid box between domain I and II. A membrane-proximal region precedes the transmembrane (TM) region. On the intracellular side, two tyrosine kinase domains that are separated by a kinase insert follow a juxtamembrane (JM) domain, and at the C-terminus is a C-tail domain. The third Ig-loop of FGF receptor 2 is involved in the chondrogenic process and can contain either the IIIa and IIIb (kgfr) or IIIa and IIIc (bek) exonic sequences (Johnson and Williams, 1993).

Using chick limb micromass culture, Szebenyi et al. (1995) have looked at changes in the expression of the FGFRs in differentiated cartilage and have found transcripts for fgfr1 in undifferentiated proliferating mesenchyme, fgfr2 in precartilage condensations, and fgfr3 in differentiating cartilage nodules suggesting spatiotemporal regulation in limb development. Binding of the FGFs to their receptors plays an important role in limb development through the regulation of cell survival, proliferation, and precartilage cell differentiation (Fallon et al., 1994; MacCabe et al., 1991; Niswander et al., 1993; Schofield and Wolpert, 1990; Szebenyi et al., 1995; Watanabe and Ide, 1993).

The messenger RNA splice variants IIIb (kgfr) and IIIc (bek) from fgfr1 and fgfr2, as well as fgfr3 were detected in nuclease protection assays on chicken limbs (Szebenyi et al., 1995). In addition, micromass cultures of stage 23-24 wing buds and in situ hybridization of stage 18, 23, 26, and 36 wings showed a spatial distribution of messenger RNA for fgfr1, 2, and 3. Furthermore, the probes for fgfr1 and 2 contained sequences for both the kgfr and bek splice variants and did not allow for the in vivo or in vitro detection of either of these isoforms. This is critical since there is a cell type specific role for the fibroblast growth factor receptor 2 isoforms, (FGFR2) kgfr (exon 8-IIIb) and bek (exon 9-IIIc), in precartilage differentiation. In addition, fibroblast growth factors (FGFs) influence cell function in a tissue-specific or developmental manner that can lead to defects such as craniosynostosis and syndactyly (Del Gatto, 1996; Mayeda and Krainer, 1992; Oldridge et al., 1999). This differential splicing of the pre-mRNAs produced by a single gene allows for the production of splice variants and results in forms that respond to the different growth factor isoforms in a highly specific manner. Cells that will differentiate into epithelia splice only the kgfr exon, while mesenchymal cells, including fibroblasts, as well as other cell types including endothelial cells, splice the 5′ distal bek exon (Del Gatto, 1996; Fallon et al., 1994; Johnson and Williams, 1993; Rubin et al., 1989; Szebenyi et al., 1995).

Mutations in the various receptors cause various skeletal defects (Oldridge et al., 1999). Pfeiffer syndrome, a mutation in the fgfr1 gene, presents with craniosynostosis as well as limb defects; Crouzon syndrome, a result of a mutation in the fgfr2 gene, presents with limb abnormalities; and type II achondroplasia or dwarfism, is caused by a mutation in the fgfr3 gene (Szebenyi et al., 1995). Apert syndrome or acrocephalosyndactyly type 1, presents with head, hand, and foot abnormalities (Anderson et al., 1999). Oldridge et al. (1999) looked at mutations in the fgfr2 gene of 260 unrelated Apert syndrome patients and found that 258 individuals have a missense mutation in exon 7 which lies between the 2nd and 3rd Ig-like loop domains. The remaining two individuals had an ˜360 bp insertion of an Alu-element either 5′ to exon 9 or within exon 9, which arose as de novo mutations in the paternal chromosome (Oldridge et al., 1999). Exon 9 corresponds to the 3rd Ig-like loop domain and contains the bek (IIIc) sequence (Oldridge et al., 1999). In early studies involving the role of the fgfr2 splice variants IIIb (exon 8) and IIIc (exon 9), Rubin et al. (1989) found that keratinocytes expressed the FGFR2 IIIb isoform and were stimulated by KGF. In addition, fibroblasts and endothelial cells expressed the FGFR2 IIIc isoform, and responded to FGF2 (bFGF) (Johnson and Williams, 1993; Rubin et al., 1989). In an RNA analysis of fibroblasts obtained from two Apert and two Pfeiffer syndrome patients having mutations in exon 9, severity of limb abnormalities directly corresponded to ectopic expression of the of the IIIc-kgfr form of the FGFR2. These data provided evidence of the role of signal transduction pathways through the KGFR form of the receptor in relation to syndactyly in Apert syndrome (Oldridge et al., 1999; Park et al., 1995; Wilkie et al., 1995).

FIG. 4 illustrates FGFR2 with positional mutations, polymorphic nucleotides, and primers using in the Oldridge study. Top shows leader sequence (L), acid box (A), three Ig-like domains (IgI, IgIII, and IgIII), a transmembrane region (TM), and a split tyrosine-kinase domain (TK1 and TK2). Exons 8 and 9 encode for the alternative splice variants of the second a half of the IgIII domain, which is depicted by the IgIIIb (kgfr isoform) and IgIIIc (bek isoform) respectively. Positional mutations of two Apert syndrome patients (1 and 2) with Alu insertions, as well as two patients with Pfeiffer syndrome (3 and 4) with nucleotide substitutions are also shown. After Oldridge et al., (1999)

Alternative Splicing

An embodiment of the present invention describes a method and reagents that influence alternative splic