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. 1997 Jul;17(7):4096–4104. doi: 10.1128/mcb.17.7.4096

HRS/SRp40-mediated inclusion of the fibronectin EIIIB exon, a possible cause of increased EIIIB expression in proliferating liver.

K Du 1, Y Peng 1, L E Greenbaum 1, B A Haber 1, R Taub 1
PMCID: PMC232263  PMID: 9199345

Abstract

Serine-arginine (SR)-rich proteins are believed to be important in mediating alternative pre-mRNA splicing. HRS/SRp40 expression is elevated in liver cell proliferation during development, regeneration, and oncogenesis. We tested whether HRS expression correlates with the appearance of alternatively spliced fibronectin transcripts during liver growth. HRS was highly expressed during the proliferative phase of liver development, correlating with expression of the fibronectin EIIIB alternative exon. In regenerating liver, HRS protein was induced in a time course consistent with the observed increase in fibronectin transcripts containing the EIIIB exon, particularly in nonparenchymal liver cells. Furthermore, in an in vivo assay, HRS, and not other SR proteins, directly mediated EIIIB exon inclusion in the fibronectin transcript. This alternative splicing was dependent on a purine-rich region within the EIIIB exon to which HRS specifically bound. We have established that HRS has the potential to contribute to the regulation of fibronectin pre-mRNA splicing during liver growth. Changes in fibronectin forms may be important in modifying liver architecture during the proliferative response, thus providing a potential mechanism by which SR proteins may participate in cellular growth control.

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Selected References

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  1. Caputi M., Melo C. A., Baralle F. E. Regulation of fibronectin expression in rat regenerating liver. Nucleic Acids Res. 1995 Jan 25;23(2):238–243. doi: 10.1093/nar/23.2.238. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Carnemolla B., Leprini A., Allemanni G., Saginati M., Zardi L. The inclusion of the type III repeat ED-B in the fibronectin molecule generates conformational modifications that unmask a cryptic sequence. J Biol Chem. 1992 Dec 5;267(34):24689–24692. [PubMed] [Google Scholar]
  3. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  4. Cáceres J. F., Stamm S., Helfman D. M., Krainer A. R. Regulation of alternative splicing in vivo by overexpression of antagonistic splicing factors. Science. 1994 Sep 16;265(5179):1706–1709. doi: 10.1126/science.8085156. [DOI] [PubMed] [Google Scholar]
  5. Diamond R. H., Cressman D. E., Laz T. M., Abrams C. S., Taub R. PRL-1, a unique nuclear protein tyrosine phosphatase, affects cell growth. Mol Cell Biol. 1994 Jun;14(6):3752–3762. doi: 10.1128/mcb.14.6.3752. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Diamond R. H., Du K., Lee V. M., Mohn K. L., Haber B. A., Tewari D. S., Taub R. Novel delayed-early and highly insulin-induced growth response genes. Identification of HRS, a potential regulator of alternative pre-mRNA splicing. J Biol Chem. 1993 Jul 15;268(20):15185–15192. [PubMed] [Google Scholar]
  7. Enrich C., Evans W. H., Gahmberg C. G. Fibronectin isoforms in plasma membrane domains of normal and regenerating rat liver. FEBS Lett. 1988 Feb 8;228(1):135–138. doi: 10.1016/0014-5793(88)80602-1. [DOI] [PubMed] [Google Scholar]
  8. Fu X. D., Maniatis T. The 35-kDa mammalian splicing factor SC35 mediates specific interactions between U1 and U2 small nuclear ribonucleoprotein particles at the 3' splice site. Proc Natl Acad Sci U S A. 1992 Mar 1;89(5):1725–1729. doi: 10.1073/pnas.89.5.1725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fu X. D. Specific commitment of different pre-mRNAs to splicing by single SR proteins. Nature. 1993 Sep 2;365(6441):82–85. doi: 10.1038/365082a0. [DOI] [PubMed] [Google Scholar]
  10. Ge H., Zuo P., Manley J. L. Primary structure of the human splicing factor ASF reveals similarities with Drosophila regulators. Cell. 1991 Jul 26;66(2):373–382. doi: 10.1016/0092-8674(91)90626-a. [DOI] [PubMed] [Google Scholar]
  11. Glück U., Rodríguez Fernández J. L., Pankov R., Ben-Ze'ev A. Regulation of adherens junction protein expression in growth-activated 3T3 cells and in regenerating liver. Exp Cell Res. 1992 Oct;202(2):477–486. doi: 10.1016/0014-4827(92)90102-e. [DOI] [PubMed] [Google Scholar]
  12. Gong Q., Chan S. J., Bajkowski A. S., Steiner D. F., Frankfater A. Characterization of the cathepsin B gene and multiple mRNAs in human tissues: evidence for alternative splicing of cathepsin B pre-mRNA. DNA Cell Biol. 1993 May;12(4):299–309. doi: 10.1089/dna.1993.12.299. [DOI] [PubMed] [Google Scholar]
  13. Greenbaum L. E., Cressman D. E., Haber B. A., Taub R. Coexistence of C/EBP alpha, beta, growth-induced proteins and DNA synthesis in hepatocytes during liver regeneration. Implications for maintenance of the differentiated state during liver growth. J Clin Invest. 1995 Sep;96(3):1351–1365. doi: 10.1172/JCI118170. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Haber B. A., Chin S., Chuang E., Buikhuisen W., Naji A., Taub R. High levels of glucose-6-phosphatase gene and protein expression reflect an adaptive response in proliferating liver and diabetes. J Clin Invest. 1995 Feb;95(2):832–841. doi: 10.1172/JCI117733. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Haber B. A., Mohn K. L., Diamond R. H., Taub R. Induction patterns of 70 genes during nine days after hepatectomy define the temporal course of liver regeneration. J Clin Invest. 1993 Apr;91(4):1319–1326. doi: 10.1172/JCI116332. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Haber B., Naji L., Cressman D., Taub R. Coexpression of liver-specific and growth-induced genes in perinatal and regenerating liver: attainment and maintenance of the differentiated state during rapid proliferation. Hepatology. 1995 Sep;22(3):906–914. [PubMed] [Google Scholar]
  17. Hodges D., Bernstein S. I. Genetic and biochemical analysis of alternative RNA splicing. Adv Genet. 1994;31:207–281. doi: 10.1016/s0065-2660(08)60399-5. [DOI] [PubMed] [Google Scholar]
  18. Hsu J. C., Bravo R., Taub R. Interactions among LRF-1, JunB, c-Jun, and c-Fos define a regulatory program in the G1 phase of liver regeneration. Mol Cell Biol. 1992 Oct;12(10):4654–4665. doi: 10.1128/mcb.12.10.4654. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Huh G. S., Hynes R. O. Elements regulating an alternatively spliced exon of the rat fibronectin gene. Mol Cell Biol. 1993 Sep;13(9):5301–5314. doi: 10.1128/mcb.13.9.5301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Huh G. S., Hynes R. O. Regulation of alternative pre-mRNA splicing by a novel repeated hexanucleotide element. Genes Dev. 1994 Jul 1;8(13):1561–1574. doi: 10.1101/gad.8.13.1561. [DOI] [PubMed] [Google Scholar]
  21. Humphrey M. B., Bryan J., Cooper T. A., Berget S. M. A 32-nucleotide exon-splicing enhancer regulates usage of competing 5' splice sites in a differential internal exon. Mol Cell Biol. 1995 Aug;15(8):3979–3988. doi: 10.1128/mcb.15.8.3979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Jarnagin W. R., Rockey D. C., Koteliansky V. E., Wang S. S., Bissell D. M. Expression of variant fibronectins in wound healing: cellular source and biological activity of the EIIIA segment in rat hepatic fibrogenesis. J Cell Biol. 1994 Dec;127(6 Pt 2):2037–2048. doi: 10.1083/jcb.127.6.2037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kimura H., Sogawa K., Sakai Y., Fujii-Kuriyama Y. Alternative splicing mechanism in a cytochrome P-450 (P-450PB-1) gene generates the two mRNAs coding for proteins of different functions. J Biol Chem. 1989 Feb 5;264(4):2338–2342. [PubMed] [Google Scholar]
  24. Kohtz J. D., Jamison S. F., Will C. L., Zuo P., Lührmann R., Garcia-Blanco M. A., Manley J. L. Protein-protein interactions and 5'-splice-site recognition in mammalian mRNA precursors. Nature. 1994 Mar 10;368(6467):119–124. doi: 10.1038/368119a0. [DOI] [PubMed] [Google Scholar]
  25. Krainer A. R., Mayeda A., Kozak D., Binns G. Functional expression of cloned human splicing factor SF2: homology to RNA-binding proteins, U1 70K, and Drosophila splicing regulators. Cell. 1991 Jul 26;66(2):383–394. doi: 10.1016/0092-8674(91)90627-b. [DOI] [PubMed] [Google Scholar]
  26. Lavigueur A., La Branche H., Kornblihtt A. R., Chabot B. A splicing enhancer in the human fibronectin alternate ED1 exon interacts with SR proteins and stimulates U2 snRNP binding. Genes Dev. 1993 Dec;7(12A):2405–2417. doi: 10.1101/gad.7.12a.2405. [DOI] [PubMed] [Google Scholar]
  27. Malim M. H., Cullen B. R. Rev and the fate of pre-mRNA in the nucleus: implications for the regulation of RNA processing in eukaryotes. Mol Cell Biol. 1993 Oct;13(10):6180–6189. doi: 10.1128/mcb.13.10.6180. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Manley J. L., Tacke R. SR proteins and splicing control. Genes Dev. 1996 Jul 1;10(13):1569–1579. doi: 10.1101/gad.10.13.1569. [DOI] [PubMed] [Google Scholar]
  29. Mohn K. L., Laz T. M., Hsu J. C., Melby A. E., Bravo R., Taub R. The immediate-early growth response in regenerating liver and insulin-stimulated H-35 cells: comparison with serum-stimulated 3T3 cells and identification of 41 novel immediate-early genes. Mol Cell Biol. 1991 Jan;11(1):381–390. doi: 10.1128/mcb.11.1.381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Norton P. A., Hynes R. O. Alternative splicing of chicken fibronectin in embryos and in normal and transformed cells. Mol Cell Biol. 1987 Dec;7(12):4297–4307. doi: 10.1128/mcb.7.12.4297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Oyama F., Hirohashi S., Sakamoto M., Titani K., Sekiguchi K. Coordinate oncodevelopmental modulation of alternative splicing of fibronectin pre-messenger RNA at ED-A, ED-B, and CS1 regions in human liver tumors. Cancer Res. 1993 May 1;53(9):2005–2011. [PubMed] [Google Scholar]
  32. Oyama F., Hirohashi S., Shimosato Y., Titani K., Sekiguchi K. Deregulation of alternative splicing of fibronectin pre-mRNA in malignant human liver tumors. J Biol Chem. 1989 Jun 25;264(18):10331–10334. [PubMed] [Google Scholar]
  33. Oyama F., Murata Y., Suganuma N., Kimura T., Titani K., Sekiguchi K. Patterns of alternative splicing of fibronectin pre-mRNA in human adult and fetal tissues. Biochemistry. 1989 Feb 7;28(3):1428–1434. doi: 10.1021/bi00429a072. [DOI] [PubMed] [Google Scholar]
  34. Pagani F., Zagato L., Vergani C., Casari G., Sidoli A., Baralle F. E. Tissue-specific splicing pattern of fibronectin messenger RNA precursor during development and aging in rat. J Cell Biol. 1991 Jun;113(5):1223–1229. doi: 10.1083/jcb.113.5.1223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Ramchatesingh J., Zahler A. M., Neugebauer K. M., Roth M. B., Cooper T. A. A subset of SR proteins activates splicing of the cardiac troponin T alternative exon by direct interactions with an exonic enhancer. Mol Cell Biol. 1995 Sep;15(9):4898–4907. doi: 10.1128/mcb.15.9.4898. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Screaton G. R., Cáceres J. F., Mayeda A., Bell M. V., Plebanski M., Jackson D. G., Bell J. I., Krainer A. R. Identification and characterization of three members of the human SR family of pre-mRNA splicing factors. EMBO J. 1995 Sep 1;14(17):4336–4349. doi: 10.1002/j.1460-2075.1995.tb00108.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Sharp P. A. Split genes and RNA splicing. Cell. 1994 Jun 17;77(6):805–815. doi: 10.1016/0092-8674(94)90130-9. [DOI] [PubMed] [Google Scholar]
  38. Stamatoglou S. C., Hughes R. C. Cell adhesion molecules in liver function and pattern formation. FASEB J. 1994 Apr 1;8(6):420–427. doi: 10.1096/fasebj.8.6.8168692. [DOI] [PubMed] [Google Scholar]
  39. Sun Q., Mayeda A., Hampson R. K., Krainer A. R., Rottman F. M. General splicing factor SF2/ASF promotes alternative splicing by binding to an exonic splicing enhancer. Genes Dev. 1993 Dec;7(12B):2598–2608. doi: 10.1101/gad.7.12b.2598. [DOI] [PubMed] [Google Scholar]
  40. Sundby C., McCaffery S., Anderson J. M. Turnover of the photosystem II D1 protein in higher plants under photoinhibitory and nonphotoinhibitory irradiance. J Biol Chem. 1993 Dec 5;268(34):25476–25482. [PubMed] [Google Scholar]
  41. Tacke R., Manley J. L. The human splicing factors ASF/SF2 and SC35 possess distinct, functionally significant RNA binding specificities. EMBO J. 1995 Jul 17;14(14):3540–3551. doi: 10.1002/j.1460-2075.1995.tb07360.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Tanaka K., Watakabe A., Shimura Y. Polypurine sequences within a downstream exon function as a splicing enhancer. Mol Cell Biol. 1994 Feb;14(2):1347–1354. doi: 10.1128/mcb.14.2.1347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Taub R. Liver regeneration 4: transcriptional control of liver regeneration. FASEB J. 1996 Mar;10(4):413–427. [PubMed] [Google Scholar]
  44. Tavian D., De Petro G., Colombi M., Portolani N., Giulini S. M., Gardella R., Barlati S. RT-PCR detection of fibronectin EDA+ and EDB+ mRNA isoforms: molecular markers for hepatocellular carcinoma. Int J Cancer. 1994 Mar 15;56(6):820–825. doi: 10.1002/ijc.2910560611. [DOI] [PubMed] [Google Scholar]
  45. Tian M., Maniatis T. A splicing enhancer complex controls alternative splicing of doublesex pre-mRNA. Cell. 1993 Jul 16;74(1):105–114. doi: 10.1016/0092-8674(93)90298-5. [DOI] [PubMed] [Google Scholar]
  46. Wang J., Takagaki Y., Manley J. L. Targeted disruption of an essential vertebrate gene: ASF/SF2 is required for cell viability. Genes Dev. 1996 Oct 15;10(20):2588–2599. doi: 10.1101/gad.10.20.2588. [DOI] [PubMed] [Google Scholar]
  47. Wu J. Y., Maniatis T. Specific interactions between proteins implicated in splice site selection and regulated alternative splicing. Cell. 1993 Dec 17;75(6):1061–1070. doi: 10.1016/0092-8674(93)90316-i. [DOI] [PubMed] [Google Scholar]
  48. Zahler A. M., Lane W. S., Stolk J. A., Roth M. B. SR proteins: a conserved family of pre-mRNA splicing factors. Genes Dev. 1992 May;6(5):837–847. doi: 10.1101/gad.6.5.837. [DOI] [PubMed] [Google Scholar]
  49. Zahler A. M., Neugebauer K. M., Lane W. S., Roth M. B. Distinct functions of SR proteins in alternative pre-mRNA splicing. Science. 1993 Apr 9;260(5105):219–222. doi: 10.1126/science.8385799. [DOI] [PubMed] [Google Scholar]
  50. Zahler A. M., Neugebauer K. M., Stolk J. A., Roth M. B. Human SR proteins and isolation of a cDNA encoding SRp75. Mol Cell Biol. 1993 Jul;13(7):4023–4028. doi: 10.1128/mcb.13.7.4023. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Zuo P., Manley J. L. The human splicing factor ASF/SF2 can specifically recognize pre-mRNA 5' splice sites. Proc Natl Acad Sci U S A. 1994 Apr 12;91(8):3363–3367. doi: 10.1073/pnas.91.8.3363. [DOI] [PMC free article] [PubMed] [Google Scholar]

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