Journal of Microbiology

The Microbiological Society of Korea (한국미생물학회)
권호 표지

Replacement of Thymidine Phosphorylase RNA with Group I Intron of Tetrahymena thermophila by Targeted Trans-Splicing

  • Park, Young-Hee (Department of Molecular Biology & Institute of Nanosensor and Biotechnology, Dankook University) ;
  • Jung, Heung-Su (Department of Molecular Biology & Institute of Nanosensor and Biotechnology, Dankook University) ;
  • Kwon, Byung-Su (Department of Molecular Biology & Institute of Nanosensor and Biotechnology, Dankook University) ;
  • Lee, Seong-Wook (Department of Molecular Biology & Institute of Nanosensor and Biotechnology, Dankook University)
  • Published : 2003.01.01
Download PDF
⟨ Previous Next ⟩

Abstract

The group I intron from Tetrahymena thermophila has been demonstrated to employ splicing reactions with its substrate RNA in the trans configuration. Moreover, we have recently shown that the transsplicing group I ribozyme can replace HCV-specific transcripts with a new RNA that exerts anti-viral activity. In this study, we explored the potential use of RNA replacement for cancer treatment by developing trans-splicing group I ribozymes, which could replace tumor-associated RNAs with the RNA sequence attached to the 3' end of the ribozymes. Thymidine phosphorylase (TP) RNA was chosen as a target RNA because it is known as a valid cancer prognostic factor. By performing an RNA mapping strategy that is based on a trans-splicing ribozyme library, we first determined which regions of the TP RNA are accessible to ribozymes, and found that the leader sequences upstream of the AUG start codon appeared to be particularly accessible. Next, we assessed the ribozyme activities by comparing trans-splicing activities of several ribozymes that targeted different regions of the TP RNA. This assessment was performed to verify if the target site predicted to be accessible is truly the most accessible. The ribozyme that could target the most accessible site, identified by mapping studies, was the most active with high fidelity in vitro. Moreover, the specific trans-splicing ribozyme reacted with and altered the TP transcripts by transferring an intended 3' exon tag sequence onto the targeted TP RNA in mammalian cells with high fidelity. These results suggest that the Tetrahymena ribozyme can be utilized to replace TP RNAs in tumors with a new RNA harboring anti-cancer activity, which would revert the malignant phenotype.

Keywords

References

  1. Brown, N.S., A. Jones, C. Rujiyama, A.L. Harris, and R. Bicknell. 2000. Thymidine phosphorylase induces carcinoma cell oxidative stress and promotes secretion of angiogenic factors. Cancer Res. 60, 6298-6302.
  2. Cech, T.R. 1993. Structure and mechanism of the large catalytic RNAs: Group I and group II introns and ribonuclease P, p. 239-269. In F. Gesteland and J.F. Atkins (eds.), The RNA World. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, New York.
  3. Feramisco, J.R., J.E. Smart, K. Burridge, D.M. Helfman, and G.P. Thomas. 1982. Co-existence of vinculin and a vinculin-like protein of higher molecular weight in smooth muscle. J. Biol. Chem. 257, 11024-11031.
  4. Fox, S.B., A. Moghaddam, and M. Westwood. 1995. Plateletderived endothelial cell growth factor/thymidine phosphorylase expression in normal tissues: an immunohistochemical study. J. Pathol. 176, 183-190.
  5. Furukawa, T., A. Yoshimura, and Y. Yamada. 1992. Angiogenic factor. Nature 356, 668.
  6. Ikeda, R., T. Furukawa, K. Yamada, and S. Akiyama. 2002. Molecular basis for the inhibition of hypoxia-induced apoptosis by 2-deoxy-D-ribose. Biochem. Biophys. Res. Commun. 291, 806-812.
  7. Ikeda, R., T. Furukawa, K. Yamada, and S. Akiyama, 2003. Thymidine phosphorylase inhibits apoptosis induced by cisplatin. Biochem. Biophys. Res. Commun. 301, 358-363.
  8. Iltzsch, M.H., M.H. Kouni, and S. Cha. 1985. Kinetic studies of thymidine phosphorylase from mouse liver. Biochemistry 24, 6799-6807.
  9. Ishikawa, F., K. Miyazono, U. Hellman, H. Drexler, C. Wernstedt, K. Hagiwara, K. Usuki, F. Takaku, W. Risau, and C.-H. Heldin. 1989. Identification of angiogenic activity and the cloning and expression of platelet-derived endothelial cell growth factor. Nature 338, 557-562.
  10. Jaeger, J., D. Turner, and M. Zuker. 1989. Improved predictions of secondary structures for RNA. Proc. Natl. Acad. Sci. USA 86, 7706-7710.
  11. Jones, J.T., S.-W. Lee, and B.A. Sullenger. 1996. Tagging ribozyme reaction sites to follow trans-splicing in mammalian cells. Nat. Med. 2, 643-648.
  12. Lan, N., R.P. Howrey, S.-W. Lee, C.A. Smith, and B.A. Sullenger. 1998. Ribozyme-mediated repair of sickle $\beta$-globin mRNAs in erythrocyte precursors. Science 280, 1593-1596.
  13. Lan, N., B.L. Rooney, S.-W. Lee, R.P. Howrey, C.A. Smith, and B.A. Sullenger. 2000. Enhancing RNA repair efficiency by combining trans-splicing ribozymes that recognize different accessible sites on a target RNA. Mol. Ther. 2, 245-255.
  14. Luccioni, C., J. Beaumatin, V. Bardot, and D. Lefrancois. 1994. Pyrimidine nucleotide metabolism in human colon carcinomas: comparison of normal tissues, primary tumors and xenografts. Int. J. Cancer 58, 517-522.
  15. Miyadera, K., T. Sumizawa, and S. Akiyama. 1995. Role of thymidine phosphorylase activity in the angiogenic effect of plateletderived endothelial cell growth factor/thymidine phosphorylase. Cancer Res. 55, 1687-1690.
  16. Miyazono, K., T. Okabe, A. Urabe, and C.H. Heldin. 1987. Purification and properties of an endothelial cell growth factor from human platelets. J. Biol. Chem. 262, 4098-4103.
  17. Mori, S., S. Takao, H. Nama, and T. Aikou. 2002. Thymidine phosphorylase suppresses Fas-induced apoptotic signal transduction independent of its enzymatic activity. Biochem. Biophys. Res. Commun. 295, 300-305.
  18. Phylactou, L.A., C. Darrah, and M.A.J. Wood. 1998. ibozymemediated trans-splicing of a trinucleotide repeat. Nat. Genet. 18, 378-381.
  19. Rogers, C.S., C.G. Vanoye, B.A. Sullenger, and A.L. George, Jr. 2002. Functional repair of a mutant chloride channel using a trans-splicing ribozyme. J. Clin. Invest. 110, 1783-1798.
  20. Ryu, K.-J., J.-H. Kim, and S.-W. Lee. 2003. Ribozyme-mediated selective induction of new gene activity in hepatitis C virus internal ribosome entry site-expressing cells by targeted transsplicing. Mol. Ther. 7, 386-395.
  21. Shin, K.-S., S.-J. Bae, E.-S. Hwang, S. Jeong, and S.-W. Lee. 2002. Ribozyme-mediated replacement of p53 RNA by targeted trans-splicing. J. Microbiol. Biotechnol. 12, 844-848.
  22. Sullenger, B.A. and T.R. Cech. 1994. Ribozyme-mediated repair of defective mRNA by targeted trans-splicing. Nature 317, 619-622.
  23. Sullenger, B.A. and T.R. Cech. 1995. RNA repair: a new possibility for gene therapy. J. NIH Res. 7, 46-47.
  24. Sullenger, B.A. and E. Gilboa. 2002. Emerging clinical applications of RNA. Nature 418, 252-258.
  25. Takebayashi, Y., K. Yamada, and K. Miyadera. 1996. The activity and expression of thymidine phosphorylase in human solid tumors. Eur. J. Cancer 32A, 1227-1232
  26. Watanabe, T. and B.A. Sullenger. 2000. Induction of wild-type p53 activity in human cancer cells by ribozymes that repair mutant p53 transcripts. Proc. Natl. Acad. Sci. USA 97, 8490-8494.