Abstract
We have succeeded in constructing an effective system for the expression of ribozymes under the control of a human tRNAVal promoter, which ensures a high level of production of ribozymes in vivo. The engineered tRNAVal-driven ribozymes, based on computer-predicted secondary structure, were relatively stable and were transported to the cytoplasm, where they could be colocalized with their target RNA. The activity of the exported ribozymes was significantly higher than that of ribozymes that remained in the nucleus. This chapter examines the methods for construction of the appropriate vector that can produce tRNA-driven ribozymes with high-level intracellular activity and for analysis of the constructs in cells.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Kawasaki, H., Eckner, R., Yao, T.-P., Taira, K., Chiu, R., Livingston, D. M., and Yokoyama, K. K. (1998) Distinct roles of the co-activators p300 and CBP in retinoic-acid-induced F9-cell differentiation. Nature 393, 284–289.
Kuwabara, T., Warashina, M., Tanabe, T., Tani, K., Asano, S., and Taira, K. (1998) A novel allosterically trans-activated ribozyme, the maxizyme, with exceptional specificity in vitro and in vivo. Mol. Cell 2, 617–627.
Tanabe, T., Kuwabara, T., Warashina, M., Tani, K., Taira K., and Asano, S. (2000) Oncogene inactivation in a mouse model. Nature 406, 473–474.
Bertrand, E., Castanotto, D., Zhou, C., Carbonnelle, C., Lee, G. P., Chatterjee, S., et al. (1997) The expression cassette determines the functional activity of ribozymes in mammalian cells by controlling their intracellular localization. RNA 3, 75–88.
Good, P. D., Krikos, A. J., Li, S. X., Lee, N. S., Gilver, L., Ellington, A., et al. (1997) Expression of small, therapeutic RNAs in human cell nuclei. Gene. Ther. 4, 45–54.
Koseki, S., Tanabe, T., Tani, K., Asano, S., Shioda, T., Nagai, Y., et al. (1999) Factors governing the activity in vivo of ribozymes transcribed by RNA polymerase III. J. Virol. 73, 1868–1877.
Kato, Y., Kuwabara, T., Warashina, M., Toda, H., and Taira, K. (2001) Relationships between the activities in vitro and in vivo of various kinds of ribozyme and their intracellular localization in mammalian cells. J. Biol. Chem. 276, 15,378–15,385.
Warashina, M., Kuwabara, T., Kato, Y., Sano, M., and Taira, K. (2001) RNA-protein hybrid ribozymes that efficiently cleave any mRNA independently of the structure of the target RNA. Proc. Natl. Acad. Sci. USA 98, 5572–5577.
Kuwabara, T., Warashina, M., Sano, M., Tang, H., Wong-Staal, F., Munekata, E. and Taira, K. (2001) Recognition of engineered tRNAs with an extended 3′ end by Exportin-t (Xpo-t) and transport of tRNA-attached ribozymes to the cytoplasm in somatic cells. Biomacromolecules 2, 1229–1242.
Arts, G.-J., Fornerod, M., and Mattaj, I. W. (1998) Identification of a nuclear export receptor for tRNA. Curr. Biol. 8, 305–314.
Kutay, U., Lipowsky, G., Izaurralde, E., Bischoff, F. R., Schwarzmaier, P., Hartmann, E., and Görlich, D. (1998) Identification of a tRNA-specific nuclear export receptor. Mol. Cell 1, 359–369.
Kuwabara, T., Warashina, M., Koseki, S., Sano, M., Ohkawa, J., Nakayama, K., and Taira, K. (2001) Significantly higher activity of a cytoplasmic hammerhead ribozyme than a corresponding nuclear counterpart: engineered tRNAs with an extended 3′ end can be exported efficiently and specifically to the cytoplasm in mammalian cells. Nucleic Acids Res. 29, 2780–2788.
Eckstein, F. (1998) Searching for the ideal partner. Nature Biotechnol. 16, 24.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2004 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Sano, M., Taira, K. (2004). Ribozyme Expression Systems. In: Sioud, M. (eds) Ribozymes and siRNA Protocols. Methods in Molecular Biology™, vol 252. Humana Press. https://doi.org/10.1385/1-59259-746-7:195
Download citation
DOI: https://doi.org/10.1385/1-59259-746-7:195
Publisher Name: Humana Press
Print ISBN: 978-1-58829-226-1
Online ISBN: 978-1-59259-746-8
eBook Packages: Springer Protocols