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Originally published In Press as doi:10.1074/jbc.M010570200 on January 30, 2001

J. Biol. Chem., Vol. 276, Issue 18, 15378-15385, May 4, 2001
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Relationships between the Activities in Vitro and in Vivo of Various Kinds of Ribozyme and Their Intracellular Localization in Mammalian Cells*

Yoshio KatoDagger §, Tomoko Kuwabara§||, Masaki WarashinaDagger §||, Hirofumi Toda§, and Kazunari TairaDagger §**

From the Dagger  Department of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, 7-3-1 Hongo, Tokyo 113-8656 and the § Gene Discovery Research Center, National Institute of Advanced Industrial Science and Technology, 1-1-4 Higashi, Tsukuba Science City 305-8562, Japan

Nineteen different functional RNAs were synthesized for an investigation of the actions of ribozymes, in vitro and in vivo, under the control of two different promoters, tRNA or U6, which localize transcripts either in the cytoplasm or in the nucleus. No relationships were found between the activities of these RNAs in cultured cells and the kinetic parameters of their respective chemical cleavage reactions in vitro, indicating that in no case was chemical cleavage the rate-limiting step in vivo. For example, a hepatitis delta virus (HDV) ribozyme, whose activity in vitro was almost 3 orders of magnitude lower than that of a hammerhead ribozyme, still exhibited similar activity in cells when an appropriate expression system was used. As expected, external guide sequences, the actions of which depend on nuclear RNase P, were more active in the nucleus. Analysis of data obtained with cultured cells clearly demonstrated that the cytoplasmic ribozymes were significantly more active than the nuclear ribozymes, suggesting that mature mRNAs in the cytoplasm might be more accessible to antisense molecules than are pre-mRNAs in the nucleus. Our findings should be useful for the future design of intracellularly active functional molecules.

* This work was supported in part by grants from the Ministry of Economy, Trade and Industry of Japan and also by a grant-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science and Techology, Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

These authors contributed equally to this work.

|| Recipient of a research fellowship for young scientists from the Japan Society for the Promotion of Science.

** To whom correspondence should be addressed: Dept. of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, Hongo, Tokyo 113-8656, Japan. Tel.: 81-3-5841-8828 or 81-298-61-3015; Fax: 81-298-61-3019; E-mail: taira@chembio.t.u-tokyo.ac.jp.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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