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