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Journal of Virology, March 1999, p. 1868-1877, Vol. 73, No. 3
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Factors Governing the Activity In Vivo of Ribozymes Transcribed by RNA Polymerase III

Shiori Koseki,1,2,3 Tsuyoshi Tanabe,4 Kenzaburo Tani,4 Shigetaka Asano,4 Tatsuo Shioda,5 Yoshiyuki Nagai,5 Takashi Shimada,6 Jun Ohkawa,1,2,3,* and Kazunari Taira1,2,3,*

National Institute for Advanced Interdisciplinary Research, AIST, MITI, Tsukuba Science City 305-8562,1 National Institute of Bioscience and Human Technology, AIST, MITI, Tsukuba Science City 305-8566,2 Department of Hepatology/Oncology4 and Department of Viral Infection,5 Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8539, Department of Biochemistry and Molecular Biology, Nippon Medical School, Bunkyo-ku, Tokyo 113-8602,6 and Institute of Applied Biochemistry, University of Tsukuba, Tsukuba Science City 305-8572,3 Japan

Received 3 September 1998/Accepted 4 December 1998

In order to determine the parameters that govern the activity of a ribozyme in vivo, we made a systematic analysis of chimeric tRNAVal ribozymes by measuring their cleavage activities in vitro as well as the steady-state levels of transcripts, the half-lives of transcribed tRNAVal ribozymes, and their activities in both HeLa and H9 cells. These analyses were conducted by the use of transient expression systems in HeLa cells and stable transformants that express ribozymes. Localization of transcripts appeared to be determined by the higher-order structure of each transcribed tRNAVal ribozyme. Since colocalization of the ribozyme with its target RNA is important for strong activity of the ribozyme in vivo, the best system for tRNA-based expression seems to be one in which the structure of the transcript is different from that of the natural tRNA precursor so that processing of the tRNAVal ribozyme can be avoided. At the same time, the structure of the transcript must be similar enough to allow recognition, probably by an export receptor, so that the transcript can be exported to the cytoplasm to ensure colocalization with its target. In the case of several tRNAVal ribozymes that we constructed, inspection of computer-predicted secondary structures enabled us to control the export of transcripts. We found that only a ribozyme that was transcribed at a high level and that had a sufficiently long half-life, within cells, had significant activity when used to withstand a challenge by human immunodeficiency virus type 1.

* Corresponding author. Mailing address: Institute of Applied Biochemistry, University of Tsukuba, Tennoudai 1-1-1, Tsukuba Science City 305-8572, Japan. Phone: 81-298-54-3015. Fax: 81-298-54-3019. E-mail: taira{at}nair.go.jp.

Journal of Virology, March 1999, p. 1868-1877, Vol. 73, No. 3
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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