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Nucleic Acids Research, 2001, Vol. 29, No. 13 2780-2788
© 2001 Oxford University Press

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

Tomoko Kuwabara1,2, Masaki Warashina1,2, Shiori Koseki1,2, Masayuki Sano1,2, Jun Ohkawa1,2, Kazuhisa Nakayama3 and Kazunari Taira1,4,* 1Gene Discovery Research Center, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-4 Higashi, Tsukuba Science City 305-8562, Japan, 2Institute of Applied Biochemistry and 3Institute of Biological Sciences, University of Tsukuba, 1-1-1 Tennoudai, Tsukuba Science City 305-8572, Japan and 4Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, Hongo, Tokyo 113-8656, Japan

Hammerhead ribozymes were expressed under the control of similar tRNA promoters, localizing transcripts either in the cytoplasm or the nucleus. The tRNAVal-driven ribozyme (tRNA-Rz; tRNA with extra sequences at the 3' end) that has been used in our ribozyme studies was exported efficiently into the cytoplasm and ribozyme activity was detected only in the cytoplasmic fraction. Both ends of the transported tRNA-Rz were characterized comprehensively and the results confirmed that tRNA-Rz had unprocessed 5' and 3' ends. Furthermore, it was also demonstrated that the activity of the exported ribozyme was significantly higher than that of the ribozyme which remained in the nucleus. We suggest that it is possible to engineer tRNA-Rz, which can be exported to the cytoplasm based on an understanding of secondary structures, and then tRNA-driven ribozymes may be co-localized with their target mRNAs in the cytoplasm of mammalian cells.

* To whom correspondence should be addressed at: Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, Hongo, Tokyo 113-8656, Japan. Tel: +81 3 5841 8828; Fax: +81 298 61 3019; Email: taira{at}chembio.t.u-tokyo.ac.jp The authors wish it to be known that, in their opinion, the first three authors should be regarded as joint First Authors


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