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Identification of genes that function in the TNF-α-mediated apoptotic pathway using randomized hybrid ribozyme libraries

Nature Biotechnology volume 20, pages 376–380 (2002)Cite this article

A Retraction to this article was published on 01 September 2006

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Abstract

Now that the sequences of many genomes are available, methods are required for the rapid identification of functional genes. We describe here a simple system for the isolation of genes that function in the tumor necrosis factor-α (TNF-α)–mediated pathway of apoptosis, using RNA helicase–associated ribozyme libraries with randomized substrate-binding arms. Because target-site accessibility considerably limits the effective use of intracellular ribozymes, the effectiveness of a conventional ribozyme library has been low. To overcome this obstacle, we attached to ribozymes an RNA motif (poly(A)-tail) able to interact with endogenous RNA helicase(s) so that the resulting helicase-attached, hybrid ribozymes can more easily attack target sites regardless of their secondary or tertiary structures. When the phenotype of cells changes upon introduction of a ribozyme library, genes responsible for these changes may be identified by sequencing the active ribozyme clones. In the case of TNF-α-mediated apoptosis, when a ribozyme library was introduced into MCF-7 cells, surviving clones were completely or partially resistant to TNF-α-induced apoptosis. We identified many pro-apoptotic genes and partial sequences of previously uncharacterized genes using this method. Our gene discovery system should be generally applicable to the identification of functional genes in various systems.

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Figure 1: Unwinding and cleavage activities of RNA–protein hybrid ribozymes and construction of hybrid ribozyme libraries.
Figure 2: Rationale for system for discovery of genes that act in TNF-α-mediated apoptosis using poly(A)-linked ribozyme libraries.
Figure 3: Expression and apoptotic functions of target genes.
Figure 4: Gene UG26, involved in TNF-mediated apoptosis.

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Acknowledgements

This research was supported by grants from the Ministry of Economy, Trade and Industry (METI) of Japan, by a grant from the New Energy and Industrial Technology Development Organization (NEDO) of Japan, and by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan.

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Authors and Affiliations

  1. Department of Chemistry and Biotechnology, School of Engineering, The University of Tokyo, Hongo, 113-8656, Tokyo, Japan

    Hiroaki Kawasaki, Eigo Suyama & Kazunari Taira

  2. Gene Discovery Research Center, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba Science City, 305-8562, Japan

    Hiroaki Kawasaki, Reiko Onuki, Eigo Suyama & Kazunari Taira

  3. Graduate School of Life and Environmental Sciences, University of Tsukuba, Tennoudai 1-1-1, Tsukuba Science City, 305-8572, Japan

    Reiko Onuki

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Correspondence to Kazunari Taira.

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Kawasaki, H., Onuki, R., Suyama, E. et al. Identification of genes that function in the TNF-α-mediated apoptotic pathway using randomized hybrid ribozyme libraries. Nat Biotechnol 20, 376–380 (2002). https://doi.org/10.1038/nbt0402-376

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