Genomics & Informatics

Korea Genome Organization (한국유전체학회)
권호 표지

Construction of an RNase P Ribozyme Library System for Functional Genomics Applications

  • Hong, Sun-Woo (Department of Chemistry and BK School of Molecular Science, Pohang University of Science and Technology) ;
  • Choi, Hyo-Jei (Department of Chemistry, Korea Advanced Institute of Science and Technology) ;
  • Lee, Young-Hoon (Department of Chemistry, Korea Advanced Institute of Science and Technology) ;
  • Lee, Dong-Ki (Department of Chemistry and BK School of Molecular Science, Pohang University of Science and Technology)
  • Published : 2007.03.31
Download PDF
⟨ Previous Next ⟩

Abstract

An RNase P ribozyme library has been developed as a tool for functional genomics studies. Each clone of this library contains a random 18-mer and the sequence of M1 RNA, the catalytic subunit of RNase P. Repression of target gene expression is thus achieved by the complementary binding of mRNA to the random guide sequence and the successive target cleavage via M1 RNA. Cellular expression of the ribozyme expression was confirmed, and EGFP mRNA was used as a model to demonstrate that the RNase P ribozyme expression system can inhibit the target gene expression. The constructed RNase P ribozyme library has a complexity of $1.4\times10^7$. This novel library system should become a useful in functional genomics, to identify novel gene functions in mammalian cells.

Keywords

References

  1. Akashi, H., Matsumoto, S., and Taira K. (2005). Gene discovery by ribozyme and siRNAlibraries. Nat. Rev. Mol. Cell. Biol. 6, 413-422 https://doi.org/10.1038/nrm1646
  2. Beger, C. et al. (2001). Identification of Id4 as a regulator of BRCA1 expression by using a ribozyme-library-based inverse genomics approach. Proc. Natl. Acad. Sci. USA 98, 130-135
  3. Brummelkamp, T. R., Bernards, R., and Agami, R. (2002). A system for stable expression of short interfering RNAs in mammalian cells. Science 296, 550-553 https://doi.org/10.1126/science.1068999
  4. Kole, R. and Altman, S. (1981). Properties of purified ribonuclease P from Escherichia coli. Biochemistry 20, 1902-1906 https://doi.org/10.1021/bi00510a028
  5. Lee, D.K., Seol, W., and Kim, J.S. (2003). Custom DNA-binding proteins and artificial transcription factors. Curr. Top. Med. Chem. 3, 645-657 https://doi.org/10.2174/1568026033452384
  6. Lee, D.K., Kim, Y., Kim, J.S., and Seol, W. (2004). Induction and characterization of a taxol-resistance phenotype with transiently expressed artificial transcription activator libraries. Nucleic Acids Res. 32, e116 https://doi.org/10.1093/nar/gnh114
  7. Li, Q. X., Robbins, J. M., Welch, P. J., Wong-Staal, F., and Barber, J. R. (2000). A novel functional genomics approach identifies mTERT as a suppressor of fibroblast transformation. Nucleic Acids Res. 28, 2605-2612 https://doi.org/10.1093/nar/28.13.2605
  8. Raj, S.M. and Liu, F. (2003). Engineering of RNase Pribozyme for gene-targeting applications. Gene 14, 59-69
  9. Suyama, E., Kawasaki, H., Nakajima, M., and Taira, K. (2003). Identification of genes involved in cell invasion by using a library of randomized hybrid ribozymes. Proc. Natl. Acad. Sci. USA 100, 5616-5621
  10. Suyama, E. et al. (2004). Identification of metastasis-related genes in a mouse model using a library of randomized ribozymes. J. Biol. Chem. 279, 38083-38086 https://doi.org/10.1074/jbc.C400313200
  11. Trang, P., Kim, K., Zhu, J., and Liu, F. (2003). Expression of an RNase P ribozyme against the mRNA encoding human cytomegalovirus protease inhibits viral capsid protein processing and growth. J. Mol. Biol. 328, 1123-1135 https://doi.org/10.1016/S0022-2836(03)00398-X
  12. Welch, P. J. et al. (2000). Identification and validation of a gene involved in anchorage-independent cell growth control using a library of randomized hairpin ribozymes. Genomics 66, 274-283 https://doi.org/10.1006/geno.2000.6230