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Development of Genome Engineering Tools from Plant-Specific PPR Proteins Using Animal Cultured Cells

  • Takehito Kobayashi
  • Yusuke Yagi
  • Takahiro Nakamura
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1469)

Abstract

The pentatricopeptide repeat (PPR) motif is a sequence-specific RNA/DNA-binding module. Elucidation of the RNA/DNA recognition mechanism has enabled engineering of PPR motifs as new RNA/DNA manipulation tools in living cells, including for genome editing. However, the biochemical characteristics of PPR proteins remain unknown, mostly due to the instability and/or unfolding propensities of PPR proteins in heterologous expression systems such as bacteria and yeast. To overcome this issue, we constructed reporter systems using animal cultured cells. The cell-based system has highly attractive features for PPR engineering: robust eukaryotic gene expression; availability of various vectors, reagents, and antibodies; highly efficient DNA delivery ratio (>80 %); and rapid, high-throughput data production. In this chapter, we introduce an example of such reporter systems: a PPR-based sequence-specific translational activation system. The cell-based reporter system can be applied to characterize plant genes of interested and to PPR engineering.

Key words

Animal cultured cell DNA-binding protein Genome editing HEK293T cell Translational activation Pentatricopeptide repeat PPR RNA-binding protein 

Notes

Acknowledgments

This work was in part supported by Cross-ministerial Strategic Innovation Promotion Program (SIP) of Council for Science, Technology and Innovation (CSTI) (T.N.) and Grants-in-Aid 26117718 and 25292219 (T.N.), and 26870430 (to Y.Y.) from the Ministry of Education, Culture, Sports, Science, and Technology. Y.Y. is supported by JSPS fellowship.

References

  1. 1.
    Gaj T, Gersbach CA, Barbas CF 3rd (2013) ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol 31:397–405CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Kim H, Kim JS (2014) A guide to genome engineering with programmable nucleases. Nat Rev Genet 15:321–334CrossRefPubMedGoogle Scholar
  3. 3.
    Hilton IB, Gersbach CA (2015) Enabling functional genomics with genome engineering. Genome Res 10:1442–1455CrossRefGoogle Scholar
  4. 4.
    Andrés C, Lurin C, Small ID (2007) The multifarious roles of PPR proteins in plant mitochondrial gene expression. Physiol Plant 129:14–22CrossRefGoogle Scholar
  5. 5.
    Nakamura T, Yagi Y, Kobayashi K (2012) Mechanistic insight into pentatricopeptide repeat proteins as sequence-specific RNA-binding proteins for organellar RNAs in plants. Plant Cell Physiol 53:1171–1179CrossRefPubMedGoogle Scholar
  6. 6.
    Barkan A, Rojas M, Fujii S, Yap A, Chong YS, Bond CS, Small I (2012) A combinatorial amino acid code for RNA recognition by pentatricopeptide repeat proteins. PLoS Genet 8:e1002910CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Yagi Y, Nakamura T, Small I (2014) The potential for manipulating RNA with pentatricopeptide repeat proteins. Plant J 78:772–782CrossRefPubMedGoogle Scholar
  8. 8.
    Van Der Kelen K, Beyaert R, Inzé D, De Veylder L (2009) Translational control of eukaryotic gene expression. Crit Rev Biochem Mol Biol 44:143–168CrossRefGoogle Scholar
  9. 9.
    Cao J, Arha M, Sudrik C, Bugaj LJ, Schaffer DV, Kane RS (2013) Light-inducible activation of target mRNA translation in mammalian cells. Chem Commun (Camb) 49:8338–8340CrossRefGoogle Scholar
  10. 10.
    Cao J, Arha M, Sudrik C, Schaffer DV, Kane RS (2014) Bidirectional regulation of mRNA translation in mammalian cells by using PUF domains. Angew Chem Int Ed Engl 53:4900–4904CrossRefPubMedGoogle Scholar
  11. 11.
    De Gregorio E, Preiss T, Hentze MW (1999) Translation driven by an eIF4G core domain in vivo. EMBO J 18:4865–4874CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Kotera E, Tasaka M, Shikanai T (2005) A pentatricopeptide repeat protein is essential for RNA editing in chloroplasts. Nature 433:326–330CrossRefPubMedGoogle Scholar
  13. 13.
    Okuda K, Nakamura T, Sugita M, Shimizu T, Shikanai T (2006) A pentatricopeptide repeat protein is a site recognition factor in chloroplast RNA editing. J Biol Chem 281:37661–37667CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Takehito Kobayashi
    • 1
  • Yusuke Yagi
    • 1
  • Takahiro Nakamura
    • 1
  1. 1.Faculty of AgricultureKyushu UniversityFukuokaJapan

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