Chromosome and Genomic Engineering in Plants pp 147-155 | Cite as
Development of Genome Engineering Tools from Plant-Specific PPR Proteins Using Animal Cultured Cells
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 proteinNotes
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.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.Kim H, Kim JS (2014) A guide to genome engineering with programmable nucleases. Nat Rev Genet 15:321–334CrossRefPubMedGoogle Scholar
- 3.Hilton IB, Gersbach CA (2015) Enabling functional genomics with genome engineering. Genome Res 10:1442–1455CrossRefGoogle Scholar
- 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.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.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.Yagi Y, Nakamura T, Small I (2014) The potential for manipulating RNA with pentatricopeptide repeat proteins. Plant J 78:772–782CrossRefPubMedGoogle Scholar
- 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.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.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.De Gregorio E, Preiss T, Hentze MW (1999) Translation driven by an eIF4G core domain in vivo. EMBO J 18:4865–4874CrossRefPubMedPubMedCentralGoogle Scholar
- 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.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