Abstract
C-to-U RNA editing in mitochondria and plastids is widespread in almost all terrestrial plants, where it mainly changes codons to encode conserved amino acids in organelle mRNAs. In flowering plants, the number of RNA editing sites reaches 400–600 in mitochondria and about 40 in plastids, respectively. To date, more than 100 factors involved in RNA editing have been identified. Since target cytidines of each factor are often distributed across multiple transcripts, comprehensive monitoring of all RNA editing sites is necessary for their characterization. Comparing the signals of C and T in the Sanger sequencing chromatogram of RT-PCR products is the most frequently employed method for quantification of RNA editing efficiency, although several methods based on next-generation sequencing have been developed. I here describe a quick and easy method for quantification of RNA editing efficiency at several hundred sites using the Sanger sequencing chromatogram data.
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References
Takenaka M, Zehrmann A, Verbitskiy D, Härtel B, Brennicke A (2013) RNA editing in plants and its evolution. Annu Rev Genet 47:335–352
Small ID, Schallenberg-Rüdinger M, Takenaka M, Mireau H, Ostersetzer-Biran O (2020) Plant organellar RNA editing: what 30 years of research has revealed. Plant J 101:1040–1056
Cheng S, Gutmann B, Zhong X, Ye Y, Fisher MF, Bai F, Castleden I, Song Y, Song B, Huang J, Liu X, Xu X, Lim BL, Bond CS, Yiu S-M, Small I (2016) Redefining the structural motifs that determine RNA binding and RNA editing by pentatricopeptide repeat proteins in land plants. Plant J 85:532–547
Takenaka M, Jörg A, Burger M, Haag S (2019) RNA editing mutants as surrogates for mitochondrial SNP mutants. Plant Physiol Biochem 135:310–321
Hayes ML, Santibanez PI (2020) A plant pentatricopeptide repeat protein with a DYW-deaminase domain is sufficient for catalyzing C-to-U RNA editing in vitro. J Biol Chem 295:3497–3505
Oldenkott B, Yang Y, Lesch E, Knoop V, Schallenberg-Rüdinger M (2019) Plant-type pentatricopeptide repeat proteins with a DYW domain drive C-to-U RNA editing in Escherichia coli. Commun Biol 2:85
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:e1002910
Takenaka M, Zehrmann A, Brennicke A, Graichen K (2013) Improved computational target site prediction for Pentatricopeptide repeat RNA editing factors. PLoS One 8:e65343
Yagi Y, Hayashi S, Kobayashi K, Hirayama T, Nakamura T (2013) Elucidation of the RNA recognition code for pentatricopeptide repeat proteins involved in organelle RNA editing in plants. PLoS One 8:e57286
Andrés-Colás N, Zhu Q, Takenaka M, De Rybel B, Weijers D, Van Der Straeten D (2017) Multiple PPR protein interactions are involved in the RNA editing system in Arabidopsis mitochondria and plastids. Proc Natl Acad Sci U S A 114:8883–8888
Bentolila S, Heller WP, Sun T, Babina AM, Friso G, van Wijk KJ, Hanson MR (2012) RIP1, a member of an Arabidopsis protein family, interacts with the protein RARE1 and broadly affects RNA editing. Proc Natl Acad Sci U S A 109:E1453–E1461
Guillaumot D, Lopez-Obando M, Baudry K, Avon A, Rigaill G, Falcon de Longevialle A, Broche B, Takenaka M, Berthomé R, De Jaeger G, Delannoy E, Lurin C (2017) Two interacting PPR proteins are major Arabidopsis editing factors in plastid and mitochondria. Proc Natl Acad Sci U S A 114:8877–8882
Shi X, Germain A, Hanson MR, Bentolila S (2016) RNA recognition motif-containing protein ORRM4 broadly affects mitochondrial RNA editing and impacts plant development and flowering. Plant Physiol 170:294–309
Sun T, Shi X, Friso G, Van Wijk K, Bentolila S, Hanson MR (2015) A zinc finger motif-containing protein is essential for chloroplast RNA editing. PLoS Genet 11:e1005028
Takenaka M, Zehrmann A, Verbitskiy D, Kugelmann M, Härtel B, Brennicke A (2012) Multiple organellar RNA editing factor (MORF) family proteins are required for RNA editing in mitochondria and plastids of plants. Proc Natl Acad Sci U S A 109:5104–5109
Bentolila S, Oh J, Hanson MR, Bukowski R (2013) Comprehensive high-resolution analysis of the role of an arabidopsis gene family in RNA editing. PLoS Genet 9:e1003584
Brehme N, Bayer-Császár E, Glass F, Takenaka M (2015) The DYW subgroup PPR protein MEF35 targets RNA editing sites in the mitochondrial rpl16, nad4 and cob mRNAs in Arabidopsis thaliana. PLoS One 10:e0140680
Liu R, Cao S-K, Sayyed A, Yang H-H, Zhao J, Wang X, Jia R-X, Sun F, Tan B-C (2020) The DYW-subgroup pentatricopeptide repeat protein PPR27 interacts with ZmMORF1 to facilitate mitochondrial RNA editing and seed development in maize. J Exp Bot 71:5495–5505
Acknowledgments
This work was supported by the JSPS Grants-in-Aid for Scientific Research [18H02462] to M.T. I am grateful to Brody Frink and Ayako Maeda for critical reading and suggestions.
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Takenaka, M. (2022). Quantification of Mitochondrial RNA Editing Efficiency Using Sanger Sequencing Data. In: Van Aken, O., Rasmusson, A.G. (eds) Plant Mitochondria. Methods in Molecular Biology, vol 2363. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1653-6_18
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DOI: https://doi.org/10.1007/978-1-0716-1653-6_18
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