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Using multiplex single-base extension typing to screen for mutants defective in RNA editing

Nature Protocols volume 7, pages 1931–1945 (2012)Cite this article

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Abstract

RNA editing is an RNA maturation process that changes the nucleotide present at particular positions (editing sites) in specific RNAs; in plant organelles, the most common nucleotide change is from cytidine (C) to uridine (U). In a mutant suspected of affecting RNA editing, all known editing sites have to be analyzed. Therefore, to screen a population of mutants, all individuals must be analyzed at every editing site. We describe a multiplex single-nucleotide polymorphism (SNP)–typing procedure to economically screen a mutant individual or population for differences at hundreds of nucleotide positions in RNA or DNA. By using this protocol, we have previously identified mutants defective in RNA editing in a randomly mutated population of Arabidopsis thaliana. The procedure requires 2–3 weeks to identify the individual plant in the mutant population. The time required to locate the mutated gene is between 3 and 24 months in Arabidopsis. Although this procedure has been developed to study RNA editing in plants, it could also be used to investigate other RNA modification processes. It could also be adapted to investigate RNA editing in other organisms.

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Figure 1: Flowchart outlining the steps of the protocol.
Figure 2: Strategy for detecting SNPs in a population of RNA molecules.
Figure 3: Detailed overview of the concept of this protocol to detect variations in RNA editing in a population of mutant individuals.
Figure 4: Example of an actual primer set targeting 20 SNPs (RNA editing events) in five RNAs and the data output.
Figure 5: Detailed setup of the pooling strategy for screening a mutant plant population to identify RNA editing mutants.

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References

  1. Nishikura, K. Functions and regulation of RNA editing by ADAR deaminases. Annu. Rev. Biochem. 79, 321–349 (2010).

    Article  CAS  Google Scholar 

  2. Koito, A. & Ikeda, T. Apolipoprotein B mRNA – editing, catalytic polypeptide cytidine deaminases and retroviral restriction. Wiley Interdiscip. Rev. RNA 3, 529–541 (2012).

    Article  CAS  Google Scholar 

  3. Göringer, H.U., Katari1, V.S. & Böhm, C. The structural landscape of native editosomes in African trypanosomes. Wiley Interdiscip. Rev. RNA 2, 395–407 (2011).

    Article  Google Scholar 

  4. Aphasizhev, R. & Aphasizheva, I. Uridine insertion/deletion editing in trypanosomes: a playground for RNA-guided information transfer. Wiley Interdiscip. Rev. RNA 2, 669–685 (2011).

    Article  CAS  Google Scholar 

  5. Rhee, A.C., Somerlot, B.H., Parimi, N. & Gott, J.M. Distinct roles for sequences upstream of and downstream from Physarum editing sites. RNA 15, 1753–1765 (2009).

    Article  CAS  Google Scholar 

  6. Wahlstedt, H. & Öhman, M. Site-selective versus promiscuous A-to-I editing. Wiley Interdiscip. Rev. RNA 2, 761–771 (2011).

    Article  CAS  Google Scholar 

  7. Garrett, S. & Rosenthal, J.J.C. RNA editing underlies temperature adaptation in K+ channels from polar octopuses. Science 335, 848 (2012).

    Article  CAS  Google Scholar 

  8. Chateigner-Boutin, A.L. & Small, I. Plant RNA editing. RNA Biol. 7, 213–219 (2010).

    Article  CAS  Google Scholar 

  9. Tillich, M., Lehwark, P., Morton, B.R. & Maier, U.G. The evolution of chloroplast RNA editing. Mol. Biol. Evol. 23, 1912–1921 (2006).

    Article  CAS  Google Scholar 

  10. Giegé, P. & Brennicke, A. RNA editing in Arabidopsis mitochondria effects 441 C to U changes in ORFs. Proc. Natl. Acad. Sci. USA 96, 15324–15329 (1999).

    Article  Google Scholar 

  11. Grewe, F. et al. A unique transcriptome: 1782 positions of RNA editing alter 1406 codon identities in mitochondrial mRNAs of the lycophyte Isoetes engelmannii. Nucleic Acids Res. 39, 2890–2902 (2011).

    Article  CAS  Google Scholar 

  12. Hecht, J., Grewe, F. & Knoop, V. Extreme RNA editing in coding islands and abundant microsatellites in repeat sequences of Selaginella moellendorffii mitochondria: the root of frequent plant mtDNA recombination in early tracheophytes. Genome Biol. Evol. 3, 344–358 (2011).

    Article  CAS  Google Scholar 

  13. Kotera, E., Tasaka, M. & Shikanai, T. A pentatricopeptide repeat protein is essential for RNA editing in chloroplasts. Nature 433, 326–330 (2005).

    Article  CAS  Google Scholar 

  14. Shikanai, T. RNA editing in plant organelles: machinery, physiological function and evolution. Cell. Mol. Life Sci. 63, 689–708 (2006).

    Article  Google Scholar 

  15. Fujii, S. & Small, I. The evolution of RNA editing and pentatricopeptide repeat genes. New Phytol. 191, 37–47 (2011).

    Article  CAS  Google Scholar 

  16. Lurin, C. et al. Genome-wide analysis of Arabidopsis pentatricopeptide repeat proteins reveals their essential role in organelle biogenesis. Plant Cell 16, 2089–2103 (2004).

    Article  CAS  Google Scholar 

  17. Andrés, C., Lurin, C. & Small, I.D. The multifarious roles of PPR proteins in plant mitochondrial gene expression. Physiol. Plant. 129, 14–22 (2007).

    Article  Google Scholar 

  18. Okuda, K., Myouga, R., Motohashi, K., Shinozaki, K. & Shikanai, T. Conserved domain structure of pentatricopeptide repeat proteins involved in chloroplast RNA editing. Proc. Natl. Acad. Sci. USA 104, 8178–1883 (2006).

    Article  Google Scholar 

  19. Chateigner-Boutin, A.-L. et al. CLB19, a pentatricopeptide repeat protein required for editing of rpoA and clpP chloroplast transcripts. Plant J. 56, 590–602 (2008).

    Article  CAS  Google Scholar 

  20. Zehrmann, A., Verbitskiy, D., van der Merwe, J.A., Brennicke, A. & Takenaka, M. A DYW domain containing pentatricopeptide repeat protein is required for RNA editing at multiple sites in mitochondria of Arabidopsis thaliana. Plant Cell 21, 558–567 (2009).

    Article  CAS  Google Scholar 

  21. Verbitskiy, D., Zehrmann, A., van der Merwe, J.A., Brennicke, A. & Takenaka, M. The PPR-protein encoded by the lovastatin insensitive 1 gene is involved in RNA editing at three sites in mitochondria of Arabidopsis thaliana. Plant J. 61, 446–455 (2010).

    Article  CAS  Google Scholar 

  22. Zhu, Q. et al. SLO2, a mitochondrial PPR protein affecting several RNA editing sites, is required for energy metabolism. Plant J. 71, 836–849 (2012).

    Article  CAS  Google Scholar 

  23. Zehrmann, A. et al. The DYW-class PPR protein MEF7 is required for RNA editing at four sites in mitochondria of Arabidopsis thaliana. RNA Biol. 9, 1–7 (2012).

    Article  Google Scholar 

  24. Heazlewood, J.L. et al. Experimental analysis of the Arabidopsis mitochondrial proteome highlights signaling and regulatory components, provides assessment of targeting prediction programs, and indicates plant-specific mitochondrial proteins. Plant Cell 16, 241–256 (2004).

    Article  CAS  Google Scholar 

  25. Klodmann, J., Senkler, M., Rode, C. & Braun, H.P. The protein complex proteome of plant mitochondria. Plant Physiol. 157, 587–598 (2011).

    Article  CAS  Google Scholar 

  26. Zehrmann, A., van der Merwe, J.A., Verbitskiy, D., Brennicke, A. & Takenaka, M. Ecotype-specific variations in the extent of RNA editing in plant mitochondria. Mitochondrion 8, 319–327 (2008).

    Article  CAS  Google Scholar 

  27. Bentolila, S., Chateigner-Boutin, A.L. & Hanson, M.R. Ecotype allelic variation in C-to-U editing extent of a mitochondrial transcript identifies RNA-editing quantitative trait loci in Arabidopsis. Plant Physiol. 139, 2006–2016 (2005).

    Article  CAS  Google Scholar 

  28. Chateigner-Boutin, A.-L. & Small, I.D. A rapid high-throughput method for the detection and quantification of RNA editing based on high-resolution melting of amplicons. Nucleic Acids Res. 35, e114 (2007).

    Article  Google Scholar 

  29. Takenaka, M. & Brennicke, A. Multiplex single base extension typing to identify nuclear genes required for RNA editing in plant organelles. Nucleic Acids Res. 37, e13 (2009).

    Article  Google Scholar 

  30. Takenaka, M. Identifying specific trans-factors of RNA editing in plant mitochondria by multiplex single base extension typing. in RNA and DNA Editing (ed. Aphasizhev, R.) 151–161 (Humana Press, 2011).

  31. Lindblad-Toh, K. et al. Large-scale discovery and genotyping of single-nucleotide polymorphisms in the mouse. Nat. Genet. 24, 381–386 (2000).

    Article  CAS  Google Scholar 

  32. Nelson, T.M., Just, R.S., Loreille, O., Schanfield, M.S. & Podini, S. Development of a multiplex single base extension assay for mitochondrial DNA haplotype typing. Croat. Med. J. 48, 460–472 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Sasaki, T., Yukawa, Y., Wakasugi, T., Yamada, K. & Sugiura, M. A simple in vitro RNA editing assay for chloroplast transcripts using fluorescent dideoxynucleotides: distinct types of sequence elements required for editing of ndh transcripts. Plant J. 47, 802–810 (2006).

    Article  CAS  Google Scholar 

  34. Takenaka, M., Verbitskiy, D., Zehrmann, A. & Brennicke, A. Reverse genetic screening identifies five E-class PPR-proteins involved in RNA editing in mitochondria of Arabidopsis thaliana. J. Biol. Chem. 285, 27122–27129 (2010).

    Article  CAS  Google Scholar 

  35. Hammani, K. et al. The pentatricopeptide repeat protein OTP87 is essential for RNA editing of nad7 and atp1 transcripts in Arabidopsis mitochondria. J. Biol. Chem. 286, 21361–21371 (2011).

    Article  CAS  Google Scholar 

  36. Sosso, D. et al. PPR2263, a DYW-subgroup pentatricopeptide repeat protein, is required for mitochondrial nad5 transcript editing, mitochondrion biogenesis and maize growth. Plant Cell 24, 676–691 (2012).

    Article  CAS  Google Scholar 

  37. Verbitskiy, D., Zehrmann, A., Härtel, B., Brennicke, A. & Takenaka, M. The DYW-E-PPR protein MEF14 is required for RNA editing at site matR-1895 in mitochondria of Arabidopsis thaliana. FEBS Lett. 585, 700–704 (2011).

    Article  CAS  Google Scholar 

  38. Takenaka, M. et al. Multiple organellar RNA editing factor (MORF) family proteins are required for RNA editing in mitochondria and plastids of plants. Proc. Natl. Acad. Sci. USA 109, 5104–5109 (2012).

    Article  CAS  Google Scholar 

  39. Schaefer, M., Pollex, T., Hanna, K. & Lyko, F. RNA cytosine methylation analysis by bisulfite sequencing. Nucleic Acids Res. 37, e12 (2009).

    Article  Google Scholar 

  40. Bianco, T., Hussey, D. & Dobrovic, A. Methylation-sensitive, single-strand conformation analysis (MS-SSCA): a rapid method to screen for and analyze methylation. Hum. Mutat. 14, 289–293 (1999).

    Article  CAS  Google Scholar 

  41. Picardi, E., Regina, T., Verbitskiy, D., Brennicke, A. & Quagliariello, C. REDIdb: an upgraded bioinformatics resource for organellar RNA editing sites. Mitochondrion 11, 360–365 (2011).

    Article  CAS  Google Scholar 

  42. Baldino, F., Chesselet, M.-F. & Lewis, M.E. High-resolution in situ hybridization histochemistry. Methods Enzymol. 168, 761–777 (1989).

    Article  CAS  Google Scholar 

  43. Takenaka, M. & Brennicke, A. RNA editing in plant mitochondria: assays and biochemical approaches. Methods Enzymol. 424, 439–458 (2007).

    Article  CAS  Google Scholar 

  44. Scholl, R., Rivero-Lepinckas, L. & Crist, D. Growth of plants and preservation of seeds. in Arabidopsis Vol. 82 (eds. Martínez-Zapater, J.M. & Salinas, J.) (Humana Press, 1998).

  45. Yoo, S.-D., Cho, Y.-H. & Sheen, J. Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis. Nat. Protoc. 2, 1565–1572 (2007).

    Article  CAS  Google Scholar 

  46. Takenaka, M. MEF9, an E subclass pentatricopeptide repeat protein, is required for an RNA editing event in the nad7 transcript in mitochondria of Arabidopsis. Plant Physiol. 152, 939–947 (2010).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank D. Pruchner for excellent experimental help. We are very grateful to C. Kubisch, Walther Vogel, C. Maier and B. Weber in the Institut für Humangenetik, Universität Ulm, for the use of the automated sequencing equipment and materials and their very constructive and helpful discussions throughout. We thank C. Leaver, University of Oxford, for his kind help with the manuscript. This work was supported by grants from the Deutsche Forschungsgemeinschaft (DFG). M.T. is a Heisenberg fellow.

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Authors and Affiliations

  1. Molekulare Botanik, Universität Ulm, Ulm, Germany

    Mizuki Takenaka & Axel Brennicke

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  1. Mizuki Takenaka
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  2. Axel Brennicke
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Contributions

M.T. had the idea, developed the concept, designed and conducted the experiments and contributed to the writing of the paper. A.B. wrote the paper.

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Correspondence to Mizuki Takenaka.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Table 1

The full set of the oligonucleotide primers designed for the reverse transcriptase (RT)- step to generate the first cDNA strand of protein coding regions in the mitochondrial and plastid transcriptomes in Arabidopsis thaliana (Step 5). The cDNAs altogether cover all RNA editing sites in the organellar mRNAs. These primers can be mixed for a single RT reaction, but you should perform the subsequent PCR amplifications in separate reactions (Supplementary Table 2). (PDF 101 kb)

Supplementary Table 2

The full set of the oligonucleotide primers designed for the PCR step in Step 9. This PCR step is used to amplify the relevant cDNA fragments from the first cDNA strand of the mitochondrial and plastid mRNAs (Supplementary Table 1). You should run each PCR amplification in a separate reaction and pool the products afterwards as given in Supplementary Table 3. (PDF 121 kb)

Supplementary Table 3

The sets of the RT-PCR products that we pool for the subsequent SNaPshot primer extension reactions (Step 14). To keep track of all these fragments we suggest you use the names and labels as indicated in the list. (PDF 246 kb)

Supplementary Table 4

The full set of the oligonucleotide primers designed for the SNaPshot primer extension reactions (Step 16). The oligonucleotide primers for the mitochondrial (m) and plastid (c) editing sites are labelled respectively. The sample set given in Table 1 and shown in Figure 4 is primer mix 12. Primers of identical length can be used in opposite directions, i.e. one is a forward primer (labelled F) and the other reverse (R). Their output is easily differentiated by the different dyes. (PDF 280 kb)

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Takenaka, M., Brennicke, A. Using multiplex single-base extension typing to screen for mutants defective in RNA editing. Nat Protoc 7, 1931–1945 (2012). https://doi.org/10.1038/nprot.2012.117

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