Skip to main content
Log in

Mitochondrial electroporation and in organello RNA editing of chimeric atp6 transcripts

  • Original Paper
  • Published:
Molecular Genetics and Genomics Aims and scope Submit manuscript

Abstract

The Sorghum bicolor atp6-1 gene and chimeric atp6 genes with additional maize sequences were introduced into isolated maize mitochondria via electroporation. Transcripts isolated after in vitro incubation of the transformed organelles were then analysed for RNA editing. Transcripts of the S. bicolor atp6-1 gene, and the RNAs obtained from most of chimeric sorghum-maize atp6 gene constructs tested, were not edited. However, the transcript of one engineered chimeric gene comprising the 5′untranslated sequence and a segment of the N-terminal ORF of the maize atp6 combined with the sorghum atp6 core ORF and 3′untranslated sequence was found to be partially edited. We were able to exclude low RNA stability or insufficient editing capacity as the reason for failure to edit in the other instances. Instead, the data indicate that the maize sequence in the edited fusion transcript provides a structural motif or binding site for a transcript-specific editing factor.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  • Araya A, Domec C, Begu D, Litvak S (1992) An in vitro system for the editing of ATP synthase subunit 9 mRNA using wheat mitochondrial extracts. Proc Natl Acad Sci USA 89:1040–1044

    Google Scholar 

  • Binder S, Marchfelder A, Brennicke A (1996) Regulation of gene expression in plant mitochondria. Plant Mol Biol 32:303–314

    CAS  PubMed  Google Scholar 

  • Brennicke A, Marchfelder A, Binder S (1999) RNA editing. FEMS Microbiol Rev 23:297–316

    Google Scholar 

  • Chaudhuri S, Carrer H, Maliga P (1995) Site-specific factor involved in the editing of the psbL mRNA in tobacco plastids. EMBO J 14:2951–2957

    Google Scholar 

  • Covello PS, Gray MW (1989) RNA editing in plant mitochondria. Nature 341:662–666

    Article  CAS  PubMed  Google Scholar 

  • Farré J-C, Araya A (2001) Gene expression in isolated plant mitochondria: high fidelity of transcription, splicing and editing of a transgene product in electroporated organelles. Nucleic Acids Res 29:2484–2491

    Google Scholar 

  • Farré JC, Leon G, Jordana X, Araya A (2001) cis recognition elements in plant mitochondrion RNA editing. Mol Cell Biol 21:6731–6737

    Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  • Gualberto JM, Lamattina L, Bonnard G, Weil JH, Grienenberger JM (1989) RNA editing in wheat mitochondria results in the conservation of protein sequences. Nature 341:660–662

    Article  CAS  PubMed  Google Scholar 

  • Handa H (2003) The complete nucleotide sequence and RNA editing content of the mitochondrial genome of rapeseed (Brassica napus L.): comparative analysis of the mitochondrial genomes of rapeseed and Arabidopsis thaliana. Nucleic Acids Res 31:5907–5916

    Article  CAS  PubMed  Google Scholar 

  • Hiesel R, Wissinger B, Schuster W, Brennicke A (1989) RNA editing in plant mitochondria. Science 246:1632–1634

    CAS  PubMed  Google Scholar 

  • Howad W, Kempken F (1997) Cell-type specific loss of atp6 RNA editing in cytoplasmic male sterile Sorghum bicolor. Proc Natl Acad Sci USA 94:11090–11095

    Google Scholar 

  • Howad W, Tang HV, Pring DR, Kempken F (1999) Nuclear genes from Tx CMS maintainer lines are unable to maintain atp6 RNA editing in any anther cell-type in the Sorghum bicolor A3 cytoplasm. Curr Genet 36:62–68

    Google Scholar 

  • Kempken F, Howad W (1996) Mitochondrial RNA editing is sequence specific and independent of transcript abundance in Sorghum bicolor. Curr Genet 30:186–190

    Google Scholar 

  • Kempken F, Mullen JA, Pring DR, Tang HV (1991) RNA editing of Sorghum mitochondrial atp6 transcripts changes 15 amino acids and generates a carboxyterminus identical to yeast. Curr Genet 20:417–422

    CAS  PubMed  Google Scholar 

  • Kempken F, Howad W, Pring DR (1998) Mutations at specific atp6 codons which cause human mitochondrial diseases also lead to male sterility in a plant. FEBS Lett 441:159–160

    Article  CAS  PubMed  Google Scholar 

  • Kempken F, Stockmeyer K, Staudinger M (2000) DNA Transfer in Mitochondrien Botanikertagung 2000 Jena. Finkdruck, Apolda, p 30

    Google Scholar 

  • Kubo T, Nishizawa S, Sugawara A, Itchoda N, Estiati N, Mikami T (2000) The complete nucleotide sequence of the mitochondrial genome of sugar beet (Beta vulgaris L.) reveals a novel gene for tRNA (Cys)(GCA). Nucleic Acids Res 28:2571–2576

    Article  CAS  PubMed  Google Scholar 

  • Kumar R, Levings CS III (1993) RNA editing of a chimeric maize mitochondrial gene transcript is sequence specific. Curr Genet 23:154–159

    CAS  PubMed  Google Scholar 

  • Levings CS III (1990) The Texas cytoplasm of maize: cytoplasmic male sterility and disease susceptibility. Science 250:942–947

    Google Scholar 

  • Lu B, Hanson MR (1992) A single nuclear gene specifies the abundance and extent of RNA editing of a plant mitochondrial transcript. Nucleic Acids Res 20:5699–5703

    Google Scholar 

  • Malek O, Lättig K, Hiesel R, Brennicke A, Knoop V (1996) RNA editing in bryophytes and a molecular phylogeny of land plants. EMBO J 15:1403–1411

    CAS  PubMed  Google Scholar 

  • Mullen JA, Pring DR, Kempken F, Ferguson J, Chase CD (1992) Sorghum mitochondrial atp6: divergent amino extensions to a conserved core polypeptide. Plant Mol Biol 20:71–79

    Google Scholar 

  • Newton KJ (1994) Procedures for isolating mitochondria and mitochondrial DNA and RNA. In: Freeling M, Walbot V (eds) The maize handbook. Springer, New York, pp 549–556

    Google Scholar 

  • Notsu Y et al. (2002) The complete sequence of the rice (Oryza sativa L.) mitochondrial genome: frequent DNA sequence acquisition and loss during the evolution of flowering plants. Mol Gen Genomics 268:434–445

    Google Scholar 

  • Pring DR, Tang HV (2001) Mitochondrial atp6 transcript editing during microgametogenesis in male-sterile sorghum. Curr Genet 39:371–376

    Google Scholar 

  • Pring DR, Mullen JA, Kempken F (1992) Conserved sequence blocks 5′to start codons of plant mitochondrial genes. Plant Mol Biol 19:313–317

    Google Scholar 

  • Rapp WD, Stern DB (1992) A conserved 11 nucleotide sequence contains an essential promoter element of the maize mitochondrial atp1 gene. EMBO J 11:1065–1073

    Google Scholar 

  • Rapp WD, Lupold S, Mack S, Stern DB (1993) Architecture of the maize mitochondrial atp1 promoter as determined by linker-scanning and point mutagenesis. Mol Cell Biol 13:7232–7238

    Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor, Cold Spring Harbor

    Google Scholar 

  • Schuster W, Ternes R, Knoop V, Hiesel R, Wissinger B, Brennicke A (1991) Distribution of RNA editing sites in Oenothera mitochondrial mRNAs and rRNAs. Curr Genet 20:397–404

    Article  Google Scholar 

  • Staudinger M, Kempken F (2003) Electroporation of higher-plant mitochondria: transcripts of an introduced cox2 gene, but not an atp6 gene, are edited in organello. Mol Gen Genomics 269:553–561

    Article  Google Scholar 

  • Staudinger M, Kempken F (2004) In organello editing of mitochondrial atp9,cox2, and nad9 transcripts. Endocyt Cell Res 15:551–560

    Google Scholar 

  • Stuart K, Panigrahi AK (2002) RNA editing: complexity and complications. Mol Microbiol 45:591–596

    Article  Google Scholar 

  • Takenaka M, Brennicke A (2003) In vitro RNA editing in pea mitochondria requires NTPor dNTP, suggesting involvement of an RNA helicase. J Biol Chem 278:47526–47533

    Article  Google Scholar 

  • Takenaka M, Neuwirt J, Brennicke A (2004) Complex cis-elements determine an RNA editing site in pea mitochondria. Nucleic Acids Res 32:4137–4144

    Article  Google Scholar 

  • Ward GC, Levings CS, III (1991) The protein-encoding gene T- urf13 is not edited in maize mitochondria. Plant Mol Biol 17:1083–1088

    Article  Google Scholar 

  • Yan B, Pring DR (1997) Transcriptional initiation sites in Sorghum mitochondrial DNA indicate conserved and variable features. Curr Genet 32:287–295

    Article  Google Scholar 

  • Yu W, Schuster W (1995) Evidence for a site-specific cytidine deamination reaction involved in C-to-U RNA editing of plant mitochondria. J Biol Chem 270:18227–18233

    Article  Google Scholar 

Download references

Acknowledgements

We thank Mrs. Ilka Braumann (Kiel) for help with vector construction, and Prof. Daryl Pring (Gainesville) for providing vector pAF1 and critical discussion. This work was supported by grants from the Deutsche Forschungsgemeinschaft to Frank Kempken (Ke409/12-1 and Ke409/15-1). The authors thank two anonymous referees for valuable suggestions for improving the manuscript.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Frank Kempken.

Additional information

Communicated by R. Hagemann

Rights and permissions

Reprints and permissions

About this article

Cite this article

Staudinger, M., Bolle, N. & Kempken, F. Mitochondrial electroporation and in organello RNA editing of chimeric atp6 transcripts. Mol Genet Genomics 273, 130–136 (2005). https://doi.org/10.1007/s00438-005-1117-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00438-005-1117-x

Keywords

Navigation