Mitochondrial Targeting of Catalytic RNAs

  • Daria Mileshina
  • Adnan Khan Niazi
  • Eliza Wyszko
  • Maciej Szymanski
  • Romain Val
  • Clarisse Valentin
  • Jan Barciszewski
  • André Dietrich
Part of the Methods in Molecular Biology book series (MIMB, volume 1265)


Genetic transformation of mitochondria in multicellular eukaryotes has remained inaccessible, hindering fundamental investigations and applications to gene therapy or biotechnology. In this context, we have developed a strategy to target nuclear transgene-encoded RNAs into mitochondria in plants. We describe here mitochondrial targeting of trans-cleaving ribozymes destined to knockdown organelle RNAs for regulation studies and inverse genetics and biotechnological purposes. The design and functional assessment of chimeric RNAs combining the ribozyme and the mitochondrial shuttle are detailed, followed by all procedures to prepare constructs for in vivo expression, generate stable plant transformants, and establish target RNA knockdown in mitochondria.

Key words

Mitochondria Plant Ribozyme RNA knockdown RNA transport Transformation tRNA 



This work has been published under the framework of the LABEX [ANR-11-LABX-0057_MITOCROSS] and benefits from a funding from the state managed by the French National Research Agency as part of the “Investments for the future” program. Further support through grants from the French National Research Agency (ANR-06-MRAR-037-02, ANR-09-BLAN-0240-01), the Polish Ministry of Science and Higher Education and the Polish National Science Center is acknowledged. Our projects are also supported by regular funding from the French National Center for Scientific Research (CNRS-UPR2357) and the University of Strasbourg (UdS).


  1. 1.
    Dhillon VS, Fenech M (2014) Mutations that affect mitochondrial functions and their association with neurodegenerative diseases. Mutat Res 759C:1–13CrossRefGoogle Scholar
  2. 2.
    Frei U, Peiretti EG, Wenzel G (2004) Significance of cytoplasmic DNA in plant breeding. In: Janick J (ed) Plant breeding reviews. Wiley, Hoboken, pp 175–210Google Scholar
  3. 3.
    Gualberto JM, Mileshina D, Wallet C, Niazi AK, Weber-Lotfi F, Dietrich A (2013) The plant mitochondrial genome: dynamics and maintenance. Biochimie 100:107–120. doi: 10.1016/j.biochi.2013.1009.1016 CrossRefPubMedGoogle Scholar
  4. 4.
    Hikosaka K, Kita K, Tanabe K (2013) Diversity of mitochondrial genome structure in the phylum Apicomplexa. Mol Biochem Parasitol 188:26–33CrossRefPubMedGoogle Scholar
  5. 5.
    Saccone C, De Giorgi C, Gissi C, Pesole G, Reyes A (1999) Evolutionary genomics in Metazoa: the mitochondrial DNA as a model system. Gene 238:195–209CrossRefPubMedGoogle Scholar
  6. 6.
    Gray MW (2012) Mitochondrial evolution. Cold Spring Harb Perspect Biol 4:a011403CrossRefPubMedCentralPubMedGoogle Scholar
  7. 7.
    Bonnefoy N, Remacle C, Fox TD (2007) Genetic transformation of Saccharomyces cerevisiae and Chlamydomonas reinhardtii mitochondria. Methods Cell Biol 80:525–548CrossRefPubMedGoogle Scholar
  8. 8.
    Zhou J, Liu L, Chen J (2010) Mitochondrial DNA heteroplasmy in Candida glabrata after mitochondrial transformation. Eukaryot Cell 9:806–814CrossRefPubMedCentralPubMedGoogle Scholar
  9. 9.
    Niazi AK, Mileshina D, Cosset A, Val R, Weber-Lotfi F, Dietrich A (2013) Targeting nucleic acids into mitochondria: progress and prospects. Mitochondrion 13:548–558CrossRefPubMedGoogle Scholar
  10. 10.
    Salinas T, Duchene AM, Marechal-Drouard L (2008) Recent advances in tRNA mitochondrial import. Trends Biochem Sci 33:320–329CrossRefPubMedGoogle Scholar
  11. 11.
    Val R, Wyszko E, Valentin C, Szymanski M, Cosset A, Alioua M, Dreher TW, Barciszewski J, Dietrich A (2011) Organelle trafficking of chimeric ribozymes and genetic manipulation of mitochondria. Nucleic Acids Res 39:9262–9274CrossRefPubMedCentralPubMedGoogle Scholar
  12. 12.
    Burchard J, Jackson AL, Malkov V, Needham RH, Tan Y, Bartz SR, Dai H, Sachs AB, Linsley PS (2009) MicroRNA-like off-target transcript regulation by siRNAs is species specific. RNA 15:308–315CrossRefPubMedCentralPubMedGoogle Scholar
  13. 13.
    Maxam AM, Gilbert W (1980) Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol 65:499–560CrossRefPubMedGoogle Scholar
  14. 14.
    Matsuda D, Dreher TW (2004) The tRNA-like structure of Turnip yellow mosaic virus RNA is a 3′-translational enhancer. Virology 321:36–46CrossRefPubMedGoogle Scholar
  15. 15.
    Dietrich A, Marechal-Drouard L, Carneiro V, Cosset A, Small I (1996) A single base change prevents import of cytosolic tRNA(Ala) into mitochondria in transgenic plants. Plant J 10:913–918CrossRefPubMedGoogle Scholar
  16. 16.
    Hammann C, Luptak A, Perreault J, de la Pena M (2012) The ubiquitous hammerhead ribozyme. RNA 18:871–885CrossRefPubMedCentralPubMedGoogle Scholar
  17. 17.
    Nelson JA, Shepotinovskaya I, Uhlenbeck OC (2005) Hammerheads derived from sTRSV show enhanced cleavage and ligation rate constants. Biochemistry 44:14577–14585CrossRefPubMedGoogle Scholar
  18. 18.
    Persson T, Hartmann RK, Eckstein F (2002) Selection of hammerhead ribozyme variants with low Mg2+ requirement: importance of stem-loop II. Chembiochem 3:1066–1071CrossRefPubMedGoogle Scholar
  19. 19.
    Knoop V (2011) When you can’t trust the DNA: RNA editing changes transcript sequences. Cell Mol Life Sci 68:567–586CrossRefPubMedGoogle Scholar
  20. 20.
    Zuker M (2003) Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31:3406–3415CrossRefPubMedCentralPubMedGoogle Scholar
  21. 21.
    Igamberdiev AU, Kleczkowski LA (2001) Implications of adenylate kinase-governed equilibrium of adenylates on contents of free magnesium in plant cells and compartments. Biochem J 360:225–231CrossRefPubMedCentralPubMedGoogle Scholar
  22. 22.
    Perrotta AT, Been MD (1991) A pseudoknot-like structure required for efficient self-cleavage of hepatitis delta virus RNA. Nature 350:434–436CrossRefPubMedGoogle Scholar
  23. 23.
    Zuo J, Niu QW, Chua NH (2000) Technical advance: an estrogen receptor-based transactivator XVE mediates highly inducible gene expression in transgenic plants. Plant J 24:265–273CrossRefPubMedGoogle Scholar
  24. 24.
    van Engelen FA, Molthoff JW, Conner AJ, Nap JP, Pereira A, Stiekema WJ (1995) pBINPLUS: an improved plant transformation vector based on pBIN19. Transgenic Res 4:288–290CrossRefPubMedGoogle Scholar
  25. 25.
    Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735–743CrossRefPubMedGoogle Scholar
  26. 26.
    Boyes DC, Zayed AM, Ascenzi R, McCaskill AJ, Hoffman NE, Davis KR, Gorlach J (2001) Growth stage-based phenotypic analysis of Arabidopsis: a model for high throughput functional genomics in plants. Plant Cell 13:1499–1510CrossRefPubMedCentralPubMedGoogle Scholar
  27. 27.
    Lloyd AL, Marshall BJ, Mee BJ (2005) Identifying cloned Helicobacter pylori promoters by primer extension using a FAM-labelled primer and GeneScan analysis. J Microbiol Methods 60:291–298CrossRefPubMedGoogle Scholar
  28. 28.
    Taylor NL, Stroher E, Millar AH (2014) Arabidopsis organelle isolation and characterization. Methods Mol Biol 1062:551–572CrossRefPubMedGoogle Scholar
  29. 29.
    Ellis J, Rogers J (1993) Design and specificity of hammerhead ribozymes against calretinin mRNA. Nucleic Acids Res 21:5171–5178CrossRefPubMedCentralPubMedGoogle Scholar
  30. 30.
    Hertel KJ, Herschlag D, Uhlenbeck OC (1996) Specificity of hammerhead ribozyme cleavage. EMBO J 15:3751–3757PubMedCentralPubMedGoogle Scholar
  31. 31.
    Borghi L (2010) Inducible gene expression systems for plants. Methods Mol Biol 655:65–75CrossRefPubMedGoogle Scholar
  32. 32.
    Kong Y, Zhu Y, Gao C, She W, Lin W, Chen Y, Han N, Bian H, Zhu M, Wang J (2013) Tissue-specific expression of SMALL AUXIN UP RNA41 differentially regulates cell expansion and root meristem patterning in Arabidopsis. Plant Cell Physiol 54:609–621CrossRefPubMedGoogle Scholar
  33. 33.
    Rodrigues MI, Bravo JP, Sassaki FT, Severino FE, Maia IG (2013) The tonoplast intrinsic aquaporin (TIP) subfamily of Eucalyptus grandis: characterization of EgTIP2, a root-specific and osmotic stress-responsive gene. Plant Sci 213:106–113CrossRefPubMedGoogle Scholar
  34. 34.
    Su X, Xu WZ, Liu X, Zhuo RF, Wang CY, Zhang X, Kakutani K, You S (2013) The isolation and identification of a light-induced protein in alfalfa sprouts and the cloning of its specific promoter. Gene 520:139–147CrossRefPubMedGoogle Scholar
  35. 35.
    Molla KA, Karmakar S, Chanda PK, Ghosh S, Sarkar SN, Datta SK, Datta K (2013) Rice oxalate oxidase gene driven by green tissue-specific promoter increases tolerance to sheath blight pathogen (Rhizoctonia solani) in transgenic rice. Mol Plant Pathol 14:910–922CrossRefPubMedGoogle Scholar
  36. 36.
    Ye R, Zhou F, Lin Y (2012) Two novel positive cis-regulatory elements involved in green tissue-specific promoter activity in rice (Oryza sativa L ssp.). Plant Cell Rep 31:1159–1172CrossRefPubMedGoogle Scholar
  37. 37.
    Li Y, Liu S, Yu Z, Liu Y, Wu P (2013) Isolation and characterization of two novel root-specific promoters in rice (Oryza sativa L.). Plant Sci 207:37–44CrossRefPubMedGoogle Scholar
  38. 38.
    Imai A, Takahashi S, Nakayama K, Satoh H (2013) The promoter of the carotenoid cleavage dioxygenase 4a-5 gene of Chrysanthemum morifolium (CmCCD4a-5) drives petal-specific transcription of a conjugated gene in the developing flower. J Plant Physiol 170:1295–1299CrossRefPubMedGoogle Scholar
  39. 39.
    Zavallo D, Lopez BM, Hopp HE, Heinz R (2010) Isolation and functional characterization of two novel seed-specific promoters from sunflower (Helianthus annuus L.). Plant Cell Rep 29:239–248CrossRefPubMedGoogle Scholar
  40. 40.
    Sunkara S, Bhatnagar-Mathur P, Sharma KK (2014) Isolation and functional characterization of a novel seed-specific promoter region from peanut. Appl Biochem Biotechnol 172:325–339CrossRefPubMedGoogle Scholar
  41. 41.
    Garwick-Coppens SE, Herman A, Harper SQ (2011) Construction of permanently inducible miRNA-based expression vectors using site-specific recombinases. BMC Biotechnol 11:107CrossRefPubMedCentralPubMedGoogle Scholar
  42. 42.
    Gupta S, Schoer RA, Egan JE, Hannon GJ, Mittal V (2004) Inducible, reversible, and stable RNA interference in mammalian cells. Proc Natl Acad Sci U S A 101:1927–1932CrossRefPubMedCentralPubMedGoogle Scholar
  43. 43.
    Henriksen JR, Lokke C, Hammero M, Geerts D, Versteeg R, Flaegstad T, Einvik C (2007) Comparison of RNAi efficiency mediated by tetracycline-responsive H1 and U6 promoter variants in mammalian cell lines. Nucleic Acids Res 35:e67CrossRefPubMedCentralPubMedGoogle Scholar
  44. 44.
    Zhang J, Wang C, Ke N, Bliesath J, Chionis J, He QS, Li QX, Chatterton JE, Wong-Staal F, Zhou D (2007) A more efficient RNAi inducible system for tight regulation of gene expression in mammalian cells and xenograft animals. RNA 13:1375–1383CrossRefPubMedCentralPubMedGoogle Scholar
  45. 45.
    Zhou H, Huang C, Xia XG (2008) A tightly regulated Pol III promoter for synthesis of miRNA genes in tandem. Biochim Biophys Acta 1779:773–779CrossRefPubMedCentralPubMedGoogle Scholar
  46. 46.
    Kim GB, Bae JH, An CS, Nam YW (2013) Single or multiple gene silencing directed by U6 promoter-based shRNA vectors facilitates efficient functional genome analysis in Medicago truncatula. Plant Mol Biol Rep 31:963–977CrossRefGoogle Scholar
  47. 47.
    Lu S, Shi R, Tsao CC, Yi X, Li L, Chiang VL (2004) RNA silencing in plants by the expression of siRNA duplexes. Nucleic Acids Res 32:e171CrossRefPubMedCentralPubMedGoogle Scholar
  48. 48.
    Wang MB, Helliwell CA, Wu LM, Waterhouse PM, Peacock WJ, Dennis ES (2008) Hairpin RNAs derived from RNA polymerase II and polymerase III promoter-directed transgenes are processed differently in plants. RNA 14:903–913CrossRefPubMedCentralPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Daria Mileshina
    • 1
  • Adnan Khan Niazi
    • 1
  • Eliza Wyszko
    • 2
  • Maciej Szymanski
    • 2
    • 3
  • Romain Val
    • 1
  • Clarisse Valentin
    • 1
  • Jan Barciszewski
    • 2
  • André Dietrich
    • 1
  1. 1.Institut de Biologie Moléculaire des PlantesCNRS and Université de StrasbourgStrasbourgFrance
  2. 2.Institute of Bioorganic ChemistryPolish Academy of SciencesPoznanPoland
  3. 3.Laboratory of Bioinformatics, Institute of Molecular Biology and BiotechnologyAdam Mickiewicz UniversityPoznanPoland

Personalised recommendations