Skip to main content
SpringerLink
Log in
Book cover

Mitochondrial Medicine pp 227–254Cite as

Mitochondrial Targeting of Catalytic RNAs

  • Protocol

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1265))

Abstract

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.

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

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   139.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Dhillon VS, Fenech M (2014) Mutations that affect mitochondrial functions and their association with neurodegenerative diseases. Mutat Res 759C:1–13

    Article  Google Scholar 

  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 175–210

    Google Scholar 

  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

    Article  PubMed  Google Scholar 

  4. Hikosaka K, Kita K, Tanabe K (2013) Diversity of mitochondrial genome structure in the phylum Apicomplexa. Mol Biochem Parasitol 188:26–33

    Article  CAS  PubMed  Google Scholar 

  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 238:195–209

    Article  CAS  PubMed  Google Scholar 

  6. Gray MW (2012) Mitochondrial evolution. Cold Spring Harb Perspect Biol 4:a011403

    Article  PubMed Central  PubMed  Google Scholar 

  7. Bonnefoy N, Remacle C, Fox TD (2007) Genetic transformation of Saccharomyces cerevisiae and Chlamydomonas reinhardtii mitochondria. Methods Cell Biol 80:525–548

    Article  CAS  PubMed  Google Scholar 

  8. Zhou J, Liu L, Chen J (2010) Mitochondrial DNA heteroplasmy in Candida glabrata after mitochondrial transformation. Eukaryot Cell 9:806–814

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  9. Niazi AK, Mileshina D, Cosset A, Val R, Weber-Lotfi F, Dietrich A (2013) Targeting nucleic acids into mitochondria: progress and prospects. Mitochondrion 13:548–558

    Article  CAS  PubMed  Google Scholar 

  10. Salinas T, Duchene AM, Marechal-Drouard L (2008) Recent advances in tRNA mitochondrial import. Trends Biochem Sci 33:320–329

    Article  CAS  PubMed  Google Scholar 

  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 39:9262–9274

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  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 15:308–315

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  13. Maxam AM, Gilbert W (1980) Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol 65:499–560

    Article  CAS  PubMed  Google Scholar 

  14. Matsuda D, Dreher TW (2004) The tRNA-like structure of Turnip yellow mosaic virus RNA is a 3′-translational enhancer. Virology 321:36–46

    Article  CAS  PubMed  Google Scholar 

  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 10:913–918

    Article  CAS  PubMed  Google Scholar 

  16. Hammann C, Luptak A, Perreault J, de la Pena M (2012) The ubiquitous hammerhead ribozyme. RNA 18:871–885

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  17. Nelson JA, Shepotinovskaya I, Uhlenbeck OC (2005) Hammerheads derived from sTRSV show enhanced cleavage and ligation rate constants. Biochemistry 44:14577–14585

    Article  CAS  PubMed  Google Scholar 

  18. Persson T, Hartmann RK, Eckstein F (2002) Selection of hammerhead ribozyme variants with low Mg2+ requirement: importance of stem-loop II. Chembiochem 3:1066–1071

    Article  CAS  PubMed  Google Scholar 

  19. Knoop V (2011) When you can’t trust the DNA: RNA editing changes transcript sequences. Cell Mol Life Sci 68:567–586

    Article  CAS  PubMed  Google Scholar 

  20. Zuker M (2003) Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31:3406–3415

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  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 360:225–231

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  22. Perrotta AT, Been MD (1991) A pseudoknot-like structure required for efficient self-cleavage of hepatitis delta virus RNA. Nature 350:434–436

    Article  CAS  PubMed  Google Scholar 

  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 24:265–273

    Article  CAS  PubMed  Google Scholar 

  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 4:288–290

    Article  PubMed  Google Scholar 

  25. Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735–743

    Article  CAS  PubMed  Google Scholar 

  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 13:1499–1510

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  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 60:291–298

    Article  CAS  PubMed  Google Scholar 

  28. Taylor NL, Stroher E, Millar AH (2014) Arabidopsis organelle isolation and characterization. Methods Mol Biol 1062:551–572

    Article  PubMed  Google Scholar 

  29. Ellis J, Rogers J (1993) Design and specificity of hammerhead ribozymes against calretinin mRNA. Nucleic Acids Res 21:5171–5178

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  30. Hertel KJ, Herschlag D, Uhlenbeck OC (1996) Specificity of hammerhead ribozyme cleavage. EMBO J 15:3751–3757

    PubMed Central  CAS  PubMed  Google Scholar 

  31. Borghi L (2010) Inducible gene expression systems for plants. Methods Mol Biol 655:65–75

    Article  CAS  PubMed  Google Scholar 

  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 54:609–621

    Article  CAS  PubMed  Google Scholar 

  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 213:106–113

    Article  CAS  PubMed  Google Scholar 

  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 520:139–147

    Article  CAS  PubMed  Google Scholar 

  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 14:910–922

    Article  CAS  PubMed  Google Scholar 

  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 31:1159–1172

    Article  CAS  PubMed  Google Scholar 

  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 207:37–44

    Article  CAS  PubMed  Google Scholar 

  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 170:1295–1299

    Article  CAS  PubMed  Google Scholar 

  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 29:239–248

    Article  CAS  PubMed  Google Scholar 

  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 172:325–339

    Article  CAS  PubMed  Google Scholar 

  41. Garwick-Coppens SE, Herman A, Harper SQ (2011) Construction of permanently inducible miRNA-based expression vectors using site-specific recombinases. BMC Biotechnol 11:107

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  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 101:1927–1932

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  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:e67

    Article  PubMed Central  PubMed  Google Scholar 

  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 13:1375–1383

    Article  PubMed Central  PubMed  Google Scholar 

  45. Zhou H, Huang C, Xia XG (2008) A tightly regulated Pol III promoter for synthesis of miRNA genes in tandem. Biochim Biophys Acta 1779:773–779

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  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 31:963–977

    Article  CAS  Google Scholar 

  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:e171

    Article  PubMed Central  PubMed  Google Scholar 

  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 14:903–913

    Article  PubMed Central  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

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).

Author information

Authors and Affiliations

  1. Institut de Biologie Moléculaire des Plantes, CNRS and Université de Strasbourg, 12 rue du Général Zimmer, 67084, Strasbourg, France

    Daria Mileshina, Adnan Khan Niazi, Romain Val, Clarisse Valentin & André Dietrich

  2. Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61-704, Poznan, Poland

    Eliza Wyszko, Maciej Szymanski & Jan Barciszewski

  3. Laboratory of Bioinformatics, Institute of Molecular Biology and Biotechnology, Adam Mickiewicz University, Umultowska 89, 61-614, Poznan, Poland

    Maciej Szymanski

Authors
  1. Daria Mileshina
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Adnan Khan Niazi
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Eliza Wyszko
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Maciej Szymanski
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Romain Val
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Clarisse Valentin
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Jan Barciszewski
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. André Dietrich
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to André Dietrich .

Editor information

Editors and Affiliations

  1. Midwestern University, Glendale, Arizona, USA

    Volkmar Weissig

  2. ISANH, Paris, France

    Marvin Edeas

Rights and permissions

Reprints and permissions

Copyright information

© 2015 Springer Science+Business Media New York

About this protocol

Cite this protocol

Mileshina, D. et al. (2015). Mitochondrial Targeting of Catalytic RNAs. In: Weissig, V., Edeas, M. (eds) Mitochondrial Medicine. Methods in Molecular Biology, vol 1265. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2288-8_17

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-2288-8_17

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2287-1

  • Online ISBN: 978-1-4939-2288-8

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation