Abstract
Although 30 years ago it was strongly suggested that some cytoplasmic ribosomes are bound to the surface of yeast mitochondria, the mechanisms and the raison d’ětre of this process are not understood. For instance, it is not perfectly known which of the several hundred nuclearly encoded genes have to be translated to the mitochondrial vicinity to guide the import of the corresponding proteins. One can take advantage of several modern methods to address a number of aspects of the site-specific translation process of messenger ribonucleic acid (mRNA) coding for proteins imported into mitochondria. Three complementary approaches are presented to analyze the spatial distribution of mRNAs coding for proteins imported into mitochondria. Starting from biochemical purifications of mitochondria-bound polysomes, we describe a genomewide approach to classify all the cellular mRNAs according to their physical proximity with mitochondria; we also present real-time quantitative reverse transcription polymerase chain reaction monitoring of mRNA distribution to provide a quantified description of this localization. Finally, a fluorescence microscopy approach on a single living cell is described to visualize the in vivo localization of mRNAs involved in mitochondria biogenesis.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Schatz, G. and Dobberstein, B. (1996) Common principles of protein translocation across membranes. Science 271, 1519–1526.
Neupert, W. (1997) Protein import into mitochondria. Annu. Rev. Biochem. 66, 863–917.
Kellems, R. E., Allison, V. F., and Butow, R. A. (1975) Cytoplasmic type 80S ribosomes associated with yeast mitochondria. IV. Attachment of ribosomes to the outer membrane of isolated mitochondria. J. Cell Biol. 65, 1–14.
Verner, K. (1993) Co-translational protein import into mitochondria: an alternative view. Trends Biochem. Sci. 18, 366–371.
Suissa, M. and Schatz, G. (1982) Import of proteins into mitochondria. Translatable mRNAs for imported mitochondrial proteins are present in free as well as mitochondria-bound cytoplasmic polysomes. J. Biol. Chem. 257, 13,048–13,055.
Marc, P., Margeot, A., Devaux, F., Blugeon, C., Corral-Debrinski, M., and Jacq, C. (2002) Genome-wide analysis of mRNAs targeted to yeast mitochondria. EMBO Rep. 3, 159–164.
Karlberg, O., Canback, B., Kurland, C. G., and Andersson, S. G. (2000) The dual origin of the yeast mitochondrial proteome. Yeast 17, 170–187.
Saint-Georges, Y., Bonnefoy, N., di Rago, J. P., Chiron, S., and Dujardin, G. (2002) A pathogenic cytochrome b mutation reveals new interactions between subunits of the mitochondrial bc1 complex. J. Biol. Chem. 277, 49,397–49,402.
Bertrand, E., Chartrand, P., Schaefer, M., Shenoy, S. M., Singer, R. H., and Long, R. M. (1998) Localization of ASH1 mRNA particles in living yeast. Mol. Cell 2, 437–445.
Femino, A. M., Fay, F. S., Fogarty, K., and Singer, R. H. (1998) Visualization of single RNA transcripts in situ. Science 280, 585–590.
Femino, A. M., Fogarty, K., Lifshitz, L. M., Carrington, W., and Singer, R. H. (2003) Visualization of single molecules of mRNA in situ. Methods Enzymol. 361, 245–304.
Long, R. M., Singer, R. H., Meng, X., Gonzalez, I., Nasmyth, K., and Jansen, R. P. (1997) Mating type switching in yeast controlled by asymmetric localization of ASH1 mRNA. Science 277, 383–387.
Long, R. M., Elliott, D. J., Stutz, F., Rosbash, M., and Singer, R. H. (1995) Spatial consequences of defective processing of specific yeast mRNAs revealed by fluorescent in situ hybridization. RNA 1, 1071–1078.
Chartrand, P., Bertrand, E., Singer, R. H., and Long, R. M. (2000) Sensitive and high-resolution detection of RNA in situ. Methods Enzymol. 318, 493–506.
Tusher, V. G., Tibshirani, R., and Chu, G. (2001) Significance analysis of microarrays applied to the ionizing radiation response. Proc. Natl. Acad. Sci. USA 98, 5116–5121.
Buck, M. J. and Lieb, J. D. (2004) ChIP-chip: considerations for the design, analysis, and application of genome-wide chromatin immunoprecipitation experiments. Genomics 83, 349–360.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2007 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Garcia, M., Darzacq, X., Devaux, F., Singer, R.H., Jacq, C. (2007). Yeast Mitochondrial Transcriptomics. In: Leister, D., Herrmann, J.M. (eds) Mitochondria. Methods in Molecular Biology™, vol 372. Humana Press. https://doi.org/10.1007/978-1-59745-365-3_35
Download citation
DOI: https://doi.org/10.1007/978-1-59745-365-3_35
Publisher Name: Humana Press
Print ISBN: 978-1-58829-667-2
Online ISBN: 978-1-59745-365-3
eBook Packages: Springer Protocols