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Isolation of mRNAs Associated with Yeast Mitochondria to Study Mechanisms of Localized Translation
Published on: March 14, 2014
Long mRNAs coding for yeast mitochondrial proteins of prokaryotic origin preferentially localize to the vicinity of
Julien Sylvestre1, Stéphane Vialette, Marisol Corral Debrinski
1Laboratoire de Génétique Moléculaire, Ecole Normale Supérieure, 46 rue d'Ulm 75230 Paris cedex O5, France. Claude.Jacq@biologie.ens.fr
Background:
Subcellular messenger RNA localization is important in most eukaryotic cells, even in unicellular organisms like yeast for which this process has been underestimated. Microarrays are rarely used to study subcellular mRNA localization at whole-genome level, but can be adapted to that purpose. This work focuses on studying the repartition of yeast nuclear transcripts encoding mitochondrial proteins between free cytosolic polysomes and polysomes bound to the mitochondrial outer membrane.
Results:
Combining biochemical fractionations with oligonucleotide array analyses permits clustering of genes on the basis of the subcellular sites of their mRNA translation. A large fraction of yeast nuclear transcripts known to encode mitochondrial proteins is found in mitochondrial outer-membrane-bound fractions. These results confirm and extend a previous analysis conducted with partial genomic microarrays. Interesting statistical relations among mRNA localization, gene origin and mRNA lengths were found: longer and older mRNAs are more prone to be localized to the vicinity of mitochondria. These observations are included in a refined model of mitochondrial protein import.
Conclusions:
Mitochondrial biogenesis requires concerted expression of the many genes whose products make up the organelle. In the absence of any clear transcriptional program, coordinated mRNA localization could be an important element of the time-course of organelle construction. We have built a 'MitoChip' localization database from our results which allows us to identify interesting genes whose mRNA localization might be essential for mitochondrial biogenesis in most eukaryotic cells. Moreover, many components of the experimental and data-analysis strategy implemented here are of general relevance in global transcription studies.
Insights
Subcellular messenger RNA (mRNA) localization is crucial for yeast mitochondrial biogenesis. This study reveals that many yeast mitochondrial protein mRNAs are translated on mitochondria, with longer, older mRNAs localizing preferentially.
Area of Science:
- Cell Biology
- Molecular Biology
- Genomics
Background:
- Subcellular mRNA localization is vital in eukaryotic cells, including yeast, though often underestimated.
- Microarray technology can be adapted for whole-genome studies of subcellular mRNA localization.
- This research investigates the distribution of yeast nuclear transcripts encoding mitochondrial proteins between free and mitochondrially-bound polysomes.
Purpose of the Study:
- To analyze the localization of yeast nuclear transcripts encoding mitochondrial proteins.
- To investigate the relationship between mRNA characteristics and subcellular localization.
- To refine models of mitochondrial protein import.
Main Methods:
- Biochemical fractionations combined with oligonucleotide array analyses.
- Whole-genome microarray analysis of mRNA distribution.
- Statistical analysis of mRNA localization, gene origin, and mRNA length.
Main Results:
- A significant portion of yeast mitochondrial protein mRNAs are found in mitochondrial outer membrane-bound fractions.
- Gene clustering based on mRNA translation sites was achieved.
- Longer and older mRNAs showed a higher propensity for localization near mitochondria.
Conclusions:
- Coordinated mRNA localization may be essential for mitochondrial biogenesis in the absence of a clear transcriptional program.
- A 'MitoChip' database was created to identify genes critical for mitochondrial biogenesis.
- The experimental and data analysis strategies are applicable to broader global transcription studies.
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