Genome-wide analysis of mRNAs targeted to yeast mitochondria

Abstract

It is agreed that nuclear-encoded mitochondrial proteins are post-translationally targeted to mitochondria, even if, in some cases, a co-translational phase can assist the import of precursor proteins. We used yeast DNA microarrays to analyse the mRNA populations associated with free and mitochondrion-bound polysomes. As expected, many mRNAs, known to encode mitochondrial proteins, are localized to free cytoplasmic polysomes, but many are localized to mitochondrion-bound polysomes. Furthermore, the 3'-UTR of six randomly chosen mitochondrion-bound mRNAs contains sufficient information to target, in vivo, non-translatable RNA to the vicinity of mitochondria. Interestingly, genes producing mRNAs that are targeted to mitochondria are mainly of ancient bacterial origin, whereas those producing mRNAs that are translated in the cytoplasm are mainly of eukaryotic origin. These observations, which support the recent hypotheses concerning the dual origin of the mitochondrial proteome, provide new insights into the biogenesis of mitochondria.

Fig. 1. Procedure for isolating and identifying mRNAs associated with mitochondrion-bound polysomes. Yeast cells were subjected to classical lysis and the mitochondrial fraction was isolated. Mitochondrion-bound polysomes were released by incubation with detergent and purified as described previously (Ades and Butow, 1980). The post-mitochondrial supernatant fractions were used to prepare free polysomes. We synthesized cDNAs from mitochondrion-bound and free polysomes and labelled them with the fluorescent dyes Cy5 and Cy3, respectively. The labelled cDNAs were hybridized to a DNA microarray. The full protocol is available on our website (www.biologie.ens.fr/yeast-publi.html).

Fig. 2. Relationship between MLR and localization of proteins. A standardized MLR value was determined for each mRNA. mRNAs enriched in mitochondria-bound polysomes have the highest MLR values. (A) Distribution of mRNA according to MLR. (B and C) Percentage of mRNAs encoding cytosolic (B) or mitochondrial (C) proteins (according to the YPD) are plotted as a function of MLR value. The blue lines indicate the percentage of all localized proteins encoding either cytosolic (B) or mitochondrial (C) proteins in the YPD.

Fig. 3. mRNAs encoding mitochondrial proteins found in the two extreme cytosolic and mitochondrion-bound fractions. Top 25 highest (A) and lowest (B) MLR values measured for mRNAs encoding mitochondrial proteins. MLR values are shown in the first column, and the ratios found in Diehn’s experiments, from cells grown in glucose, are shown in the second column. Colour scales are provided for MLR and for ratios, ranging from red (mitochondrial or membrane localization) to green (cytosolic localization). The clear asymmetric mRNA localization observed in cells grown with galactose (the present work) contrast with the results observed when the carbon source is glucose (Diehn et al., 2000).

Fig. 4. 3′-UTR of mRNAs with highest MLR can target a chimeric RNA to the vicinity of mitochondria. The 3′-UTR of six ORFs with high MLR values (84–98) and unknown function were analysed by the ‘green RNA method’ (Bertrand et al., 1998; Bloom et al., 1999). As expected, the gRNA-induced fluorescent speckles are co-localised with mitochondria when 3′-UTR is cloned in the 5′3′ orientation. ATM1/3′-UTR is the positive control. BGL2 is a cell wall protein, its 3′-UTR constitute a negative control. Each 3′-UTR was cloned in both the 3′5′ and 5′3′ orientation (control shown only for COQ4). For each construct, gRNA (g), Hoechst staining of the DNA (H) and Nomarski (N) view are provided.

Fig. 5. The dual origin of the mitochondrial proteome matches the dual location of mRNAs. mRNAs encoding mitochondrial proteins were separated according to MLR values in six classes containing an equivalent number of ORFs. For each class, the proportions of genes of eukaryotic origin, including yeast genes, are displayed as black bars, and those of prokaryotic origin are displayed as blue bars (Karlberg et al., 2000). The mRNAs associated with mitochondrion-bound polysomes (high MLR values) correspond mainly to genes of ancient prokaryotic origin, whereas cytosolically translated mRNAs correspond to genes of eukaryotic origin.