A Case of Gene Fragmentation in Plant Mitochondria Fixed by the Selection of a Compensatory Restorer of Fertility-Like PPR Gene.

The high mutational load of mitochondrial genomes combined with their uniparental inheritance and high polyploidy favors the maintenance of deleterious mutations within populations. How cells compose and adapt to the accumulation of disadvantageous mitochondrial alleles remains unclear. Most harmful changes are likely corrected by purifying selection, however, the intimate collaboration between mitochondria- and nuclear-encoded gene products offers theoretical potential for compensatory adaptive changes. In plants, cytoplasmic male sterilities are known examples of nucleo-mitochondrial coadaptation situations in which nuclear-encoded restorer of fertility (Rf) genes evolve to counteract the effect of mitochondria-encoded cytoplasmic male sterility (CMS) genes and restore fertility. Most cloned Rfs belong to a small monophyletic group, comprising 26 pentatricopeptide repeat genes in Arabidopsis, called Rf-like (RFL). In this analysis, we explored the functional diversity of RFL genes in Arabidopsis and found that the RFL8 gene is not related to CMS suppression but essential for plant embryo development. In vitro-rescued rfl8 plantlets are deficient in the production of the mitochondrial heme-lyase complex. A complete ensemble of molecular and genetic analyses allowed us to demonstrate that the RFL8 gene has been selected to permit the translation of the mitochondrial ccmFN2 gene encoding a heme-lyase complex subunit which derives from the split of the ccmFN gene, specifically in Brassicaceae plants. This study represents thus a clear case of nuclear compensation to a lineage-specific mitochondrial genomic rearrangement in plants and demonstrates that RFL genes can be selected in response to other mitochondrial deviancies than CMS suppression.

The translational landscape of Arabidopsis mitochondria.

Messenger RNA translation is a complex process that is still poorly understood in eukaryotic organelles like mitochondria. Growing evidence indicates though that mitochondrial translation differs from its bacterial counterpart in many key aspects. In this analysis, we have used ribosome profiling technology to generate a genome-wide snapshot view of mitochondrial translation in Arabidopsis. We show that, unlike in humans, most Arabidopsis mitochondrial ribosome footprints measure 27 and 28 bases. We also reveal that respiratory subunits encoding mRNAs show much higher ribosome association than other mitochondrial mRNAs, implying that they are translated at higher levels. Homogenous ribosome densities were generally detected within each respiratory complex except for complex V, where higher ribosome coverage corroborated with higher requirements for specific subunits. In complex I respiratory mutants, a reorganization of mitochondrial mRNAs ribosome association was detected involving increased ribosome densities for certain ribosomal protein encoding transcripts and a reduction in translation of a few complex V mRNAs. Taken together, our observations reveal that plant mitochondrial translation is a dynamic process and that translational control is important for gene expression in plant mitochondria. This study paves the way for future advances in the understanding translation in higher plant mitochondria.

The pentatricopeptide repeat protein MTSF2 stabilizes a nad1 precursor transcript and defines the 3΄ end of its 5΄-half intron.

RNA expression in plant mitochondria implies a large number of post-transcriptional events in which transcript processing and stabilization are essential. In this study, we analyzed the function of the Arabidopsis mitochondrial stability factor 2 gene (MTSF2) and show that the encoded pentatricopeptide repeat protein is essential for the accumulation of stable nad1 mRNA. The production of mature nad1 requires the assembly of three independent RNA precursors via two trans-splicing reactions. Genetic analyses revealed that the lack of nad1 in mtsf2 mutants results from the specific destabilization of the nad1 exons 2-3 precursor transcript. We further demonstrated that MTSF2 binds to its 3΄ extremity with high affinity, suggesting a protective action by blocking exoribonuclease progression. By defining the 3΄ end of nad1 exons 2-3 precursor, MTSF2 concomitantly determines the 3΄ extremity of the first half of the trans-intron found at the end of the transcript. Therefore, binding of the MTSF2 protein to nad1 exons 2-3 precursor evolved both to stabilize the transcript and to define a 3΄ extremity compatible with the trans-splicing reaction needed to reconstitute mature nad1. We thus reveal that the range of transcripts stabilized by association with protective protein on their 3΄ end concerns also mitochondrial precursor transcripts.

The MTL1 Pentatricopeptide Repeat Protein Is Required for Both Translation and Splicing of the Mitochondrial NADH DEHYDROGENASE SUBUNIT7 mRNA in Arabidopsis.

Mitochondrial translation involves a complex interplay of ancient bacteria-like features and host-derived functionalities. Although the basic components of the mitochondrial translation apparatus have been recognized, very few protein factors aiding in recruiting ribosomes on mitochondria-encoded messenger RNA (mRNAs) have been identified in higher plants. In this study, we describe the identification of the Arabidopsis (Arabidopsis thaliana) MITOCHONDRIAL TRANSLATION FACTOR1 (MTL1) protein, a new member of the Pentatricopeptide Repeat family, and show that it is essential for the translation of the mitochondrial NADH dehydrogenase subunit7 (nad7) mRNA. We demonstrate that mtl1 mutant plants fail to accumulate the Nad7 protein, even though the nad7 mature mRNA is produced and bears the same 5' and 3' extremities as in wild-type plants. We next observed that polysome association of nad7 mature mRNA is specifically disrupted in mtl1 mutants, indicating that the absence of Nad7 results from a lack of translation of nad7 mRNA. These findings illustrate that mitochondrial translation requires the intervention of gene-specific nucleus-encoded PPR trans-factors and that their action does not necessarily involve the 5' processing of their target mRNA, as observed previously. Interestingly, a partial decrease in nad7 intron 2 splicing was also detected in mtl1 mutants, suggesting that MTL1 is also involved in group II intron splicing. However, this second function appears to be less essential for nad7 expression than its role in translation. MTL1 will be instrumental to understand the multifunctionality of PPR proteins and the mechanisms governing mRNA translation and intron splicing in plant mitochondria.

The use of amphipols for solution NMR studies of membrane proteins: advantages and constraints as compared to other solubilizing media.

Solution-state nuclear magnetic resonance studies of membrane proteins are facilitated by the increased stability that trapping with amphipols confers to most of them as compared to detergent solutions. They have yielded information on the state of folding of the proteins, their areas of contact with the polymer, their dynamics, water accessibility, and the structure of protein-bound ligands. They benefit from the diversification of amphipol chemical structures and the availability of deuterated amphipols. The advantages and constraints of working with amphipols are discussed and compared to those associated with other non-conventional environments, such as bicelles and nanodiscs.