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 ccmF_N2 gene encoding a heme–lyase complex subunit which derives from the split of the ccmF_N 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.     

Dopamine-induced α-synuclein oligomers show self- and cross-propagation properties

Amyloid aggregates of α-synuclein (αS) protein are the predominant species present within the intracellular inclusions called Lewy bodies in Parkinson’s disease (PD) patients. Among various aggregates, the low-molecular weight ones broadly ranging between 2 and 30 mers are known to be the primary neurotoxic agents responsible for the impairment of neuronal function. Recent research has indicated that the neurotransmitter dopamine (DA) is one of the key physiological agents promoting and augmenting αS aggregation, which is thought to be a significant event in PD pathologenesis. Specifically, DA is known to induce the formation of soluble oligomers of αS, which in turn are responsible for inducing several important cellular changes leading to cellular toxicity. In this report, we present the generation, isolation, and biophysical characterization of five different dopamine-derived αS oligomers (DSOs) ranging between 3 and 15 mers, corroborating previously published reports. More importantly, we establish that these DSOs are also capable of replication by self-propagation, which leads to the replication of DSOs upon interaction with αS monomers, a process similar to that observed in mammilian prions. In addition, DSOs are also able to cross-propagate amyloid-β (Aβ) aggregates involved in Alzheimer’s disease (AD). Interestingly, while self-propagation of DSOs occur with no net gain in protein structure, cross-propagation proceeds with an overall gain in β-sheet conformation. These results implicate the involvement of DSOs in the progression of PD, and, in part, provide a molecular basis for the observed co-existence of AD-like pathology among PD patients.     

Photoproduction of molecular hydrogen by Rhodospirillum rubrum immobilized in composite agar layer/microporous membrane structures

Summary Viable cells of Rhodospirillum rubrum were immobilized by entrapment in a planar agar matrix bounded by a microporous membrane filter and were tested for H₂ photoproduction in synthetic waste water provided with malate and glutamate. Optimum H₂ production was obtained at 15 klx for a C/N ratio of 7–8. Production rates as high as 565 mm³ H₂ · h^-1 per cubic centimetre of agar were recorded. The composite structures, however, suffered from high diffusion limitations which increased with the population of immobilized bacteria. The H₂-evolving activity could be maintained over several months by periodically incubating the biocatalytic structures in a rich nutrient broth. No contamination of the nutrient broth due to leakage of photosynthetic organisms from the gel appeared during incubation of the structures.     

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.Translation is the fundamental process decoding the genetic message present on mRNAs into proteins. In plant cells, mRNA translation occurs in the cytoplasm but also in two organelles, mitochondria and plastids. Because of their prokaryotic origin, the translation machineries operating in these two organelles share many characteristics with the bacterial translation apparatus (Bonen, 2004; Barkan, 2011). However, most of these bacteria-like features have been modified throughout evolution, and current organellar translation systems cooperate with numerous nucleus-encoded eukaryotic trans-factors. The divergence from bacteria is particularly obvious in plant mitochondria, notably because mitochondrial mRNAs lack the typical Shine and Dalgarno (SD) motif in their 5′ leaders and alternative start codons other than AUG are often used to initiate translation (Bonen, 2004). Proteomic and bioinformatic analyses allowed the identification of most proteins and RNA factors forming the core of the plant mitochondrial translation machinery, including translation initiation and elongation factors as well as ribosomal proteins (Bonen, 2004; Bonen and Calixte, 2006). However, the dynamics of this machinery remains largely obscure. In particular, nothing is known about the recruitment of mitochondrial ribosomes on 5′ untranslated regions in the absence of the SD motif and about the recognition of the correct translation initiation codon by the small ribosomal subunit. The high degree of sequence divergence among 5′ leaders of mitochondrial genes suggests a ribosome recruitment mechanism involving gene-specific cis-sequences and trans-factors (Hazle and Bonen, 2007; Choi et al., 2012). Up to now, only two proteins belonging to the Pentatricopeptide Repeat (PPR) family have been found to promote mitochondrial translation in higher plants (Uyttewaal et al., 2008b; Manavski et al., 2012). How they facilitate translation is still unclear, as for the few characterized PPR proteins shown to participate in plastid translation (Fisk et al., 1999; Schmitz-Linneweber et al., 2005; Cai et al., 2011; Zoschke et al., 2012, 2013). The plastid PENTATRICOPEPTIDE REPEAT PROTEIN10 (PPR10) protein of maize (Zea mays) is the only one for which the function has been elucidated at the molecular level. It was shown that, upon binding, PPR10 impedes the formation of a stem-loop structure in the 5′ leader of the ATP synthase subunit c (atpH) mRNA, permitting the recruitment of ribosomes through the liberation of an SD motif (Prikryl et al., 2011).PPR proteins represent a large family of RNA-binding proteins that has massively expanded in terrestrial plants (Barkan and Small, 2014). Most eukaryotes encode a handful of these proteins, whereas plant nuclear genomes express over 400 PPR proteins that are almost exclusively predicted to target mitochondria and/or plastids (Lurin et al., 2004; O’Toole et al., 2008). This family of proteins is characterized by the succession of tandem degenerate motifs of approximately 35 amino acids (Small and Peeters, 2000; Lurin et al., 2004). Based on the length of these repeats, the PPR family has been divided into two groups of roughly equal size in higher plants. P-type PPR proteins contain only successions of canonical 35-amino acid repeats (P), whereas PLS PPR proteins are composed of sequential repeats of P, short (S), and long (L) PPR motifs. P-type PPR proteins were shown to participate in various aspects of organellar RNA processing, whereas PLS PPR proteins have been almost exclusively associated with C-to-U RNA editing (for review, see Barkan and Small, 2014; Hammani and Giegé, 2014). Recent crystal structures showed that PPR motifs adopt an antiparallel helix-turn-helix fold whose repetition forms a solenoid-like structure (Ringel et al., 2011; Howard et al., 2012; Ban et al., 2013; Yin et al., 2013; Coquille et al., 2014; Gully et al., 2015). PPR tracks organize highly specific interaction domains that were shown to associate with single-stranded RNAs (Schmitz-Linneweber et al., 2005; Beick et al., 2008; Uyttewaal et al., 2008a; Williams-Carrier et al., 2008; Pfalz et al., 2009; Cai et al., 2011; Hammani et al., 2011; Prikryl et al., 2011; Khrouchtchova et al., 2012; Manavski et al., 2012; Zhelyazkova et al., 2012; Ke et al., 2013; Yin et al., 2013). The mechanism of sequence-specific RNA recognition by PPR proteins was recently uncovered, and combinations involving amino acid 6 of one motif and amino acid 1 of the subsequent motif correlate strongly with the identity of the RNA base to be bound (Barkan et al., 2012; Takenaka et al., 2013; Yagi et al., 2013).Besides those involved in RNA editing, few mitochondria-targeted PPR proteins have been characterized to date. Thus, our knowledge of the mechanisms governing the production and the expression of mitochondrial RNAs in higher plants is very limited. In this analysis, we describe the function of a novel mitochondria-targeted PPR protein of Arabidopsis (Arabidopsis thaliana) called MITOCHONDRIAL TRANSLATION FACTOR1 (MTL1). Genetic and biochemical analyses indicate that MTL1 is essential for the translation of the mitochondrial NADH dehydrogenase subunit7 (nad7) mRNA. Effectively, the Nad7 protein does not accumulate to detectable levels in mtl1 mutants, and this absence correlates with a lack of association of nad7 mature mRNA with mitochondrial polysomes. Interestingly, a partial but significant decrease in nad7 intron 2 splicing was also detected in mtl1 mutants, suggesting that the MTL1 protein is also involved in group II intron splicing. Since the decrease in splicing was only partial, this second function of MTL1 appears less essential for nad7 expression than its role in translation.     

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.