Defects of mitochondrial RNA turnover lead to the accumulation of double-stranded RNA in vivo

The RNA helicase SUV3 and the polynucleotide phosphorylase PNPase are involved in the degradation of mitochondrial mRNAs but their roles in vivo are not fully understood. Additionally, upstream processes, such as transcript maturation, have been linked to some of these factors, suggesting either dual roles or tightly interconnected mechanisms of mitochondrial RNA metabolism. To get a better understanding of the turn-over of mitochondrial RNAs in vivo, we manipulated the mitochondrial mRNA degrading complex in Drosophila melanogaster models and studied the molecular consequences. Additionally, we investigated if and how these factors interact with the mitochondrial poly(A) polymerase, MTPAP, as well as with the mitochondrial mRNA stabilising factor, LRPPRC. Our results demonstrate a tight interdependency of mitochondrial mRNA stability, polyadenylation and the removal of antisense RNA. Furthermore, disruption of degradation, as well as polyadenylation, leads to the accumulation of double-stranded RNAs, and their escape out into the cytoplasm is associated with an altered immune-response in flies. Together our results suggest a highly organised and inter-dependable regulation of mitochondrial RNA metabolism with far reaching consequences on cellular physiology

Mitofusin mutations associated with CMT2A neuropathy trigger neuronal alterations by enhancing mitochondrial fusion

Les mitochondries forment un réseau très dynamique remodelé par deux processus antagonistes appelés : fusion et fission mitochondriales. Chez l’homme, une altération de ces processus, sont à l’origine de nombreuses maladies qui affectent essentiellement le système nerveux. L'objectif principal des travaux de ma thèse était de caractériser l'impact d'un déséquilibre entre la fusion et la fission mitochondriale dans le contexte d'une neuropathie héréditaire : la maladie de Charcot-Marie-Tooth de type 2A (CMT2A), qui est causée par des mutations dominantes dans la mitofusine MFN2. Dans le but d’étudier les mécanismes à l’origine de cette maladie, j’ai développé le premier modèle drosophile de CMT2A en exprimant dans les neurones de mouches quatre allèles de mitofusine retrouvés fréquemment chez les patients. De manière surprenante, les différents allèles altèrent très différemment la morphologie mitochondriale. En effet, alors que les mutations associées au domaine GTPase inhibent la fusion et agrègent les mitochondries, les mutations du domaine dit HB1 induisent au contraire un excès de fusion. J’ai pu ensuite déterminer que l’agrégation des mitochondries et l’excès de fusion, conduisent de manière commune à un défaut de transport des mitochondries au niveau des synapses et à une altération du métabolisme oxydatif associée à une accumulation de mutation dans l’ADN mitochondrial. Chez les drosophiles exprimant des allèles dominants actifs de mitofusine, tous ces dysfonctionnements disparaissent lorsqu’on augmente la fission suggérant que la pathogénicité des allèles du domaine HB1 résulte d’un déséquilibre de la balance entre fusion et fission en faveur de la fusion.Mitochondria form a dynamic network remodeled by two antagonistic processes called mitochondrial fusion and fission. While mitochondrial fusion creates interconnections between mitochondria, mitochondrial fission result in fragmentation. These processes are mediated by Dynamin-related GTPases, the outer-membrane fusion protein mitofusin, and the fission factor DPR1.The main aim of my resaearch was to characterize the impact of an imbalance between mitochondrial fusion and fission in neurons in the context of a severe hereditary neuropathy called Charcot-Marie-Tooth type 2A (CMT2A). Indeed, this disease is caused by dominant mutations in the mitofusinMFN2.In order to dissect the mechanisms by which these mutations alter mitofusin properties and neuronal function, we developed four drosophila models of CMT2A expressing the two most frequent substitutions (R94Q, R364W) and two others localizing to similar domains (T105M, L76P). The four alleles resulted in mitochondrial depletion at neuromuscular junctions, decreased oxidative metabolism, increased mtDNA mutations, and impaired locomotion that were associated with aberrant mitochondrial morphology. Interestingly, while GTPase domain-associated mutations (R94Q, T105M) aggregate unfused mitochondria, mutations within helix bundle 1 (R364W, L76P) unexpectedly enhance mitochondrial fusion, as demonstrated by rescue of mitochondrial morphology and locomotion provided by the DRP1 fission factor. In conclusion, we show that both dominant negative and dominant active forms of mitofusin can cause CMT2A, and propose for the first time that excessive mitochondrial fusion drives CMT2A pathogenesis in a large number of patients

Mitofusin gain and loss of function drive pathogenesis in Drosophila models of CMT2A neuropathy

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The Drosophila inner-membrane protein PMI controls crista biogenesis and mitochondrial diameter.

International audienceCristae are mitochondrial inner-membrane structures that concentrate respiratory chain complexes and hence regulate ATP production. Mechanisms controlling crista morphogenesis are poorly understood and few crista determinants have been identified. Among them are the Mitofilins that are required to establish crista junctions and ATP-synthase subunits that bend the membrane at the tips of the cristae. We report here the phenotypic consequences associated with the in vivo inactivation of the inner-membrane protein Pantagruelian Mitochondrion I (PMI) both at the scale of the whole organism, and at the level of mitochondrial ultrastructure and function. We show that flies in which PMI is genetically inactivated experience synaptic defects and have a reduced life span. Electron microscopy analysis of the inner-membrane morphology demonstrates that loss of PMI function increases the average length of mitochondrial cristae in embryonic cells. This phenotype is exacerbated in adult neurons in which cristae form a dense tangle of elongated membranes. Conversely, we show that PMI overexpression is sufficient to reduce crista length in vivo. Finally, these crista defects are associated with impaired respiratory chain activity and increases in the level of reactive oxygen species. Since PMI and its human orthologue TMEM11 are regulators of mitochondrial morphology, our data suggest that, by controlling crista length, PMI influences mitochondrial diameter and tubular shape