The mitochondrial (mt) DNA depletion syndromes (MDDS) are genetic disorders characterized by a severe, tissue-specific decrease of mtDNA copy number, leading to organ failure. There are two main clinical presentations: myopathic (OMIM 609560) and hepatocerebral (OMIM 251880). Known mutant genes, including TK2, SUCLA2, DGUOK and POLG, account for only a fraction of MDDS cases. We found a new locus for hepatocerebral MDDS on chromosome 2p21-23 and prioritized the genes on this locus using a new integrative genomics strategy. One of the top-scoring candidates was the human ortholog of the mouse kidney disease gene Mpv17. We found disease-segregating mutations in three families with hepatocerebral MDDS and demonstrated that, contrary to the alleged peroxisomal localization of the MPV17 gene product, MPV17 is a mitochondrial inner membrane protein, and its absence or malfunction causes oxidative phosphorylation (OXPHOS) failure and mtDNA depletion, not only in affected individuals but also in Mpv17-/- mice.

Mitochondrial protein translation is a complex process performed within mitochondria by an apparatus composed of mitochondrial DNA (mtDNA)-encoded RNAs and nuclear DNA-encoded proteins. Although the latter by far outnumber the former, the vast majority of mitochondrial translation defects in humans have been associated with mutations in RNA-encoding mtDNA genes, whereas mutations in protein-encoding nuclear genes have been identified in a handful of cases. Genetic investigation involving patients with defective mitochondrial translation led us to the discovery of novel mutations in the mitochondrial elongation factor G1 (EFG1) in one affected baby and, for the first time, in the mitochondrial elongation factor Tu (EFTu) in another one. Both patients were affected by severe lactic acidosis and rapidly progressive, fatal encephalopathy. The EFG1-mutant patient had early-onset Leigh syndrome, whereas the EFTu-mutant patient had severe infantile macrocystic leukodystrophy with micropolygyria. Structural modeling enabled us to make predictions about the effects of the mutations at the molecular level. Yeast and mammalian cell systems proved the pathogenic role of the mutant alleles by functional complementation in vivo. Nuclear-gene abnormalities causing mitochondrial translation defects represent a new, potentially broad field of mitochondrial medicine. Investigation of these defects is important to expand the molecular characterization of mitochondrial disorders and also may contribute to the elucidation of the complex control mechanisms, which regulate this fundamental pathway of mtDNA homeostasis.

A peculiar form of hepatocerebral mtDNA depletion syndrome is caused by mutations in the MPV17 gene, which encodes a small hydrophobic protein of unknown function located in the mitochondrial inner membrane. In order to define the molecular basis of MPV17 variants associated with the human disorder, we have previously taken advantage of S. cerevisiae as a model system thanks to the presence of an MPV17 ortholog gene, SYM1. We demonstrate here that the SYM1 gene product is essential to maintain OXPHOS, glycogen storage, mitochondrial morphology and mtDNA stability in stressing conditions such as high temperature and ethanol-dependent growth. To gain insight into the molecular basis of the Sym1-less phenotype, we identified and characterized multicopy suppressor genes and metabolic suppressor compounds. Our results suggest that (i) metabolic impairment and mtDNA instability occur independently from each other as a consequence of SYM1 ablation; (ii) ablation of Sym1 causes depletion of glycogen storage, possibly due to defective anaplerotic flux of tricarboxylic acid (TCA) cycle intermediates to the cytosol; (iii) flattening of mitochondrial cristae in Sym1-defective organelles suggests a role for Sym1 in the structural preservation of the inner mitochondrial membrane, which could in turn control mtDNA maintenance and stability.

In this work the structural variations of Terminal Inverted Repeats (TIR) of Bari like transposons in Drosophila species has been studied. The aim is to try and assess the relevance of different variants in the evolutionary distribution of Bari elements. Bari is a member of the widespread Tc1 superfamily of transposable elements that has colonized most species of the Drosophila genus. We previously reported the structure of two related elements that differ in their TIR organization: Bari1 harbouring 26-bp TIR (short TIRs) and Bari2 with about 250-bp TIR (long TIIR). While elements with short TIRs are complete and potentially autonomous, long ones are invariably composed of defective copies. The results show that in D. pseudobscura, D. persimilis and D. mojavensis, there is a third class of Bari elements, Bari3, that exhibit a long TIR structure and are not defective. Phylogenetic relationships among reconstructed transposases are consistent with the three subfamilies sharing a common origin. However, the final TIR organization into long or short structure is not related by descent but appears to be lineage-specific. Furthermore, we show that, independently of origin and organization, within the 250-bp terminal sequences there are three regions that are conserved in both sequence and position suggesting they are under functional constraint.

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Transposable elements (TEs) are constitutive components of both eukaryotic and prokaryotic genomes. The role of TEs in the evolution of genes and genomes has been widely assessed over the past years in a variety of model and non-model organisms. Drosophila is undoubtedly among the most powerful model organisms used for the purpose of studying the role of transposons and their effects on the stability and evolution of genes and genomes. Besides their most intuitive role as insertional mutagens, TEs can modify the transcriptional pattern of host genes by juxtaposing new cis-regulatory sequences. A key element of TE biology is that they carry transcriptional control elements that fine-tune the transcription of their own genes, but that can also perturb the transcriptional activity of neighboring host genes. From this perspective, the transposition-mediated modulation of gene expression is an important issue for the short-term adaptation of physiological functions to the environmental changes, and for long-term evolutionary changes. Here, we review the current literature concerning the regulatory and structural elements operating in cis provided by TEs in Drosophila. Furthermore, we highlight that, besides their influence on both TEs and host genes expression, they can affect the chromatin structure and epigenetic status as well as both the chromosome's structure and stability. It emerges that Drosophila is a good model organism to study the effect of TE-linked regulatory sequences, and it could help future studies on TE-host interactions in any complex eukaryotic genome.

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Bari elements are members of the Tc1-mariner superfamily of DNA transposons, originally discovered in Drosophila melanogaster, and subsequently identified in silico in 11 sequenced Drosophila genomes and as experimentally isolated in four non-sequenced Drosophila species. Bari-like elements have been also studied for their mobility both in vivo and in vitro. We analyzed 23 Drosophila genomes and carried out a detailed characterization of the Bari elements identified, including those from the heterochromatic Bari1 cluster in D. melanogaster. We have annotated 401 copies of Bari elements classified either as putatively autonomous or inactive according to the structure of the terminal sequences and the presence of a complete transposase-coding region. Analyses of the integration sites revealed that Bari transposase prefers AT-rich sequences in which the TA target is cleaved and duplicated. Furthermore evaluation of transposon’s co-occurrence near the integration sites of Bari elements showed a non-random distribution of other transposable elements. We also unveil the existence of a putatively autonomous Bari1 variant characterized by two identical long Terminal Inverted Repeats, in D. rhopaloa. In addition, we detected MITEs related to Bari transposons in 9 species. Phylogenetic analyses based on transposase gene and the terminal sequences confirmed that Bari-like elements are distributed into three subfamilies. A few inconsistencies in Bari phylogenetic tree with respect to the Drosophila species tree could be explained by the occurrence of horizontal transfer events as also suggested by the results of dS analyses. This study further clarifies the Bari transposon’s evolutionary dynamics and increases our understanding on the Tc1-mariner elements’ biology.