Renè Massimiliano Marsano

Sym1, the yeast ortholog of the MPV17 human disease protein, is a stress-induced bioenergetic and morphogenetic mitochondrial modulator

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.

The complete Tirant transposable element in Drososphila melanogaster shows a structural relationship with retrovirus-like retrotransposons

We have determined the structure and organization of Tirant, a retrotransposon of Drosophila melanogaster reported in literature to be responsible for four independent mutations. Tirant is a long terminal repeat (LTR) retrotransposon 8527bp long. It possesses three open reading frames (ORF) encoding Gag, Pol and Env proteins with a strong similarity with ZAM, a recently identified member of the gypsy class of retrovirus-like mobile elements. Molecular analysis of the Tirant genomic copies present in four D. melanogaster strains revealed that most of them are defective, non-autonomous elements that differ in the position and extension of the conserved internal portion. Defective elements lacking the Gag ORF but retaining the Env ORF are abundant in heterochromatin. Four discrete Tirant transcripts are observed during embryogenesis in the strain Oregon-R, the smaller of which, 1.8kb in size, originates from the splicing of a primary transcript and leads to a subgenomic RNA coding for the Env product.

Mitochondrial glutamate carriers from Drosophila melanogaster: biochemical, evolutionary and modeling studies.

The mitochondrial carriers are members of a family of transport proteins that mediate solute transport across the inner mitochondrial membrane. Two isoforms of the glutamate carriers, GC1 and GC2 (encoded by the SLC25A22 and SLC25A18 genes, respectively), have been identified in humans. Two independent mutations in SLC25A22 are associated with severe epileptic encephalopathy. In the present study we show that two genes (CG18347 and CG12201) phylogenetically related to the human GC encoding genes are present in the D. melanogaster genome. We have functionally characterized the proteins encoded by CG18347 and CG12201, designated as DmGC1p and DmGC2p respectively, by overexpression in Escherichia coli and reconstitution into liposomes. Their transport properties demonstrate that DmGC1p and DmGC2p both catalyze the transport of glutamate across the inner mitochondrial membrane. Computational approaches have been used in order to highlight residues of DmGC1p and DmGC2p involved in substrate binding. Furthermore, gene expression analysis during development and in various adult tissues reveals that CG18347 is ubiquitously expressed in all examined D. melanogaster tissues, while the expression of CG12201 is strongly testis-biased. Finally, we identified mitochondrial glutamate carrier orthologs in 49 eukaryotic species in order to attempt the reconstruction of the evolutionary history of the glutamate carrier function. Comparison of the exon/intron structure and other key features of the analyzed orthologs suggests that eukaryotic glutamate carrier genes descend from an intron-rich ancestral gene already present in the common ancestor of lineages that diverged as early as bilateria and radiata.

FB elements can promote exon shuffling: a promoter-less white allele can be reactivated by FB mediated transposition in Drosophila melanogaster

Foldback (FB) elements are transposable elements found in many eukaryotic genomes; they are thought to contribute significantly to genome plasticity. In Drosophila melanogaster, FBs have been shown to be involved in the transposition of large chromosomal regions and in the genetic instability of some alleles of the white gene. In this report we show that FB mediated transposition of w67C23, a mutation that deletes the promoter of the white gene and its first exon, containing the start codon, can restore expression of the white gene. We have characterized three independent events in which a 14-kb fragment from the w67C23 locus was transposed into an intron region in three different genes. In each case a local promoter drives the expression of white, producing a chimeric mRNA. These findings suggest that, on an evolutionary timescale, FB elements may contribute to the creation of new genes via exon shuffling.

Organization and possible origin of the Bari-1 cluster in the heterochromatic h39 region of Drosophila melanogaster

The molecular organization of the heterochromatic h39 region of the Drosophila melanogaster second chromosome has been investigated by studying two BAC clones identified both by Southern blotting and by FISH experiments as containing tandem arrays of Bari1, a transposable element present only in this region. Such BAC clones appear to contain different portions of the h39 region since they differ in the DNA sequences flanking the Bari1 repeats on both sides. Thus, the 80 Bari1 copies estimated to be present in the h39 region are split into at least two separated subregions. On the basis of the analysis of the flanking sequences a possible mechanism depending on an aberrant activity of the Bari1 transposase is proposed for the genesis of the heterochromatic tandem arrays of the element.

MAX, a novel retrotransposon of the BEL-Pao family, is nested within the Bari 1 cluster at the heterochromatic h39 region of chromosome 2 in Drosophila melanogaster

A homogeneous array of 80 tandem repeats of the Bari1 transposon is located in the pericentromeric h39 region of chromosome 2 of Drosophila melanogaster. Here, we report that the Bari1 cluster is interrupted by an 8556-bp insertion. DNA sequencing and database searches identified this insertion as a previously unannotated retrotransposon that we have named MAX. MAX possesses two ORFs; ORF1 putatively encodes a polyprotein comprising GAG and RT domains, while ORF2 could encode a 288-amino acid protein of unknown function. Alignment with the RT domains of known LTR retrotransposons shows that MAX belongs to the BEL-Pao family, which remarkable for its widespread presence in different taxa, including lower chordates. We have analyzed the distribution of MAX elements within representative species of the Sophophora subgroup and found that they are restricted to the species of the melanogaster complex, where they are heavily represented in the heterochromatin of all autosomes and on the Y chromosome.

Functional Characterization of the Bari1 Transposition System

The transposons of the Bari family are mobile genetic elements widespread in the Drosophila genus. However, despite a broad diffusion, virtually no information is available on the mechanisms underlying their mobility. In this paper we report the functional characterization of the Bari elements transposition system. Using the Bari1 element as a model, we investigated the subcellular localization of the transposase, its physical interaction with the transposon, and its catalytic activity. The Bari1 transposase localized in the nucleus and interacted with the terminal sequences of the transposon both in vitro and in vivo, however, no transposition activity was detected in transposition assays. Profiling of mRNAs expressed by the transposase gene revealed the expression of abnormal, internally processed transposase transcripts encoding truncated, catalytically inactive transposase polypeptides. We hypothesize that a post-transcriptional control mechanism produces transposase-derived polypeptides that effectively repress transposition. Our findings suggest further clues towards understanding the mechanisms that control transposition of an important class of mobile elements, which are both an endogenous source of genomic variability and widely used as transformation vectors/biotechnological tools.

Evidences for insulator activity of the 5'UTR of the Drosophila melanogaster LTR-retrotransposon ZAM.

AbstractInsulators or chromatin boundary are DNA elements that organize the genome into ndiscrete regulatory domains by limiting the actions of enhancers and silencers through a n“positional-blocking mechanism”. The role of these sequences, both in modulation of the nenhancers range of action (enhancer–promoter selectivity) and in the organization of the nchromatin in functional domains, is emerging strongly in these last years. There is a great ninterest in identifying new insulators because deeper knowledge of these elements can nhelp understand how cis-regulatory elements coordinate the expression of the target genes. However, while insulators are critical in gene regulation and genome functioning, only a few have been reported so far. nHere, we describe a new insulator sequence that is located in the 5′UTR of the Drosophila nretrotransposon ZAM. We have used an “enhancer–blocking assay” to test its effects on nthe activity of the enhancer in transiently transfected Drosophila S2R+ cell line. Moreover, we show that the new insulator is able to affect significantly the enhancer–promoter interaction in the human cell line HEK293. nThese results suggest the possibility of employing the ZAM insulator in gene transfer nprotocols from insects to mammals in order to counteract the transgene positional and ngenotoxic effects.

Role of somatomedin-B-like domains on ENPP1 inhibition of insulin signaling

The exact mechanism by which ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1) inhibits insulin signaling is not known. ENPP1 contains two somatomedin-B-like domains (i.e. SMB 1 and 2) involved in ENPP1 dimerization in animal cells. The aim of the present study was to investigate if these domains modulate ENPP1 inhibitory activity on insulin signaling in human insulin target cells (HepG2). ENPP1 (ENPP1-3myc), ENPP1 deleted of SMB 1 (ENPP1-ΔI-3myc) or of SMB 2 (ENPP1-ΔII-3myc) domain were cloned in frame with myc tag in mammalian expression vector pRK5. Plasmids were transiently transfected in human liver HepG2 cells. ENPP1 inhibitory activity on insulin signaling, dimerization and protein-protein interaction with insulin receptor (IR), reported to mediate the modulation of ENPP1 inhibitory activity, were studied. As compared to untransfected cells, a progressive increase of ENPP1 inhibitory activity on insulin-induced IR β-subunit autophosphorylation and on Akt-S(473) phosphorylation was observed in ENPP1-3myc, ENPP1-ΔI-3myc and ENPP1-ΔII-3myc cells. Under non reducing conditions a 260 kDa homodimer, indicating ENPP1 dimerization, was observed. The ratio of non reduced (260 kDa) to reduced (130 kDa) ENPP1 was significantly decreased by two thirds in ENPP1-ΔII-3myc vs. ENPP1-3myc but not in ENPP1-ΔI-3myc. A similar ENPP1/IR interaction was detectable by co-immunoprecipitation in ENPP1-3myc, ENPP1-ΔI-3myc and ENPP1-ΔII-3myc cells. In conclusion, SMB 1 and SMB 2 are negative modulators of ENPP1 inhibitory activity on insulin signaling. For SMB 2 such effect might be mediated by a positive role on protein dimerization.

Mosquitoes LTR retrotransposons: a deeper view into the genomic sequence of Culex quinquefasciatus.

A set of 67 novel LTR-retrotransposon has been identified by in silico analyses of the Culex quinquefasciatus genome using the LTR_STRUC program. The phylogenetic analysis shows that 29 novel and putatively functional LTR-retrotransposons detected belong to the Ty3/gypsy group. Our results demonstrate that, by considering only families containing potentially autonomous LTR-retrotransposons, they account for about 1% of the genome of C. quinquefasciatus. In previous studies it has been estimated that 29% of the genome of C. quinquefasciatus is occupied by mobile genetic elements. The potential role of retrotransposon insertions strictly associated with host genes is described and discussed along with the possible origin of a retrotransposon with peculiar Primer Binding Site region. Finally, we report the presence of a group of 38 retrotransposons, carrying tandem repeated sequences but lacking coding potential, and apparently lacking “master copy” elements from which they could have originated. The features of the repetitive sequences found in these non-autonomous LTR retrotransposons are described, and their possible role discussed. These results integrate the existing data on the genomics of an important virus-borne disease vector.