Functional analysis of two alternative transcripts from porin1 gene of Drosophila melanogaster and involvement of corresponding 5'UTR sequences in the translation control

Abstract

VDAC (Voltage Dependent Anion-selective channel) is a voltage-dependent anion selective channel, a pore forming protein located in the outer mitochondrial membrane (OMM) of all eukaryotic organisms. This protein allows the passage of nucleotides, ions and small metabolites between cytosol and mitochondria. VDAC has a beta-barrel structure with its N-terminal forming an a-helix inserted into the pore and involved in the gating process. VDAC takes a maximum open state at voltage around 0mV; while at voltages greater than 20mV, for positive and negative values, VDAC switches to a closed state. The crucial role of this channel is dictated by its strategic position, able to interact with many enzymes, proteins or metabolites directly or indirectly involved in several cellular pathways, explaining thus its involvement in many diseases. In Drosophila melanogaster the gene encoding for the principal isoform of VDAC is porin1,which is made up by five exons, of which exon 1A and exon 1B, being 5 UTR sequences, are alternative between them. In fact, by means an alternative splicing process two transcripts are produced containing at the 5 -end or the exon 1A or the exon 1B, followed by the same coding sequence. The alternative transcripts 1A-VDAC and 1B-VDAC are produced in all developmental stage of fly and in any tissue. Thanks to a previous work from our team we know that 1B-VDAC transcript is unproductive because it is not translated. This result allowed us to speculate about a different cellular function for this 1B-VDAC transcript, respect the canonical 1A-VDAC mRNA. Considering all data known, the main objectives of my thesis work were: 1) understanding the molecular mechanisms responsible of the failing of 1B-VDAC mRNA translation; 2) investigate about the meaning in D. melanogaster of the alternative unproductive 1B-VDAC mRNA. Using several organism models, such as Saccharomyces cerevisiae Dpor1, HeLa cells and Drosophila melanogaster embryonic SL2 cell, we obtained important results that allow us to formulate the following hypothesis: the inhibitor role played by the 5 UTR 1B sequence on translation in yeast is probably associated to the action of specific RBPs able to bind the inner sequence 16-31. In yeast this mechanism is itself sufficient to guarantee the translational repression of the coding sequence of a gene reporter as well as the full-length mRNA of porin1 gene, demonstrating in this way that the 5 UTR 1B contains all necessary information for inducing inhibition of protein synthesis in yeast; in Drosophila the 3 UTR sequence of 1B-VDAC transcript is indispensable for carry out the translational repression mechanism of the same transcript. In fly indeed, the 1B-Luc construct is never expressed while the same 5 UTR-1B fused to the porin1 coding sequence does not influence translation of the same porin; the 5 UTR 1A represents in general a sequence able to amplify translation of any coding sequence fused to it. Indeed, fusing the 5 UTR 1A with coding sequences of gene reporters we obtained always a noticeable increase in the expression of the relative protein. This effect is not detectable in fly cells where, after transfection with the heterologous transcript 1A-porin, an increase of the endogenous amount of VDAC protein is not obtained. In Drosophila the 3 UTR sequence of 1A-VDAC transcript plays probably a role in controlling endogenous levels of VDAC. Indeed, by transfecting fly cells with the 1A-VDAC transcript which does not contain the 3 UTR sequence, the VDAC protein is only weakly translated.