The Melanoma Antigens MELOE-1 and MELOE-2 Are Translated from a <i>Bona Fide</i> Polycistronic mRNA Containing Functional IRES Sequences

<div><p>Our previous studies on melanoma antigens identified two new polypeptides, named MELOE-1 and MELOE-2, that are involved in immunosurveillance. Intriguingly, these antigens are coded by distinct open reading frames (ORF) of the <i>meloe</i> mRNA which is significantly expressed only in the melanocytic lineage. In addition, MELOE-1 and -2 specific T cell clones recognized melanoma cells but very poorly normal melanocytes suggesting differential translation of <i>meloe</i> in normal <i>vs</i> tumor cells. This prompted us to elucidate the mechanisms of translation of these antigens in melanoma cells. We first demonstrated that no splicing event or cryptic promoter could generate shorter <i>meloe</i> transcripts containing only one of the two ORFs. Triggering <i>meloe</i> RNA degradation with a siRNA close to the ORF coding for MELOE-2 abrogated expression of both MELOE-1 and MELOE-2, thus confirming that the two ORFs are always associated. Next we showed, in a bicistronic reporter system, that IRES activities could be detected upstream of MELOE-1 and MELOE-2 and finally confirmed their translation from full length <i>meloe</i> cDNA in melanoma cells with eGFP constructs. In conclusion, <i>meloe</i> is a polycistronic mRNA that generates both MELOE-1 and MELOE-2 antigens through IRES-dependent translation in melanoma cells and that may explain their tumor specificity.</p>