DROSOPHILA: A MODEL SYSTEM FOR RNA SECONDARY STRUCTURE DEPENDENT DICISTRONIC GENE EXPRESSION

Eukaryotic genes generally contain a single open reading frame (ORF) per mRNA\ud transcript, wherein translation is facilitated by a modified 5??? cap. The D. melanogaster\ud genome comprises over 100 identified dicistronic genes that contain two non-overlapping ORFs per transcript. Downstream gene translation is non-canonical due to lack of a 5??? cap. We hypothesize that embedded within the ICR are secondary structures that facilitate internal ribosomal entry. We have examined this hypothesis in the D.\ud melanogaster Tim9b/CG12788 dicistronic gene pair and its orthologs through two aims:\ud 1) use comparative genomics and bioinformatics for annotation and identification of\ud putative IRESes, and 2) conduct functional assays to validate predicted IRESes in\ud Drosophilid S2 and HEK-293T cells. These studies provide the first annotation of\ud Tim9b/CG12788 orthologs in eleven Drosophilid species, reveal that the gene structure is conserved over evolutionary time, and demonstrate the first practical test of putative\ud IRES elements for this gene pair