Abstract
Here we are interested in finding structure corresponding to a potential internal ribosomal entry site (IRES) in the 5’ UTR of the Gurken (Grk) mRNA from Drosophila melanogaster. Grk protein is an EGFR ligand essential for determining polarity and eggshell patterning in D. melanogaster development. Grk is believed to have an IRES for several reasons, including steady regulation of Grk translation under starvation conditions when canonical cap-dependent translation is repressed and the necessity of a RNA helicase for cap-dependent translation to occur under non-starvation conditions. Selective 2’-hydroxyl acylation analyzed by primer extension (SHAPE) chemistry is a powerful tool used to elucidate secondary structure in RNA molecules. In this method, N-methlyisatoic anhydride (NMIA) is incubated with folded RNA molecules and reacts with unconstrained (i.e. non base paired) 2’-hydroxyl groups, forming an adduct which blocks primer extension. Reverse transcription then gives a mixed population to be visualized via polyacrylamide gel, similar to Sanger sequencing. This information combined with the sequence of the RNA, is used to calculate probable base pairing and secondary structure of the RNA strand. In collaboration with in vitro translation Luciferase assays and selective deletion or mutation of structural features, individual secondary structural features can be selectively analyzed and included or excluded as a potential IRES. To date, multiple stem loops have been analyzed for IRES structure and activity, though none have surfaced as a strong IRES candidate. Work is continuing on additional regions of the Grk 5’-UTR.
