The SHAPE of an IRES: Secondary Structure Determination of the Internal Ribosomal Entry Site in the 5’UTR of the gurken mRNA Using SHAPE Chemistry

Abstract

Internal Ribosomal Entry Sites (IRESs) are conserved secondary structural elements present in the 5’ untranslated regions (UTR) of some essential eukaryotic mRNAs and many viral RNA genomes. IRESs allow the mRNA or viral RNA to bypass canonical cap-dependent translation initiation and entice the ribosome to assemble directly onto the RNA strand and initiate translation. Viruses utilize this method of translation initiation to hijack cellular translation machinery and eukaryotes utilize this to maintain levels of critical proteins when most translation is shut down due to cellular stress. Gurken (Grk) protein is an EGFR ligand essential for determining polarity and eggshell patterning in Drosophila melanogaster development. The gurken mRNA is believed to have an IRES for several reasons, including steady regulation of grk translation under nutrient limited 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. Here we are interested in finding structural features corresponding to a potential internal ribosomal entry site (IRES) in the 5’ UTR of the gurken mRNA from D. melanogaster. Selective 2’-hydroxyl acylation analyzed by primer extension (SHAPE) chemistry is a powerful tool used to investigate secondary structure in RNA molecules. We used this procedure to probe the grk 5’ UTR secondary structure and then compare the predicted structure to known IRES structural motifs. 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. Here I present the wild-type structure of the gurken 5’ UTR and correlations between the structural elements present there and known IRES structural features.