Abstract
Cells under stress shift their proteome by repressing cap-dependent translation initiation. RNA elements called internal ribosome entry sites (IRES) allow key cellular transcripts to remain efficiently translated to support an effective stress response. Well- characterized IRESes depend on RNA structures that reduce the protein requirements for translation initiation, thus circumventing translation inhibition. We have previously determined that the insulin receptor 5’ untranslated region (Insr 5’UTR) possesses a capacity for IRES activity that is conserved from insects to mammals. There are several prominent examples of viral IRES structures solved in solution; however, the RNA secondary structures of cellular IRESes remain mostly elusive, especially in vivo. Here we probe the secondary structure of the Insr 5’UTR IRES in tandem with two well-studied viral IRESes from Hepatitis C virus (HCV) and Encephalomyocarditis virus (EMCV) using dimethyl sulfate mutational profiling by sequencing (DMS-MaPseq) in cells and in vitro. Additionally, preliminary DMS-MaPseq data for two other cellular IRESes are presented. One is from the 5’UTR of insulin-like growth factor 1 receptor (Igf1r), which operates in the same signaling pathway as Insr and exhibits a similar capacity for cap-independent translation initiation but differs vastly in sequence. The second is from an abundant circular RNA in Drosophila that has previously been shown to translate a small peptide. We find that the viral IRES structures in cells are consistent with their known in vitro structures and that significant linearization of these well-studied IRESes occurs in the region surrounding their translation start codon in cells. Using concurrent DMS-MaPseq probing as a constraint, we present a model of the mouse insulin receptor 5’UTR. With this model as a guide, we employed a mutation strategy which allowed us to identify a conserved segment distal from the translation start codon as critical for Insr IRES function. This knowledge informed the design of a minimal IRES element with equivalent activity to the full-length Insr 5’UTR across translation contexts.