Structure-informed mutagenesis identifies a conserved region critical for mouse insulin receptor 5'UTR IRES function

Cells under stress shift their proteome by repressing cap-dependent translation initiation. RNA elements called internal ribosome entry sites (IRES) can 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 (5UTR) 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 5UTR 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. 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 the concurrent DMS- MaPseq probing as a constraint, we present a model of the mouse insulin receptor (Insr) 5UTR. 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 5UTR across translation contexts. BackgroundThe RNA structural requirements for cap-independent translation initiation facilitated by the Mus musculus insulin receptor (Insr) 5 untranslated region (5UTR) are unknown. ResultsRNA secondary structure probing of the Insr 5UTR in cells provides a folding model used to identify elements required for cap-independent translation initiation. ConclusionA small region of the Insr 5UTR distal to the translation start codon is necessary for cap-independent translation initiation and is fully sufficient in the proper structural context.