Structure-informed mutagenesis identifies combinatorial contributions to mouse insulin receptor 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. We previously determined that the 5' untranslated region (5'UTR) of the insulin receptor mRNA possesses a capacity for IRES activity that is conserved from insects to mammals. Well-characterized IRESes depend on RNA structures that reduce the protein requirements for translation initiation, thus circumventing translation inhibition. While there are several examples of viral IRES structures solved in vitro, the RNA secondary structures of cellular IRESes remain elusive and little information exists about the secondary structures of these RNAs in vivo. Here we probe the secondary structure of the Insr 5'UTR IRES along with two well-studied viral IRESes from hepatitis C virus and encephalomyocarditis virus using dimethyl sulfate mutational profiling by sequencing (DMS-MaPseq) in vitro and in cells. We find that the structures of viral IRESes in a cellular environment are largely consistent with their known in vitro structures. Using DMS-MaPseq probing as a constraint, we generated a model of the RNA secondary structure 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 of RNA, distal from the translation start codon, that is 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.