Structure-informed mutagenesis of the Insr 5′UTR IRES. (A) Strategic overview of concurrent probing of three IRES elements in HEK293T cells. Two viral IRESs from hepatitis C virus (HCV) and encephalomyocarditis virus (EMCV) 5′UTRs were probed with dimethyl sulfate (DMS) alongside the mouse insulin receptor (Insr) 5′UTR from cotransfected plasmids. These RNA structures were also pooled and probed in vitro. Prediction of the Insr 5′UTR RNA secondary structure informed a mutation strategy aimed at identifying functional components. (B) Diagram of plasmid expression cassette for this strategy. (C) Diagram of transcripts probed, with number of tiled RT-PCR amplicons annotated for each 5′UTR.
