a, Western blot showing PKR expression levels in WT 293T compared to EIF2AK2-KO 293T cells. β-actin is shown for loading control. b, Western blot analysis for eIF2α phosphorylation levels in WT 293T (left panel) or EIF2AK2-KO 293T (right panel) cells transfected with UTP or m1Ψ modified mRNA. dsRNA was used as a positive control for PKR induction. Vinculin is shown. c, Western blot analysis for ATF4 levels in cells transfected with UTP or m1Ψ modified mRNA and treated with ISRIB. Tunicamycin was used as a control for ATF4 induction and ISRIB activity, vinculin loading control is shown. d, Relative spike mRNA levels at various time points following transfection with mRNAs containing UTP, Ψ, or m1Ψ. Levels were measured by RT-qPCR and normalized to housekeeping gene (ANXA5). Data represent mean and s.d. of three biological replicates. e, Bar plots depicting firefly luciferase luminescence normalized to Renilla control of UTP (red) or m1Ψ modified (blue) mRNA prepared with two different capping mechanisms - vaccinia virus capping enzyme (VCE, left) or co-transcriptional cap1 analog (AG, right). Error bars depict s.d. of three biological replicates. f, Bar plot depicting luciferase mRNA levels in cytosol fraction compared to total cell lysate as measured by RT-qPCR and normalized to ANXA5 gene. Data represent average and s.d. of 3 biological replicates. Statistical significance was calculated by two-sided standard t. test. g, Relative U99 RNA expression normalized to ANXA5 gene as measured by RT-qPCR from each fraction (total, cytosol, membrane-associated) in cells transfected with UTP or m1Ψ luciferase IVT mRNAs after sub cellular cell fractionation. Error bars depict s.d. of three biological replicates. High levels of the nuclear U99 in the membrane-associated compartment illustrate successful fractionation. h, Western blot analysis of luciferase expression at indicated time points following cycloheximide (CHX) treatment in cells transfected with either UTP or m1Ψ-modified luciferase mRNA. i, Quantification of luciferase protein levels normalized to vinculin loading control from panel h. Values are plotted as a function of time post CHX treatment (hours) and fitted to an exponential decay model. Protein half-lives derived from the fitted curves are shown below the graph. j, Scatter plots depicting ribosome densities, calculated as the ratio of ribosome footprints to mRNA levels, for human genes (grey) and no uridine in both A and P sites along the spike mRNA (red). The left panel compares cells transfected with UTP or Ψ-modified spike mRNA, and the right panel compares cells transfected with UTP and m1Ψ-modified spike mRNA. k, Bar plot showing CCND1 endogenous mRNA levels (control) in input and eIF4E-IP samples from cells transfected with UTP- or m1Ψ-modified luciferase mRNAs, measured by RT-qPCR. Error bars show s.d. of 3 biological replicates. Statistical significance was calculated using linear regression. Fold change between input and IP for each sample is indicated on the graph.