Figure 4.

Translation of in vitro transcribed mRNA in the absence of PKR activity. (A) HEK293T cells were transfected with plasmids encoding protein inhibitors of PKR: swinepox C8L protein, wt vaccinia K3L, hyperactive K3L-H47R, inactive K3L-Y76A, or pG5 empty vector. Twenty-four hours later, unmodified or Ψ-modified in vitro transcribed mRNAs encoding firefly luciferase were delivered by lipofection, and luciferase activity was measured 4 h later. Data were normalized to values obtained when cells were first transfected with empty vector then with unmodified RNA. Presented data are mean values from three replicates ± SEM. (B) MEF cell lines derived from wild-type (WT) or transgenic mice that do not express functional PKR (PKR^−/−) were transfected with unmodified or Ψ-containing in vitro transcribed mRNAs encoding firefly luciferase. Data were normalized to values obtained when cells were transfected with unmodified RNA and expressed as fold increase in translation of Ψ-containing mRNA over unmodified RNA. Values are from three replicate wells ± SEM, and are representative of at least three independently performed experiments. (C) WT and PKR^−/– MEF cells were transfected with unmodified or Ψ-containing in vitro transcribed mRNAs encoding firefly luciferase, or mock transfected with no RNA (–). Cells were lysed 2 h following RNA transfection; proteins were then separated by SDS–PAGE and assayed for eIF-2α phosphorylation by western blotting. Relative phosphorylation is indicated above each gel lane, calculated as phosphorylated band density divided by total band density and then normalized to the phosphorylation induced by unmodified RNA in wild-type cells. Absence of PKR was also confirmed by western blotting.