PKR activation by noncanonical ligands: a 5′-triphosphate requirement versus antisense contamination

PKR is activated by in vitro transcribed SNORD113, but not chemically synthesized SNORD113. Each SNORD113 species was initially purified by denaturing PAGE prior to the PKR autophosphorylation assay. (A) Representative PhosphorImage of an autophosphorylation assay with in vitro transcribed SNORD113 (5′-ppp) and chemically synthesized SNORD113 (5′-OH). SNORD113 concentrations were 50, 100, 250, and 500 nM; perfectly duplexed RNA 106 bp in length (ds106) was 10 nM. (B) Activation data as assayed in A were normalized to ds106 and averaged values were plotted. Error bars ± SD; n = 3 with at least two biological replicates per sample.