IFIT3 and IFIT2/3 promote IFIT1-mediated translation inhibition by enhancing binding to non-self RNA

Renata C Fleith; Harriet V Mears; Edward Emmott; Stephen C Graham; Daniel S Mansur; Trevor R Sweeney

In vitro translation IFIT proteins or complexes were diluted in bovine serum albumin (BSA) diluent buffer (0.5 mg/mL BSA, 20 mM Tris pH 7.5, 160 mM KCl, 5 % glycerol, 2 mM DTT, 1U/μL RNaseOUT), and incubated with 125 ng reporter RNA for 15 minutes at 37 C to allow RNA binding. In vitro translation was performed using the Flexi Rabbit Reticulocyte Lysate (RRL) System (Promega) for 90 minutes at 30 C. Reactions were terminated by incubation on ice, followed by addition of 50 volumes of passive lysis buffer (Promega). After addition of an equal volume of firefly luciferase substrate (20 mM Tricine, 2.67 mM MgSO4, 0.1 mM EDTA, 33.3 mM DTT, 530 μM ATP, 270 μM Acetyl-CoA, 30 mg/mL luciferin, 250 μM magnesium carbonate hydroxide), luciferase signal was measured by GloMax (Promega). Luciferase values were normalised to the diluent buffer-only control for each experiment.