Realtime 2-5A kinetics suggests interferons ß and ? evade global arrest of translation by RNase L

Alisha Chitrakar; Sneha Rath; Jesse Donovan; Kaitlin Demarest; Yize Li; Raghavendra Rao Sridhar; Susan R. Weiss; Sergei V. Kotenko; Ned S. Wingreen; Alexei Korennykh

To test whether IFN arises from actively ongoing translation rather than from other potential mechanisms (e.g. delayed secretion of pre-translated IFN stores), we used pulse-treatment with a translation inhibitor, anisomycin (Fig. 5E ). In this setting, cells were first treated with poly-IC for three hours, which stopped protein synthesis but did not yet activate a strong transcriptional IFN response. Next, anisomycin was added to arrest all protein synthesis and the cells were kept for three additional hours. Control cells were kept for the same duration without anisomycin. During the last hour, media was changed to remove poly-IC, but anisomycin treatment was continued to keep the cells translationally arrested. When IFN activity in the media was assayed, we found The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/476341 doi: bioRxiv preprint S8C), but before the IFN response, blocked IFN production (Fig. 5E) . A control experiment showed that anisomycin was compatible with IFN sensing by naïve cells (Fig. 5F ). Of note, anisomycin had a mild stimulatory effect on ISG mRNAs due to an unknown mechanism; this effect acted in the opposite direction from blocking IFNs and thus did not affect the suitability of anisomycin as a control in our tests. Together, our experiments indicated that IFNs are indeed translated when the bulk of protein synthesis remains silenced by 2-5A.