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

Realtime 2-5A dynamics in live cells . CC-BY 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.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 In the absence of methods to monitor 2-5A without cell disruption, the cellular dynamics of this second messenger are poorly understood. Here we developed a biosensor for continuous and non-invasive 2-5A monitoring in live cells. The biosensor was designed based on the crystal structures of the cellular 2-5A receptor RNase L 22, 24 ( Fig. 1A) , which indicate that the N-terminal ANK domain of RNase L is sufficient for 2-5A sensing and provides a minimal dimerization module 24 . In a single ANK protomer, the N-and C-termini in cis are separated, but upon dimerization of two ANK domains, which bind notably head-to-tail, the C-/N-termini become positioned in trans next to each other (Fig. 1B) . We employed this in cis vs in trans N-/C-distance decrease to drive a dual-split-luciferase sensor.