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

All of the effects of 2-5A arise from the action of a single mammalian 2-5A receptor, pseudokinase-endoribonuclease L (RNase L) 20 . 2-5A binds to the ankyrinrepeat (ANK) domain of RNase L and promotes its oligomerization and formation of a dimeric endoribonuclease active site [21] [22] [23] . This dimer further assembles into high-order oligomers 24 that cleave viral RNAs 16, 18 and all components of the translation apparatus, including mRNAs 25 , tRNAs 26 and 28S/18S rRNAs 27, 28 . The resulting action of RNase L inhibits global translation, which puts all proteins, including IFNs, at risk of arrest during a cellular response to dsRNA. The impact of translational shutdown by RNase L on IFN synthesis and paracrine IFN signaling is unknown. Measurements of 2-5A/RNase L activity have been limited by the need for biochemical analysis, which are incompatible with live cells. To address this challenge, we developed a realtime 2-5A biosensor and used it to elucidate the kinetics of 2-5A-mediated RNase L activation and translational arrest taking place during the cellular response to immuno-stimulatory dsRNAs. Our biosensor can detect in situ 2-5A synthesis in mammalian cells and thereby it establishes a heretofore missing platform for cell-based applications. These applications can range from live cell screens for modulators of innate immune responses to mechanistic analysis of dsRNA sensing, which we describe in our present work.