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 determine whether V6 was sufficiently bright and stable in live cells, we expressed FLAG-tagged V6 in HeLa cells and stimulated these reporter cells with poly-IC. Poly-IC treatment conditions were selected to produce cleavage of 28S rRNA in the conventional end-point assay using cell disruption 22 . 28S rRNA cleavage was noticeable after one hour of poly-IC treatment and further increased after three and four hours ( Further evidence for specific 2-5A detection was obtained in cells with 2-5A synthetases knocked out. It has been shown that knockout of OAS3 is sufficient to inhibit 2-5A synthesis in human cells 11 . We found that biosensor exhibited a robust response to poly-IC in OAS1-KO and OAS2-KO cells. In contrast, the response was lost in OAS3-KO cells ( fig. S2 ), providing a confirmation that 2-5A synthesis gave rise to the reporter activity. RNase L may be activated by local production of 2-5A 30 , at secrete sites inside cells (the cytosol). Notably, OASs are present not only in the cytosol, but also in the nucleus [31] [32] [33] , which further supports that 2-5A accumulation could be nonuniform with subcellular spaces. The availability of live cell 2-5A sensor offers an opportunity to evaluate 2-5A accumulation in individual cellular compartments in situ.