Concerted 2-5A-Mediated mRNA Decay and Transcription Reprogram Protein Synthesis in dsRNA Response

Sneha Rath; Eliza Prangley; Jesse Donovan; Kaitlin Demarest; Yigal Meir; Ned Wingreen; Alexei Korennykh

Cells in 10 cm dishes were flash frozen in liquid nitrogen, scraped in cold PBS with 100 g/ml CHX and pelleted at 500 x g for 5 minutes at 4°C. The cell pellet was lysed in 5 mM HEPES, 1.5 mM KCl, 2.5 mM MgCl 2 , 100 g/ml CHX, 1x Protease inhibitor cocktail, 100 U/mL RNase inhibitor (NEB), 0.5 % Triton X-100, and 0.5 % Na-Deoxycholate. The lysate was vortexed, rotated end-over-end for 7 minutes at 4°C and centrifuged at 10,000 x g for 10 minutes at 4 °C. Clarified lysate was layered over a 12 ml 10-50 % sucrose gradient made by GradientMaster (BioComp). The 10 % and 50 % sucrose solutions were made with 20 mM HEPES, 100 mM KCl, 5 mM MgCl 2 , 100 µg/mL CHX, 1x Protease inhibitor cocktail, and 100 U/ml RNase inhibitor. In experiments designed to distinguish mRNA bound 80S vs empty 80S complexes, both lysis and sucrose gradient buffers were adjusted to a final concentration of either 100 mM or 500 mM KCl. To create empty 80S as a control, WT cells were pre-treated with 50 µg/mL puromycin for 20 min and the sucrose gradient buffer also contained 50 µg/ml puromycin in place of CHX. The lysate was spun through the gradient in an SW41Ti rotor in an Optima XE-100 Ultracentrifuge (Beckman Coulter) at 200,000 x g for two hours at 4 °C. BioComp Gradient Fractionator was used to fractionate the gradients and the 254 nm absorbance was read by an EM-1 ultraviolet monitor (BioRad).