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

An internal ribosome entry site (IRES)-containing dual luciferase plasmid was a gift from Dr. Paul Copeland (Rutgers University). Monocistronic firefly luciferase was obtained by PCR amplification of the coding region from the dual luciferase construct and cloning into BamHI/NotI digested pcDNA3.1. Plasmids were linearized with AgeI (firefly luciferase) or BamHI (dual luciferase) and purified by phenol extraction and ethanol precipitation. Capped mRNAs were transcribed using reagents from the MEGA ShortScript Kit, except for nucleoside triphosphates, and 12 mM anti-reverse cap analog (NEB). NTPs were added using a custom 10X mixture containing 75 mM each of ATP, UTP, and CTP, and 15 mM GTP. Transcription was carried out for two hours at 37 °C followed by addition of Turbo DNase I and incubation for 20 minutes at 37 °C.