Rabbit reticulocyte lysate in vitro translation

mRNAs were in vitro translated with Flexi Rabbit Reticulocyte Lysate System from Promega, that is supplemented with calf liver tRNA³⁶. Reactions for luminescence assays were programmed with 3 nM mRNA and contained 30% RRL, 10 mM amino-acid mix minus methionine, 10 mM amino acid mix minus leucine, 0.5 mM MgOAc, 100 mM KCl, and 0.8 U µL⁻¹ Murine RNAse Inhibitor (NEB), and incubated at 30 °C for 30 min before termination by incubation at 4 °C. Reactions were then diluted 1:7 in Glo Lysis Buffer (Promega), and incubated 1:1 for 5 min in the dark in opaque 96-well plates with NanoGlo Substrate freshly diluted 1:50 in NanoGlo Buffer (Promega). Luminescence was measured on a GloMax 96 Microplate Luminometer.

For comparison of translation levels between m⁷G- and A-capped reporters, seven-molar excess of m⁷G-capped and polyadenylated FLuc mRNA was added to reactions as this has been shown to better recapitulate the endogenous cap and poly(A) synergy⁶⁸. For eIF4E competition assays, 250 μM free ARCA (m⁷G-cap) or A-cap was added to reaction mixture. For eIF4A inhibition, RRL mix was pre-incubated with 4 μM hippuristanol (a kind gift from Jerry Pelletier, McGill University), prior to addition of NLuc reporters and seven-molar excess FLuc mRNA.

Reactions for western blot assays were performed as above, except 50 ng mRNA was used. 10 μL reactions were mixed with 40 μL sample buffer and heated at 70 °C for 15 min, and 20 μL was run on a 12% polyacrylamide gel.

For precision protease (PSP) site cleavage, 4 μL RRL reaction was mixed either with 17.78 μM cycloheximide, 4 μL RNase-free water, and either 2 U PSP (GE Health Sciences) or vehicle, and incubated for 30 min at 30 °C, prior to processing for luminescence or western blot analysis.