Polysome fractionation

HeLa cells were seeded in four to eight 10‐cm dishes per condition. Twenty‐four hours after plating, cells were transfected with siRNAs against DDX3X or EGFP at 1.6 nM using RNAiMAX, as above, with the media exchanged at 5 h post‐transfection. When cells reached 70–90% confluent, 24–36 h post‐knockdown, they were treated with 100 μg/ml cycloheximide (CHX) for 5 min at 37°C. Cells were then transferred to ice and washed with 2.5 ml ice‐cold PBS containing 100 μg/ml CHX, collected by scraping in 2.5 ml cold PBS + CHX, and pelleted at 234 g and 4°C for 5 min. PBS was aspirated and pellets re‐suspended in polysome‐profiling lysis buffer (20 mM Tris–HCl (pH 7.5), 150 mM NaCl, 15 mM MgCl₂, 8% (vol/vol) glycerol, 20 U/ml SUPERase, 80 U/ml murine RNase inhibitor, 0.1 mg/ml heparin, 100 μg/ml CHX, 1 mM DTT, 1× EDTA‐free protease inhibitor cocktail, 20 U/ml Turbo DNase, 1% Triton X‐100) 113. Lysates were passed through a 20G needle 10× and incubated on ice for 5 min. Cellular debris was pelleted at 14,000 g and 4°C for 5 min, and supernatant transferred to a fresh tube. Total lysate RNA was estimated by NanoDrop. Lysates were flash‐frozen in liquid N2 and stored at −80°C until fractionation.

Sucrose gradients were prepared by successively freezing equal volumes of 50, 36.7, 23.3, and 10% sucrose (wt/vol) in 12‐ml Seton tubes. Sucrose‐gradient buffer consisted of 20 mM Tris–HCl (pH 7.5), 150 mM NaCl, 15 mM MgCl₂, 10 U/ml SUPERase, 20 U/ml murine RNase inhibitor, 100 μg/ml CHX, and 1 mM DTT (Simsek et al 113). Prior to use, gradients were allowed to thaw and linearize overnight at 4°C (Luthe, Analytical Biochemistry, 1983). For fractionation, approximately 90 (trial 1 with four 10‐cm dishes), 220, and 250 μg (trials 2 and 3, respectively, with eight 10‐cm dishes) total RNA was applied to the top of the sucrose gradient. Gradients were spun at 151,263 g and 4°C for 3 h using a Beckman Coulter Optima L‐90K ultracentrifuge and SW 41 Ti swinging‐bucket rotor.

Gradients were fractionated with Brandel's Gradient Fractionation System, measuring absorbance at 254 nm. The detector was base‐lined with 60% sucrose chase solution, and its sensitivity set to 0.5 for trial 1, and 1.0 for trials 2 and 3. For fractionation, 60% sucrose was pumped at a rate of 1.5 ml/min. Brandel's PeakChart software was used to collect data, overlay profiles, and calculate the area under the curve for monosome and polysome fractions.