Alu RNA regulates the cellular pool of active ribosomes by targeted delivery of SRP9/14 to 40S subunits

Ivanova, Elena; Berger, Audrey; Scherrer, Anne; Alkalaeva, Elena; Strub, Katharina

HEK 293T cells were transfected with TransIT-T2020 reagent (Mirus) according to manufacturer's protocol. Luciferase assays were performed 24 h post-transfection using Nucleic Acids Research, 2015 , Vol. 43, No. 5 2877 Luciferase Assay Kit (Promega). For estradiol-induced luciferase expression, HEK 293T cells were transiently transfected with a GAL-responsive luciferase reporter plasmid and with a plasmid expressing the protein GAL4 fused to the hormone binding domain of human estrogen receptor. Cells were stimulated with 100 nM estradiol at 24 h post-transfection and bioluminescence was recorded continuously for 24 h. Metabolic labeling with [ 35 S]methionine/cysteine was performed 48 h post-transfection. After 15 min, pulse labeling cells were washed twice with ice-cold phosphate buffered saline and lysed in buffer containing 20 mM Tris-HCl pH 7.5, 300 mM KCl, 1% Triton X-100, 0.1% SDS, 1 mM EGTA and protease inhibitors. Aliquots of lysates were precipitated with 10% TCA, pellets were boiled for 15 min washed twice with 10% TCA followed by acetone, dissolved in 1% SDS and analyzed by scintillation counting. Where indicated, cells were treated with 500 M sodium arsenite for 30 min then the media was changed and cells were allowed to recover for 0, 30, 60, 90 and 120 min prior to pulse labeling.