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

RRL (Green Hectares, Oregon, USA) was treated with micrococcal nuclease as described (49) with some modifications: micrococcal nuclease concentration was decreased to 75 U/ml final and incubation time was reduced to 10 min at 20 • C. Translation reactions containing L-[ 35 S]-methionine (Anawa, Zurich, Switzerland) were carried out in the presence of 100 mM potassium acetate and 1.5 mM magnesium acetate in a final volume of 10 l at 30 • C for 20 min. Translation mix was preincubated with Alu RNPs, Alu RNAs or h9/14 for 3 min at 30 • C prior to addition of 125 ng mRNA. For edeine-synchronized translation the reaction programmed with 125 ng of uncapped pPL mRNA was first incubated for 3 min at 30 • C and then the initiation was arrested by the addition of edeine to 0.5 M. The reaction was incubated one more minute before the addition of 100 nM Alu RNPs and translation continued for 15 min. Synthesized polypeptides were resolved by SDS-PAGE and quantified by phosphorimager analysis. For toeprint assays, the mRNA was incubated with fluorescently labeled oligonucleotide for 10 min at 37 • C before addition to the assembly reaction. The 80S complex was assembled by incubating the 48S complex with 200 nM eIF5, 200 nM eIF5B and 100 nM 60S subunits at 37 • C for 10 min. Peptide elongation reaction and assembly of preTC was performed by incubation of the 80S complex with 300 nM eEF1, 50 nM eEF2 and 100 nM aminoacylated total calf liver tRNA for 10 min at 37 • C. All assembly reactions were performed in buffer containing 20 mM Tris-HCl pH 7.5, 120 mM KCl, 2.5 mM MgCl 2 , 2 mM DTT, 0.25 mM spermidine, 0.1 mM ethylenediaminetetraacetic acid (EDTA), 2 mM adenosine triphosphate (ATP )and 0.2 mM guanosine triphosphate (GTP).