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

Next, we examined whether the inactive scAluY A 14A5 and scAluY A 9-3A RNPs were able to mediate binding of the mutated proteins to the 40S subunit. Both Alu RNPs were incubated individually with 40S subunits and the complexes analyzed by sucrose gradients ( Figure 5E ). As observed in similar experiments with the protein alone (24), h9/14A5 was absent from the 40S fractions whereas h9-3A/14 bound as efficiently as the wild type protein to 40S subunits. These results indicated that SRP9/14 binding to the 40S subunit was required but not sufficient for Alu RNP-mediated inhibition of 48S complex formation. Supplementing the translation reactions containing scAluY A RNP with equimolar amounts of h9/14A5 did not significantly change the inhibitory activity of the scAluY A RNP ( Figure 5F, right panel) . However, a 2-fold excess of h9/14A5 over the scAluY A RNP diminished the inhibitory effect. Binding of h9/14 to Alu RNAs is characterized by high on and off-rates (25) . In the presence of an excess of h9/14A5 over the wild-type protein, the latter was presumably exchanged with the mutated protein forming the inactive scAluY A 14A5 RNP, which lead to a loss in the inhibitory activity of the RNP.