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

Upon addition of the scAluY A RNP to either of the two subunits, we found one third of h9/14 comigrating with 40S subunits whereas less than 10% comigrated with the 60S subunits ( Figure 5A) . Remarkably, the scAluY A RNA migrated exclusively in the top fractions consistent with the notion that the 40S-bound h9/14 was dissociated from the Alu RNA. In contrast, free h9/14 bound quantitatively and nonselectively to both the 40S and the 60S subunits (Figure 5B) . We next examined the binding of the scAluY A RNP and h9/14 to 80S ribosomes. To this end, 80S ribosomes were allowed to associate from high salt purified 40S and 60S subunits for 10 min before the addition of the Alu RNP or h9/14. Only a fraction of the subunits assembled into 80S ribosomes in the presence of 1.5 mM magnesium acetate and individual subunits were therefore still present. Upon the addition scAluY A RNP we found h9/14 to be exclusively associated with 40S subunits whereas in the absence of the Alu RNA the protein bound nonselectively and was equally distributed between 40S and 60S containing fractions ( Figure 5C ). Hence, the presence of the Alu RNA appears to confer binding specificity to h9/14. This is further supported by the fact that all h9/14 bound quantitatively Nucleic Acids Research, 2015 , Vol. 43, No. 5 2883 to 40S subunits even when present in a 2-fold molar excess over 40S indicating that it has more than one binding site on the 40S subunit (Supplementary Figure S6) . In contrast the presence of 2-fold molar excess of scAluY A RNP over 40S did not increase the amount of the 40S-bound h9/14 suggesting that in this case the binding is saturable and occurs most likely to one specific site on the ribosome.