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

Only the L1-dependent Fluc expression was still strongly repressed. Presumably, low levels of scAlu RNPs might be sufficient to inhibit translation of this mRNA. We next studied the effect of Alu expression on global cellular translation by measuring total [ 35 S]methionine/cysteine incorporation following a 15 min pulse, and somewhat surprisingly, no significant changes were seen in [ 35 S]-incorporation in response to scAlu RNA expression ( Figure 6E , lower panel, −Ars). Additionally, protein analysis by SDS-PAGE did not reveal any noticeable changes in the expression of specific proteins at high levels of scAluY NF1 RNA (Supplementary Figure S8B) . We therefore reasoned that the Alu RNP-mediated decrease in active 40S subunits might severely hamper protein synthesis from mRNAs starting translation de novo such as the newly synthesized reporter mRNAs whereas protein synthesis from polysome-associated mRNAs remained largely unaffected. Ribosomal subunits are presumably recycled via a closed-loop structure in already existing polysomes (44) . To strengthen this hypothesis we performed two experiments. First, we expressed an estradiol-inducible luciferase reporter plasmid in HEK 293T cells along with the scAluY NF1 RNA-expressing or the control plasmid. Fluc mRNA synthesis was induced by the addition of estradiol to the culture medium at 24 h post-transfection and luciferase activity was monitored during the next 24 h. While luciferase expression increased rapidly in control cells, it was strongly delayed in scAluY NF1 RNA-expressing cells ( Figure 6D ) consistent with our working hypothesis that reduced concentrations of active 40S subunits hampered de novo initiation ( Figure 6D ).