Figure 3. The ribosomal protein RACK1 is required for IRES-mediated translation.

(A) Stable S2 transformants expressing a shRNA targeting the 5′ UTR of RACK1 were transfected with vectors expressing three versions of RACK1 (WT, D108Y or R38D/K40E). Expression of the transfected RACK1 was monitored by western blot using an antibody recognizing the N-terminal tag HA. The cells were infected with CrPV for 16h, and viral RNA loads were determined by qRT-PCR. Data represent the mean and s.e.m. from three independent experiments. (B) RACK1 is required for translation regulated by the 5′ IRES, but not the intergenic (IGR) IRES, of CrPV. S2 cells were treated with dsRNAs corresponding to GFP (control), AGO2, eIF4E or RACK1 for 3 days, before transfection of the indicated Luciferase reporters (5′CAP, IRES_CrPV-IGR or IRES_CrPV-5′; see Fig. S2). Luciferase activity was monitored 48h later. The ratio of the activity of the IRES-dependent luciferase and the 5′ cap-dependent luciferase is plotted and normalized to the control for the three reporters. Data represent the mean and s.d. from six independent experiments. (C) Polysome profiles from S2 cells expressing or not a shRNA targeting the 5′ UTR of RACK1. The position of the peaks corresponding to the 80S ribosomes and the polysomes are indicated. (D) In vitro translation of capped and IRES-dependent reporters using cell free extracts prepared from control or RACK1-silenced S2 cells. Data represent the mean and s.d. from three independent experiments. ns: non significant, * p<0.05, ** p<0.01. See also Figures S2, S3.