RanBP2/Nup358 Potentiates the Translation of a Subset of mRNAs Encoding Secretory Proteins
Figure 6
RanBP2 is required for ALREX-mediated translation enhancement.
(A) U2OS cells were infected with lentivirus containing shRNA1 directed against RanBP2 or control virus. Four days post-infection, cell lysates were collected, separated by SDS-PAGE on a 6% acrylamide gel, and immunostained for RanBP2 or α-tubulin. (B–E) U2OS cells were infected with lentivirus that delivered shRNA1 against RanBP2 or control virus. Three days post-infection, cells were transfected with plasmids containing various ftz constructs and H1B-GFP. 18–24 h post-transfection the level of protein was analyzed by immunoblot (B, quantification in C) and mRNA by northern blot (D, quantification in E). Each bar represents the average and standard error of six independent experiments. *, p<0.025; **, p<0.01. (F) Three days post-infection, U2OS cells were transfected with plasmid containing MHC-ftz. 18–24 hr post-transfection the amounts of MHC-ftz mRNA found in the cytoplasm and nucleus was determined by FISH. One bar represents an average of three experiments, each of which consists of 20–30 cells. Error bars represent the standard deviation between the three experiments. (G) U2OS cells were treated with various lentiviruses. Four days post-infection, cells were fixed and stained for poly(A) mRNA by FISH. The average levels of poly(A) mRNA in the cytoplasm or nucleus were plotted. One bar represents an average of three experiments, each of which consists of 20–30 cells. Error bars represent the standard deviation between the three experiments. (H) UAP56 and URH49 were simultaneously depleted in U2OS cells by lentiviral mediated delivery of shRNAs. Four days post-infection, cell lysates collected, separated by SDS-PAGE, and immunostained for these two proteins and for α-tubulin. (I–K) U2OS cells were infected with lentivirus containing shRNA directed against the 3′ UTR of RanBP2 (shRNA3) or control virus. (I) Three days post-infection, cells were transfected with plasmids containing GFP-RanBP2, or GFP-RanBP2Δ6ZFR or without plasmid (“Mock”). After allowing expression for 48 h, cell lysates were collected and detected by immunoblot using antibodies against GFP, RanBP2, and α-tubulin. Note that the GFP-RanBP2 constructs do not contain the endogenous UTRs and are resistant to depletion by shRNA3. (J–K) Three days post-infection, cells were co-transfected with plasmids containing MHC-ftz and either GFP-RanBP2, GFP-RanBP2Δ6ZFR, or H1B-GFP and allowed to express for 48 h. Cells were immunostained using anti-GFP and anti-HA primary antibodies, and the appropriate fluorescent secondary antibodies. (J) For cells co-expressing GFP and MHC-ftz, the HA-immunofluorescence intensity was tabulated, averaged, and then normalized to the level of expression in mock, control shRNA-treated cells expressing MHC-ftz. Each bar represents the average and standard error between four independent experiments, each of which consists of 30–50 cells. Examples of cells depleted of endogenous RanBP2 with shRNA3 and expressing various GFP-RanBP2 constructs and MHC-ftz are shown in (K). Each row is a single field co-immunostained for GFP and HA. Note that cells expressing GFP-RanBP2 (top row, arrows), express higher levels of MHC-ftz than neighboring cells that express MHC-ftz alone (top row, arrowheads). In contrast, cells expressing GFP-RanBP2Δ6ZFR (bottom row, arrows) express about as much MHC-ftz protein as cells expressing MHC-ftz alone (bottom row, arrowheads). Un-transfected cells are denoted by asterisks. Scale bar = 20 µM.