Skip to main content
. 2016 Jan 12;291(11):5721–5739. doi: 10.1074/jbc.M115.682161

FIGURE 4.

FIGURE 4.

Knockdown of ribosomal proteins reduces ribosomal biogenesis depleting neuronal ribosomes. A, COS-7 cells were co-transfected with the anti-ribosomal shRNAs together with the expression vectors for their respective targets that were tagged with the GFP variants EGFP or Venus (2 + 2 μg of plasmid DNA/60-mm plate). Control shRNA was against Renilla luciferase (shLuc); to normalize for transfection efficiency, a β-galactosidase (β-gal) expression plasmid was also included (1 μg of plasmid DNAs/60-mm plate). Western blot for GFP revealed efficient knockdown of the targets 48 h post-transfection. B, DIV6, hippocampal neurons were co-transfected with the indicated shRNAs together with expression vectors for their tagged targets as well as β-gal (0.4 + 0.2 + 0.2 μg of plasmid DNAs/2 × 105 neurons, respectively). Equimolar mixes of individual shRNAs against S6 or L4 were used in these and all further experiments unless indicated otherwise. At DIV9, transfected cells were visualized by immunostaining for β-gal, and their EGFP- or Venus-positive fraction was determined. Note a decrease in EGFP-/Venus-positive cells after knockdowns of the respective RPs; means ± S.D. of four sister cultures from two independent experiments are depicted. C and D, effects of anti-ribosomal shRNAs on ribosome biogenesis. DIV6 hippocampal neurons were co-transfected with the shS14 and S6-EGFP or shS6 and L4-EGFP as indicated (0.4 + 0.15 μg of plasmid DNA/2·105 neurons, respectively). On DIV9 the cells were fixed, and ribosome distribution was monitored with GFP immunostaining; Hoechst 33258 was used to visualize nuclei. C, representative images of S6-EGFP- or L4-EGFP- expressing cells. In an shLuc-transfected neuron, RPs are present in the nucleolus, the perikaryal cytoplasm and proximal dendrites as expected for a ribosome marker (compare with Fig. 2C). The shS14 or shS6 increased nuclear retention of RP-EGFP fusion proteins. D, quantification of GFP fluorescence intensity in the nucleus and the perikaryal cytoplasm. Note reduced cytoplasmic/nuclear ratio of ribosomal markers after transfection of shS14 or shS6. Data represent means ± S.E. of at least 30 cells from two independent experiments; **, p < 0.01 (ANOVA, effects of shS14 or shS6, F1,99 = 7 or F1,58 = 10.4, respectively).