Figure 7.

Nucleolin and Importin β1 Localization Regulate Protein Synthesis

(A) The translational activity of DRG neurons in culture was assessed by puromycin incorporation. Cultures were grown in the presence of AS1411 or control aptamer for 48 hr, and then they were replated and cultured for an additional 24 hr in fresh medium without aptamer. Neurons were then pulsed with 5 mM puromycin for 10 min at 37°C or preincubated with 40 mM anisomycin for 30 min followed by the 5 mM puromycin pulse, and then they were fixed. Fixed cultures were immunostained for NFH (green) and α-puromycin (red). Scale bar, 100 μm. For anisomycin control, see Figures S7E and S7F.

(B) Representative high-sensitivity zoom images of the boxed regions in (A) reveal protein synthesis in axon tips. Scale bar, 20 μm. Quantification reveals a significant decrease in protein synthesis in axon tips of AS1411-treated WT neurons, as well as in importin β1 3′ UTR^−/− neurons. Axon tip synthesis was quantified as ratios of cell body values and then normalized to WT control. Mean ± SEM; n ≥ 80 cells from three independent cultures; ^∗∗p < 0.01 and ^∗∗∗p < 0.001, Student’s t test.

(C) Representative images of cultured 3T3 cells treated with 10 μM control or AS1411 aptamer for 48 hr and then replated and cultured for an additional 24 hr in fresh medium without aptamer. The cells subsequently were incubated with puromycin with or without anisomycin as described above, and then they were fixed and stained for F-Actin, DAPI, and α-puromycin. Scale bar, 50 μm.

(D) Quantification of puromycin labeling in the cytoplasm of 3T3 cells from the experiment described in (C) reveals a significant decrease in protein synthesis at the cell periphery in AS1411-treated cells. Mean ± SEM; n ≥ 200 cells from five independent cultures; ^∗p < 0.05, one-way ANOVA with Bonferroni post hoc test.

(E) Schematic model of the mechanism proposed in this study. Nucleolin binds importin β1 and likely other mRNAs, and the complex is transported by a kinesin motor to the axon in a neuron or the cell cortex in cycling cells. Upon arrival at the end of the microtubules, the complex is dissassembled, with nucleolin likely docking to the plasma membrane. Local translation of the cargo RNAs generates proteins that are retrogradely transported with dynein to influence protein synthesis in the soma. The dashed line indicates a negative feedback loop postulated in the original model (Rishal et al., 2012), the details of which are still unknown.

See also Figure S7.