The Translation Regulatory Subunit eIF3f Controls the Kinase-Dependent mTOR Signaling Required for Muscle Differentiation and Hypertrophy in Mouse
Figure 7
eIF3f regulates cap-dependent translation.
(A) Structure of the bicistronic reporter plasmid allowing cap-dependent expression of renilla luciferase and expression of firefly luciferase dependent on HCV IRES. (B) Overexpression of eIF3f modulates cap-dependent translation. Mouse primary cultured satellite cells were cotransfected with the bicistronic reporter vector and expression vectors encoding HA-tagged eIF3f wt and the mutant eIF3f K5–10R. Twenty-four hours posttransfection cells were grown for an additional 24h in 20% serum (control), stimulated with insulin or pretreated with rapamycin and stimulated with insulin for and additional 24 h. Cells transfected to express eIF3f wt or the mutant eIF3f K5–10R were grown in 20% serum. Luciferase activities were measured by a dual-luciferase assay. The ratio of Renilla (Cap-dependent) to Firefly (IRES-dependent) luciferase activity was calculated. Data are presented as the mean ± standard error from three independent experiments carried out in triplicate, *P<0,05 compared to control; #P<0,05 compared to Insulin + rapamycin. (C) Mouse primary cultured satellite cells were transfected with expression vectors as described in (B) and/or subjected to shRNAi-mediated silencing of eIF3f prior to labeling new protein synthesis with 35S methionine. Newly synthesized proteins were separated by SDS-PAGE, and visualized by autoradiography. (D) Newly synthesized proteins from three experiments as in (C) were quantified. *P = 0,002 and #P<0,001 compared to control; †P>0,001 compared to shRNAi eIF3f. (E). Model depicting the central role of eIF3f in the signaling pathways controlling skeletal muscle mass.