Expression of proteins encoded by mRNAs with long 5′ UTRs, but not those harboring short 5′ UTRs/TISU elements, is dependent on EIF4A1. (A) HEK293E and MCF7 cells were mock-transfected or transfected with siRNA targeting EIF4A1 (si-EIF4A1), EIF4E (si-EIF4E), or scrambled control siRNA (Scr). Levels of indicated proteins were determined 48 h post-transfection by Western blotting. ACTB served as a loading control. Experiments were carried out in independent triplicate and quantified using densitometry (Supplemental Fig. 9). (B) Total levels of indicated mRNAs isolated from cells described in A were determined by RT-qPCR. Data were log2 transformed, and values obtained for indicated mRNAs were normalized to those obtained for ACTB and to the mean expression per gene. (C) Subpolysomal, light polysome, and heavy polysome fractions were obtained from cytosolic extracts from cells described in A by ultracentrifugation using 5%–50% sucrose gradients. During fractionation, UV absorbance at 254 nm (Abs 254 nm) was continuously monitored to obtain absorbance tracings. Positions of 40S and 60S ribosomal subunits, monosome (80S), and polysomes are indicated. (D) Amount of indicated mRNAs in subpolysomal, light polysome, and heavy polysome fractions isolated from cells described in C were determined by RT-qPCR. Experiments in panels B and D were carried out in independent duplicates, each consisting of a triplicate. Data are expressed as a percentage of a given mRNA in each fraction. Bars represent SD values. P-values from one-way ANOVAs for heavy polysomes are indicated.
