Translational repression of Fer mRNA and efficient utilization of ALAS-E mRNA in differentiating mouse erythroblasts. Self-renewing (designated “proliferation” in this and the following panels) or differentiating (labeled “48h diff”) primary mouse erythroblasts were incubated with the iron chelator desferrioxamine (Des, 50 μM) or physiologic concentrations of iron-loaded human transferrin (Tf, 12.5 μM) for 24 hours prior to harvesting. (A) Polysome gradient analysis. Cytoplasmic extracts were separated in linear 15% to 40% sucrose gradients³⁹  and the RNA isolated from 18 fractions analyzed by Northern blotting. Fraction 1, top, fraction 18, bottom of the gradient. Filters were sequentially hybridized with [³²P]-labeled probes specific for mouse FerH, FerL, ALAS-E, and (in the case of differentiating cells) α-globin mRNA as control. Bottom panel, loading control; methylene blue stain of total RNA. The constant molar ratio between 28S and 18S RNA (top and bottom band, respectively) around fraction 9 indicates the assembly of 80S initiation complexes and marks the approximate boundary between the ribosome-free, untranslated, and polyribosome-bound, translated mRNA compartment, as schematically depicted at the bottom. (B) Quantification of polysome-bound, translated mRNA. Bar diagrams depict the sum of the percentage of mRNA in fractions 9-18 as determined by PhosphoImage analysis. (C) Fer protein expression in proliferating and differentiating cells. The antibody used (see “Materials and methods”) recognizes both FerH and FerL. (D) ALAS-E expression as determined by immunoprecipitation of cell extracts (normalized to equal number of counts per sample) pulse labeled for 20 minutes with [³⁵S]-methionine; to visualize the ALAS-E band in proliferating cells, this signal was amplified electronically 5 times. (E) Total mRNA levels for ALAS-E, FerH, and FerL mRNAs. Loading and quality control, 28S rRNA stained with methylene blue.