FIG. 6.
Differential regulation of the synthesis of the 51- and 48-kDa FLI-1 isoforms by the conserved region −49/−33 is mediated by upstream codons, AUG −41 and GUG −37. Capped or uncapped versions of Fli-1 mRNA with or without mutations of AUG −41 or GUG −37 codons or with both mutations were synthesized by in vitro transcription. Equal amounts of each of these synthetic Fli-1 mRNAs were used to program rabbit reticulocyte lysates in the presence of [35S]methionine. Translation products were separated by SDS-PAGE. Labeled translation products were then visualized by autoradiography, and radioactive signals corresponding to the 51- and 48-kDa proteins were quantified using a Molecular Imager. (A) Schematic representation of synthetic Fli-1 mRNA. Point mutations are underlined. The two short 5′ uORFs beginning at AUG −41 or GUG −37 and ending, respectively, at UGA +35 and UAA +15 are indicated by arrows under the drawing of the wild-type mRNA. Similarly, the two in-frame FLI-1 ORF starting at AUG +1 or AUG +100 and ending at UAG +1356 are indicated by arrows above the drawing of the wild-type mRNA. (B) Autoradiogram of the gel. (C and D) Graphical representation of the results obtained with uncapped and capped versions of the same mRNA. Results are expressed as the percentage of 51-kDa (white bars) or 48-kDa (hatched bars) isoforms produced by the indicated mutated mRNA compared to the wild type (means and standard deviations of three different experiments).