Figure 8.

ESEs and recombinant SF2/ASF stimulate translation of reporter mRNAs in vitro. (A,B) Insertion of the EDA ESE into the luciferase ORF stimulates translation in vitro. Constructs described in Figure 3A were transcribed in vitro. Reporter mRNAs (200 ng) containing either one or three copies of a wild-type or mutant version of the EDA ESE were incubated in a HeLa translation extract. Following incubation at 37°C, luciferase assays were performed (Promega). (C) Recombinant SF2/ASF stimulates expression of LCS reporter mRNAs in vitro. In vitro translation reactions were performed in the presence (black bars) or absence (white bars) of recombinant SF2/ASF (200 ng), as described above. (D) Insertion of the EDA ESE into the 3′UTR enhances translation of the luciferase reporter mRNA in vitro. Reporter mRNAs (100 ng) containing either six copies of a wild-type or mutant EDA ESE in the 3′UTR were incubated in HeLa translation extract. Following incubation at 37°C, luciferase assays were performed.