FIG. 6.
Rabbit α1-globin52-57/human CK791-96 SEQIKA binds to, prevents decay of, and blocks translation of HPV16 E7 mRNA. (A) SEQIKA fragment binds to E7 mRNA. PEMSA experiments were run by incubating unlabeled c-myc, eIF-4E, or HPV16 E7 mRNA with biotinylated control peptide LPGALS or SEQIKA. Following electrophoresis, the biotinylated peptide-mRNA complex was visualized by ECL. (B) SEQIKA fragment promotes E7 mRNA stability. c-myc, eIF-4E, and HPV16 E7 mRNA decay was assayed in vitro with the p32-depleted rabbit reticulocyte lysate obtained following incubation with E7 mRNA-conjugated magnetic beads plus the 6-mer peptide SEQIKA or LPGALS. (C) Quantification of mRNA half-life by densitometric analysis. ODu, optical density units. (D) SEQIKA fragment prevents HPV16 E7 mRNA translation promoted by anti-p32 PAbs in complete rabbit reticulocyte lysate. Translation reaction mixtures were preincubated with anti-p32 PAbs (50 μg) and/or peptide (10 μg) for 10 min prior to mRNA addition. Translation reactions were electrophoresed in SDS-12% PAGE. Following electroblotting onto a polyvinylidene difluoride membrane, E7 oncoprotein expression was analyzed by Western blotting with anti-E7 MAb.