Fig. 1. Tethered PAB causes translational stimulation. (A) The assay has two components: a luciferase reporter mRNA with binding sites for the MS2 coat protein within its 3′-UTR (Luc-MS2), and a fusion between MS2 coat protein and Xenopus PAB. Binding of coat protein to its sites tethers PAB to the mRNA. The effects of fusion proteins are measured by luciferase assay. (B) A luciferase reporter mRNA with MS2-binding sites within its 3′-UTR (Luc-MS2) was utilized. mRNAs encoding β-galactosidase (β-Gal) or luciferase (Luc-ΔMS2) that lacked MS2-binding sites were used as control mRNAs. Luciferase mRNAs lack poly(A). (C) Oocytes expressing MS2 or MS2–PAB mRNA were co-injected with Luc-MS2 and β-Gal mRNAs as a mixture. The translation of the reporter mRNAs was determined by luciferase and β-galactosidase assays. Luciferase activity (normalized for differences in the amount of β-galactosidase activity) was plotted. A representative experiment is shown. (D) The stability of ³²P-radiolabeled luciferase mRNA after incubation for 14 h in oocytes expressing either MS2 or MS2–PAB from (C) is shown (lanes 3 and 4). RNA was also harvested from oocytes immediately following injection of luciferase mRNA (lanes 1 and 2). 18S rRNA (lower panel) is shown as a loading control.