FIG. 4.

xSLBP1 stimulates the translation of the Luc-SL and Luc-SLA8 mRNAs in vitro. (A) The seven reporter mRNAs used in the various translation experiments are represented at the top. A diagram of the in vitro translation assay (taken from reference 25) is shown in the lower panel. SLBP1 or SLBP2 was synthesized from capped synthetic mRNA in a reticulocyte lysate in the presence of [³⁵S]methionine. Aliquots of lysates containing xSLBP1 or xSLBP2 were added to fresh lysate together with luciferase reporter mRNA and an uncapped polyadenylated CAT mRNA and incubated for 90 min. (B) The uncapped Luc-TL (lanes 1 and 4), Luc-SL (lanes 2 and 5), or Luc-SLA8 (lanes 3 and 6) mRNAs were added to reticulocyte lysates containing xSLBP1 (lanes 1 to 3) or xSLBP2 (lanes 4 to 6) together with uncapped polyadenylated CAT mRNA. The translation products were resolved by SDS-polyacrylamide gel electrophoresis and detected by autoradiography. There was a slight increase in the translation of the Luc-TL mRNA in the presence of xSLBP2 in the experiment whose results are shown, but this was not observed in multiple other experiments; in those experiments, there was essentially no effect seen on translation of either the Luc-TL or Luc-SL mRNAs by SLBP2 in the reticulocyte lysate (25). SL, stem-loop. (C) The reaction mixtures described for panel B were analyzed for luciferase activity; the activity levels are expressed relative to the Luc-TL mRNA levels.