FIG. 3.

SLBP stimulates translation of Luc-SL mRNA in vivo. (A) Schematic of the in vivo assay. Xenopus oocytes were injected either with buffer or with a synthetic mRNA encoding an SLBP (T₀). At 12 to 16 h later (T₁), the oocytes were injected with the reporter firefly luciferase mRNA (Luc-test) mixed with a synthetic mRNA encoding Renilla luciferase ending in the histone stem-loop (R-Luc-SL). After incubation for an additional 12 to 16 h (T₂), the oocytes were harvested and assayed for both luciferase activity and SLBP expression by Western blotting and by mobility shift assay with a radiolabeled stem-loop as the probe. The stability of reporter mRNAs was determined by S1 nuclease protection with a radiolabeled DNA fragment complementary to the 3′ end of the Luc-SL mRNA. (B) Oocytes were injected at T₀ with buffer (lanes 3, 4, 7, and 8) or with xSLBP1 (lanes 1and 2) or xSLBP2 (lanes 5 and 6) and then at T₁ with either the Luc-SL mRNA (lanes 1, 3, 5, and 7) or Luc-TL mRNA (lanes 2, 4, 6, and 8), together with R-Luc-SL mRNA. Oocytes were collected and lysed 8, 16, and 24 h later (T₂ = 8, 16, and 24 h). Total oocyte protein was resolved by gel electrophoresis, transferred to nitrocellulose, and xSLBP1 (lanes 1 to 4) or xSLBP2 (lanes 5 to 8) was detected with appropriate antibodies. Panel B shows the Western blots of samples collected at T₂ = 16 h. (C) Oocytes were injected with buffer (top), xSLBP1 mRNA (middle), or xSLBP2 mRNA (bottom). After 16 h, the oocytes were injected with the R-Luc-SL mRNA together with the Luc-SL, Luc-TL, or Luc-polyA mRNA. At 8, 16, or 24 h later, oocytes were harvested and assayed for Renilla and firefly luciferase activities. The ratio of Luc-TL luciferase activity to Renilla luciferase activity was determined, and this value was set at 1. The activity of the Luc-SL and Luc-polyA mRNAs was expressed relative to that of the Luc-TL mRNA.