FIG. 7.

Effect of xSLBP1 deletion mutant forms on translation activation in vivo. (A) Oocytes were injected with xSLBP1 deletion mutant mRNAs (Fig. 6A) or buffer at T₀. At T₁, they were injected with the R-Luc-SL mRNA together with the Luc-SL or Luc-TL mRNA. Oocytes were harvested at T₂ and assayed for Renilla and firefly luciferase activities as described in Fig. 3C. The results shown are the averages of two experiments with two different batches of oocytes, with the error bars representing the standard deviations. (B) One oocyte equivalent of lysate from oocytes injected with mRNAs encoding the mutant SLBPs described in panel A was mixed with 5 ng of radiolabeled stem-loop RNA and analyzed by gel electrophoresis. The xSLBP/SL complex was detected by autoradiography. (A) Oocytes injected with buffer (lanes 9 to 11) or mRNAs encoding mutant SLBPs containing the intact C-terminal region of xSLBP1 (lanes 3 to 8) were analyzed by Western blotting as described in Fig. 3B. (B) Total RNA from oocytes injected as described in panel A and harvested at T₂ = 32 h was hybridized to 3′-end-radiolabeled DNA complementary to the 3′ end of Luc-SL mRNA and subjected to S1 nuclease treatment. The protected DNA fragment was resolved by PAGE and detected by autoradiography. The Luc-SL mRNA protects a fragment 523 nt long that maps to the expected 3′ end, whereas Luc-TL mRNA protects a 508-nt fragment that maps to the start of the loop, where the sequences of Luc-SL and Luc-TL diverge.