Pum2 represses translation in a W344-dependent manner. RNAs encoding Pum2 WT and W344G fused to λ N protein were injected into 10 oocytes. Following an overnight incubation, the oocytes were then injected with luciferase RNA containing 5 B boxes in the 3′ UTR. The oocytes were then homogenized and luciferase activity was determined (histogram) as was expression of the fusion proteins by Western blotting (bottom). The top portion shows an autoradiogram indicating that the relative levels of 32P-UTP trace-labeled luciferase RNAs at the end of the incubation period were similar. Other RNAs encoding Pum2 WT and W344G (no fusion) were injected into oocytes with luciferase RNA, whose 3′ UTR contained or lacked a PBE. Luciferase activity, expression of the heterologous proteins, and relative amount of the luciferase reporter RNAs that remained after the incubation period were determined as above. The amount of luciferase activity in the absence of heterologous Pum2 was used as the standard against which the other values were normalized. Each experiment was performed three times; the bars on the histograms refer to SEM. The data are statistically significant (P < 0.05, Student's t-test).
