FIGURE 5.

Mutations in catalytic amino acids of Xenopus CAF1 abolish deadenylase activity but not translational repression. A and B, the relative translation of luciferase activity in response to HA:MS2:CAF1a (A) or HA:MS2:CAF1b (B) wild type or catalytic mutant was quantified as the ratio of firefly to Renilla luciferase activity. The reporter used in the experiment is indicated. Normalization and error bars were determined as for Fig. 1B. Bottom panel, lysates equivalent to two oocytes were loaded in each lane and analyzed by Western blotting with α-HA-11 and α-actin. C and D, poly(A) status of reporter RNA. Lanes 1 and 3 show the ³²P RNA without a poly(A) before and after injection into oocytes, respectively. Lanes 2 and 4 show the ³²P RNA with a poly(A) tail before and after injection into oocytes, respectively. Lanes 5 and 6 indicate poly(A) tail length of the ³²P RNA when tethered to HA:MS2:CAF1a (C) or HA:MS2:CAF1b (D) wild type or catalytic mutant, respectively. A t test was performed on at least four replicate experiments in A and B. No statistical difference was found for any of the values except CAF1b wild type and mutant forms with the adenylated reporter mRNA (p value < 0.005).