Fig. 3. Poly(A) polymerase activity of Wisp in a tethering assay and during oogenesis. (A,B) Tethering assay in Xenopus oocytes. (A) Wisp and a control protein, HsGLD-2, were tethered to luciferase reporter mRNA using MS2. β-galactosidase-encoding mRNA lacking MS2 binding sites was used as an internal control. Translational stimulation was assayed by measuring luciferase activity. Wild-type and point-mutant (DA) proteins were expressed at similar levels, as determined by western blotting with anti-HA₁₁ (bottom). (B) 130 nt, ³²P-labeled RNA was injected and then purified from oocytes. (Lanes 1-4) Tethered HsGLD-2 and Wisp added long poly(A) tails onto labeled RNA. Active site mutations disrupted elongation. (Lanes 5-8) Tails added by wild-type Wisp were removed by RNase H/oligo(dT) treatment, confirming that they were poly(A). -, RNase H only; +, RNase H plus oligo(dT). (Lanes 9-12) RNAs elongated by Wisp were bound to an oligo(dT) column. -, RNAs that did not bind; +, RNAs that bound. (C) PAT assays measuring osk, CycB, nos and bcd poly(A) tail lengths in Drosophila egg chambers of different stages (germarium to stage 8, stages 9 and 10, and stage 14) from wild-type and wisp^KG5287 ovaries. sop mRNA, which encodes a ribosomal protein and is not regulated by cytoplasmic polyadenylation, was used as a control.