Fig. 5.

Identification of poly(A) polymerases. (A) Translational activity. Assays were performed as in Fig. 2 A. Relative luciferase activity (ratio of luciferase to β-galactosidase activity) was normalized to that of GLD-2(mut). (B) Protein levels. Cell lysate equivalent to three oocytes was loaded onto each lane and analyzed by Western blotting using anti-HA antibody. Shown on the left are positions of protein markers used to calibrate molecular mass. (C) Poly(A) status of reporter mRNAs. ³²P-labeled luciferase mRNA was injected into oocytes expressing the indicated fusion proteins. RNA was extracted from oocytes after 16 h and fractionated by using biotinylated oligo(dT). –, RNAs that did not bind the resin; +, RNAs that bound the resin. (D) Polyadenylation of short RNA substrates. A ³²P-labeled RNA containing three MS2 sites was injected into oocytes expressing the MS2 fusion proteins indicated above each lane. RNA was extracted after 16 h and analyzed by electrophoresis. Shown on the left are positions of RNAs with various lengths of poly(A) (determined by comparison to markers). (E) Oligo(dT) retention. RNAs containing three MS2 sites were injected into oocytes expressing the indicated fusion proteins. Extracted RNAs were fractionated by using biotinylated oligo(dT). –, RNAs that did not bind the resin; +, RNAs that bound the resin. Left, positions of RNAs with various lengths of poly(A) (determined by comparison to markers). Input, RNA before injection. (F) Oligo(dT)/RNaseH. RNAs containing three MS2 sites were injected into oocytes expressing the indicated fusion proteins. Extracted RNAs were incubated with oligo(dT) and RNaseH. –, no RNaseH; +, RNaseH added. Arrowheads indicate positions of the injected RNA and of the same RNA with a 300-nt tail. Input, RNA before injection.