FIG. 1.

pol II ΔCTD does not support cotranscriptional pre-mRNA processing and does not inhibit processing uncoupled from transcription in Xenopus oocytes. (A) Maps of the CMV β-globin gene and capped (7MeGpppG) pre-mRNAs with and without synthetic poly(A) site (SPA, striped box) in exon 3. Poly(A) cleavage sites βpA and SPA are marked by downward arrows. Capped CAT pre-mRNA containing the SV40 t-intron is shown at the bottom. Antisense RNase protection probes are marked below the maps. (B) Western blot of Rpb1WT^R (WT^R) and Rpb1ΔCTD^R (ΔCTD^R) expressed in oocytes detected with antibody against the B10 epitope tag. Hyper- and hypophosphorylated wild-type pol II0 and pol IIA are indicated. (C) RPA of CMV β-globin transcripts in α-amanitin-injected oocytes expressing α-amanitin-sensitive (WT^S) or -resistant Rpb1 with full-length (WT^R) or deleted CTD (ΔCTD^R) with intron 1 and poly(A) site probes. VA RNA transcribed by pol III is a control for injection efficiency. The CMV β-globin (0.2 ng in 4.6 nl) and VA plasmids were coinjected with α-amanitin 15 h after injection of the Rpb1 expression plasmids (0.2 ng in 4.6 nl). Percent spliced or cleaved relative to total RNA (see Materials and Methods) is noted below lane numbers. Values too small to be accurately determined are labeled ND. (D) RPA of β-globin SPA pre-mRNA coinjected with α-amanitin into oocytes expressing Rpb1WT^R or Rpb1ΔCTD^R with intron 1 and SPA probes. Note that expression of Rpb1ΔCTD^R has no effect on processing uncoupled from transcription. (E) RPA of CMV β-globin SPA (lanes 1 to 3) or injected β-globin SPA transcripts with SPA probe (lanes 4 to 6). Oocytes were coinjected with either no antibody (−), anti-CstF p77 (p77) (5.3 ng/oocyte), or irrelevant control (C) rabbit immunoglobulin G (5.3 ng/oocyte).