Capping, splicing, and 3' processing are independently stimulated by RNA polymerase II: different functions for different segments of the CTD

Abstract

Capping, splicing, and cleavage/polyadenylation of pre-mRNAs are interdependent events that are all stimulated in vivo by the carboxy-terminal domain (CTD) of RNA Pol II. We show that the CTD independently enhances splicing and 3' processing and that stimulation of splicing by enhancers is facilitated by the CTD. We provide evidence that stimulation of 3' processing by the CTD requires contact with the 50-kD subunit of the cleavage stimulation factor, CstF. Overexpression of the CTD-binding domain of CstF p50 had a dominant-negative effect on 3' processing without disrupting the CstF complex. The CTD comprises 52 heptad repeats. The CTD carboxyl terminus including heptads 27-52 supported capping, splicing, and 3' processing but the amino terminus supported only capping. We conclude that the CTD independently stimulates all three major pre-mRNA processing steps and that different regions of the CTD can serve distinct functions in pre-mRNA processing.

Figure 1

The CTD is required for enhancer-dependent splicing independent of 3′ processing. (A) Mutation of the poly(A) site and deletion of the CTD inhibit splicing of β-globin introns 1 and 2. RNase protection of RNA from α-amanitin-treated 293 cells transfected with pSVβ128 AAUAAA or pSVβ128 AAGAAA β-globin with wild-type or mutant poly(A) sites (closed arrow) and expression vectors for α-amanitin-resistant full-length, 1–52 [HA-WT, (Gerber et al. 1995)] or CTD-truncated large subunit with 5 heptads (HA-Δ5). (*) Undigested probe; (open arrows) RNase protection probes. Ratios of spliced to unspliced transcripts for introns 1 and 2 were calculated from PhosphorImager data corrected for the [³²P]uridine content of the protected fragments. (B) CTD-dependent splicing driven by the FN EDI enhancer. (Lanes 1–4) RNase protection of pSVβ128 AAGAAA or pSVβ128 AAGAAA-FN transcripts made by α-amanitin-resistant Pol II (1–52) or Δ0. Spliced to unspliced ratios were calculated as in A. (Lanes 5–8) poly(A)⁺ and poly(A)⁻ RNAs analyzed with β-globin intron2, GPDH and VA probes. Note that most spliced β-globin transcripts are poly(A)⁻.

Figure 2

CTD-dependent 3′ processing independent of splicing. RNase protection of TK–βIFN transcripts from α-amanitin-treated cells cotransfected with a CMV-neo control (C) or cDNA expression vectors for the full-length (1–52) or CTD-deleted (Δ0) Pol II large subunit. VA transcripts served as a control for transfection efficiency.

Figure 3

Interaction between the CTD and the amino terminus of CstF p50. (A) A mix of partially purified baculoviral His-tagged CstF p50, p64, and p77 was incubated with glutathione–Sepharose beads containing immobilized GST or fusions with wild-type (1–52) or mutant CTD (mut) with 15 repeats of YSPTAPS. High-salt eluates were immunoblotted with anti-Xpress antibody. 20% of the load (L) and 10% of the flowthrough (FT) and the eluates were loaded. Diagram of CstF p50 with its amino-terminal CTD-binding domain (black) and WD40 repeats (stippled). (B) The CTD-binding domain of CstF p50 maps to the amino-terminal 95 residues. [³⁵S]methionine-labeled fragments of rat p50 were made by in vitro translation and incubated with wild-type (1–52) or mutant (mut) GST–CTD as in A. 10% of the load (L) and 50% of the high-salt eluates were loaded.

Figure 4

Dose-dependent dominant-negative effect of overexpressed CstF p50 amino terminus on 3′ processing and capping. (A) (Top) RNase protection of Gal5HIV2CATΔt reporter transcripts activated by GAL4–VP16 (see diagram). Cells were cotransfected with 0.1, 0.5, or 2.0 μg of EFpLinkCstFp50(1–95) or EFpLink vector (C). Ratios of cleaved to uncleaved RNA at the SV40 late poly(A) site are given. (Bottom) Western blotting of p50 (1–95) with antibody against the amino terminus. (B) RNase protection as in A of Gal5HIV2CATΔt transcripts that were separated into capped and uncapped fractions by binding to GST–eIF4E. Cells were cotransfected with 40 μg of either EFpLinkTag globin control or EFpLinkTagCstFp50(1–95). Yeast Rp51A RNA served as a control for the GST–eIF4E selection procedure. Capped to uncapped ratios are given for the sum of cleaved plus uncleaved transcripts. CstF p50 1–95 overexpression inhibited both cleavage at the poly(A) site and the extent of capping.

Figure 5

Overexpression CstF p50 amino terminus does not disrupt the CstF complex. Cells were cotransfected with EFpLinkTag p50(1–176) (1μg/plate). Gal5HIV2CATΔt reporter, GAL4–VP16, and GFP expression vectors. Transfected cells were selected by FACS sorting for GFP expression. Extracts from the transfected cells (p50 1–176) and untransfected controls (C) were immunoprecipitated with anti-CstFp77 (lanes 2,3) or anti-GST as a negative control (lane 1). Immunoprecipitates and total extracts were immunoblotted with anti-CstF p77, anti-p50, and anti-Myc, which recognizes p50 (1–176). Note that overexpression of p50 (1–176) does not disrupt the association of p77 with p50 and that p50 (1–176) does not form a stable complex with p77 (lane 3).

Figure 6

Binding of CstF and capping enzyme to different segments of the CTD. (A) Binding of partially purified baculoviral CstF p50 to GST, GST–mut CTD (Fig. 3A), and fusions with segments of the CTD. 2.5% of the load (L) and 50% of the eluates were immunoblotted with anti-Xpress antibody. Note binding to heptads 27–42 but not to 1–15 or 27–39. (B) Binding of HeLa CstF (p50, p77) and capping enzyme guanylyltransferase (GT) to phosphorylated and unphosphorylated fragments of the CTD. HeLa nuclear extract was chromatographed on columns of immobilized GST fusion proteins. 1% of the load (L) and 8% of the eluates were analyzed by Western blotting. Note GT binds heptads 1–15-PO₄ but CstF does not.

Figure 7

The carboxyl terminus of the CTD is necessary and sufficient to enhance 3′ processing. (A) Expression of B10 epitope-tagged CTD deletion mutants of α-amanitin-resistant Pol II large subunit. (Top) Western blot of extracts from transfected 293 cells and untransfected control (C) with anti-B10 antibody. (Bottom) Western blot of anti-B10 immunoprecipitates probed with rabbit anti-CTD antibody. (B) Cleavage at the β-globin poly(A) site is supported by heptads 27–52 but not by heptads 1–25. RNase protection of pSVβ128Rpbex5 transcripts from α-amanitin-treated cells cotransfected with CMV-neo (C), or expression vectors for α-amanitin-resistant Pol II large subunits with full-length (1–52) or truncated CTDs. Ratios of cleaved to uncleaved transcripts are given. (C) Cleavage at the β-IFN poly(A) site is supported by heptads 27–52 but not by heptads 1–25. RNase protection analysis of intronless TK-βIFN transcripts made by full-length and CTD-deleted Pol II. Ratios of cleaved to uncleaved transcripts are given.

Figure 8

The carboxyl terminus of the CTD is necessary and sufficient to enhance splicing. (A) Efficient splicing of β-globin intron 1 is supported by heptads 27–52 but not by heptads 1–25. RNAs are the same as those analyzed in Fig. 7B. (*) Irrelevant undigested probe. (B) Enhancer-dependent splicing, independent of 3′ processing, is supported by heptads 27–52 but not by heptads 1–25 or 1–15. RNase protection of transcripts from pSVβ128 AAGAAA-dsx and pSVβ128 AAGAAA-TnT, which have mutant poly(A) sites and splicing enhancers inserted into exon 3 as diagrammed.

Figure 9

Either the amino or carboxyl terminus of the CTD can enhance capping. (A) The Gal5HIV2CATΔt reporter activated by GAL4–VP16 was transcribed by full-length (1–52) and CTD-truncated α-amanitin-resistant mutants of Pol II. Capped and uncapped RNA was analyzed with probe complementary to the SV40 late poly(A) site. (*) Undigested probes. Ratios of cleaved to uncleaved and capped to uncapped transcripts are shown. Capped to uncapped ratios are for the sum of cleaved plus uncleaved transcripts and were normalized to Rp51A as in Fig. 4B. (B) Heptads 1–15 support efficient capping but not 3′ processing. Gal5HIV2CAT transcription was activated by GAL4–SW6 and HIV-1 Tat. Capped and uncapped RNA was analyzed with a probe complementary to the SV40 early poly(A) site. Capped to uncapped ratios are for the sum of cleaved plus uncleaved transcripts as in A.

Figure 10

Amino acid sequence of the human CTD with heptad repeats numbered.