The Mammalian RNA Polymerase II C-Terminal Domain Interacts with RNA to Suppress Transcription-Coupled 3′ End Formation  Syuzo Kaneko, James L. Manley  Molecular Cell  Volume 20, Issue 1, Pages 91-103 (October 2005) DOI: 10.1016/j.molcel.2005.08.033 Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 1 The RNAP II CTD Binds Pre-mRNA (A) Nonspecific polyadenylation assays were carried out with an SV40 late pre-mRNA fragment in a reaction mixture containing Mn2+ and PAP for 30 min at 30°C (lane 1) with 200 ng of GST (lane 2) or increasing amounts (50, 100, and 200 ng) of GST-CTD (lanes 3–5). RNA products were isolated and fractionated on a 5% denaturing gel. (B) Polyadenylation assays were performed with increasing amounts (50, 100, and 200 ng) of GST-CTD (lanes 2–4), except that GST-CTD (200 ng) was preheated from 40°C to 70°C for 5 min (lanes 5–8). (C) GST-CTD was cleaved by thrombin, and CTD was separated from GST by glutathione beads. Increasing amounts (200 and 400 ng) of CTD (lanes 2 and 3) and preheated (70°C for 5 min) CTD (400 ng) (lane 4) were used in the nonspecific polyadenylation assay. (D) SV40 pre-mRNA was utilized in binding reactions with no protein (lane 2), increasing amounts of GST (lanes 3–5), or GST-CTD (lanes 6–8) bound to glutathione beads. After washing, bound RNA was analyzed by denaturing PAGE. 10% input RNA is shown (lane 1). (E) Reaction mixtures that were the same as for the polyadenylation assay, except that PAP was omitted, were incubated with the RNAs indicated above each lane in the absence (lane 1) or presence (lane 2) of 200 ng of GST or increasing amounts (100 and 200 ng) of GST-CTD (lanes 3 and 4). RNA-protein complexes were separated by 5% nondenaturing PAGE. (F) Gel-shift assays were performed with SV40 and β-globin pre-mRNAs and pGEM3 vector RNA by using increasing amounts (50, 100, or 200 ng) of GST-CTD and were analyzed as in (E). Molecular Cell 2005 20, 91-103DOI: (10.1016/j.molcel.2005.08.033) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 2 CTD Requirements for RNA Interaction (A) Silver staining of SDS-PAGE gel containing mock-phosphorylated (lane 2), phosphorylated (lane 3), and untreated GST-CTD (lane 1). Yeast CTD and all-consensus CTD (CON52) are shown in lanes 4 and 5. (B) Gel-shift assays with the SV40 RNA and 400 or 800 ng of nonphosphorylated (lanes 1 and 2) or phosphorylated (lanes 3 and 4) GST-CTD. (C) Increasing amounts (200, 400, or 800 ng) of CON52 (lanes 2–4) or 200 ng wild-type CTD (lane 1) were incubated with SV40 pre-mRNA and analyzed by nondenaturing PAGE. (D) Gel-shift assays were performed with 200 ng of mouse CTD (lane 1) or 200 and 400 ng of yeast CTD (lanes 2 and 3). (E) Gel-shift assays with deletion mutants of GST-CTD. Equal amounts (200 ng) of C-terminal truncations removing four or eight heptad repeats (lanes 5 and 6) and N-terminal truncations removing 23, 32, or 40 repeats (lanes 7–9) were incubated with SV40 pre-mRNA. Free-RNA, RNA with GST, and 100 or 200 ng of wild-type GST-CTD are shown in lanes 1–4. (F) Nonspecific polyadenylation was carried out as in Figure 1A. Reaction mixtures contained 50 or 200 ng of full-length GST-CTD (lanes 1 and 2), C-terminal truncations (lanes 3–6), and N-terminal truncations (lanes 7–12). Molecular Cell 2005 20, 91-103DOI: (10.1016/j.molcel.2005.08.033) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 3 RNA Binding Specificity of the CTD (A) Sequences selected by GST-CTD after six (6.1–6.19) and seven (7.1–7.16) rounds of SELEX. Overrepresented C/A-rich sequences are in bold. (B) Diagram of tandem repeat of the selected RNA sequence number 6–18. (C) GST-CTD (1.7 μM) was incubated with ∼0.6 nM RNA substrate in the presence of increasing salt concentrations (0–100 mM KCl) and was applied onto nitrocellulose filters. RNA bound GST-CTD and free RNA were separated by repetitive washing. The graph shows the ratio of the amount of C/A-rich RNA to control RNA bound to the CTD at each salt concentration. (D) Filter binding was done with increasing amounts (0.2–1.4 μM) of GST-CTD with RNA or dsDNA in the presence of 50 mM KCl. (E) End-labeled RNA (39 pM), dsDNA (68 pM), and heat-denatured dsDNA (ssDNA) were incubated with 1.7 μM GST-CTD in the presence of 50 mM KCl and were analyzed by filter binding. (F) Filter binding with RNA and dsDNA was carried out with increasing amounts of salt (50–100 mM KCl) and 1.7 μM GST-CTD. The signal of bound RNA or DNA was set at 100% for the 50 mM salt condition. Molecular Cell 2005 20, 91-103DOI: (10.1016/j.molcel.2005.08.033) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 4 The CTD Interacts with Nascent RNA In Vivo (A) Diagram of reporter plasmids. Plasmids contained the CMV promoter element, a partial β-globin gene, and SV40 early (SVE) poly(A) signal. The inserted CBS and poly(A) site are indicated. PCR primers are shown. (B) Diagram of inserts. Tandem repeats and variable sequences of CBS and round 0 sequence are shown. (C) Brief procedure for RNA-ChIP. (D) Separation of the CTD from RNAP II body. Supernatants were treated with 0, 0.2, 1.0, or 2.0 μg/ml proteinase K (lanes 1–4). Proteolysis of the CTD was monitored by Western blot analysis with anti-CTD antibodies. Intact RNAP II and CTD are indicated. (E) RNA-ChIP analysis. The results of real-time PCR with the indicated antibodies in the presence or absence of reverse transcriptase are shown and are the average of the results from three sets of PCR experiments from one immunoprecipitation assay. We observed very similar patterns of signals in multiple sets of immunoprecipitation assays. Molecular Cell 2005 20, 91-103DOI: (10.1016/j.molcel.2005.08.033) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 5 The CTD-CBS Interaction Inhibits 3′ End Processing (A) Diagram of reporter plasmids and RNA probe. Poly(A) site and inserts are shown, and the length and position of RNA probes, read-through, and mRNA products are indicated. (B) Transient transfection of reporter plasmids followed by RNase protection analysis. Shown are RNA products detected when the CBS was inserted into the reporter plasmid (A) (lane 3), or with no insert (lane 2), insertion of the tandem repeat of round 0 sequence (lane 4), and inverted CBS (lane 5). Mock transfection is shown in lane 1. The ratios of read-through to mRNA products are indicated at the bottom. Protected RNA was fractionated by 5% denaturing PAGE. (C) Diagram of reporter plasmid containing weak poly(A) signal. Poly(A) site and inserts are shown, and the length and position of RNA probes, read-through, and mRNA products are indicated. (D) Sequence of SVE poly(A) signal. Wild-type (black) and weak poly(A) site (white) are indicated as inverted triangles, and deleted sequences are underlined. (E) RNase protection analysis of RNA from cells transfected with the plasmid shown in (C) containing the CBS (lane 4), no insert (lane 3), tandem repeats of round 0 sequence (lane 5), or C2 pausing element (lane 6). Mock transfection is shown in lane 2. Ratios of mRNA to read-through products are indicated at the bottom. (F) In vitro cleavage reactions with HeLa NE and RNA containing the adenovirus L3 poly(A) site were performed in the presence of increasing amounts (0, 200, or 400 ng) of CBS RNA (lanes 3–5), 400 ng of CBS RNA with 800 ng GST (lane 6) or GST-CTD (lane 7), or increasing amounts (200 and 400 ng) of anti-sense CBS RNA (lanes 8 and 9). Positions of pre-mRNA and 5′ and 3′ cleavage products are indicated. Molecular Cell 2005 20, 91-103DOI: (10.1016/j.molcel.2005.08.033) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 6 The CBS Suppresses Transcription Termination (A) Diagram of reporter plasmids and RNA probe. The plasmid utilized for transfection is the same as shown in Figure 5A. The length and position of RNA probe, read-through products, and initiated transcripts are indicated. (B) RNase protection analysis of RNA isolated after transfection of plasmids containing a tandem repeat of the CBS (lane 3), no insert (lane 2), or insertion of a tandem repeat of round 0 (lane 4) or inverted CBS (lane 5). Mock transfection is shown in lane 1. The positions of mRNA and unprocessed RNA are indicated. RNA products indicated by an asterisk were not initiated transcripts as confirmed by primer extension analysis (data not shown). Molecular Cell 2005 20, 91-103DOI: (10.1016/j.molcel.2005.08.033) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 7 Model of Inhibition of 3′ End Processing by Interaction between the RNAP II CTD and Newly Transcribed C/A-rich RNA (A) Polyadenylation factors assembled on the RNAP II CTD recognize the poly(A) signal and catalyze 3′ end formation in a transcription-coupled manner. Poly(A) factors (CPSF, CstF, CFI, CFII, and PAP) and N-terminal half (N-CTD) and C-terminal half (C-CTD) of the RNAP II CTD are shown. (B) When C/A-rich RNA is transcribed, the C-terminal half of the RNAP II CTD interacts with this RNA, interfering with poly(A) factor assembly. Improperly assembled factors inefficiently recognize the poly(A) signal and the efficiency of 3′ end formation is decreased. Molecular Cell 2005 20, 91-103DOI: (10.1016/j.molcel.2005.08.033) Copyright © 2005 Elsevier Inc. Terms and Conditions