The RNA Tether from the Poly(A) Signal to the Polymerase Mediates Coupling of Transcription to Cleavage and Polyadenylation  Frank Rigo, Amir Kazerouninia, Anita Nag, Harold G. Martinson  Molecular Cell  Volume 20, Issue 5, Pages 733-745 (December 2005) DOI: 10.1016/j.molcel.2005.09.026 Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 1 Transcription-Coupled 3′ End Processing Is Fast, Efficient, Accurate, and Reproducible (A) Circular plasmid DNA was transcribed for increasing lengths of time, and the RNA was displayed on a gel. The % poly(A) refers to the ratio of polyadenylated RNA to all RNA extending past the poly(A) site. This assay contained PVA, and the [MgCl2] was 5 mM. (B) Wt and mt refer to a poly(A) signal with intact or mutated hexamer, respectively. For lanes 2–7, RNA was isolated from a 15 min, 5-fold transcription-processing reaction, as for (A), that had 2 μM of cold CTP in place of [α-32P] CTP. One third of the RNA was set aside as the “Input” and the remainder was oligo(dT) selected using a Poly(A) Purist MAG kit (Ambion). The “Free” RNA was taken from a first round of selection, and the “Bound” RNA was from the second. The Input, Free, and Bound fractions were then digested with DNase I (Roche), hybridized at 65°C, and subjected to RNase protection with RNase T1 (Chao et al., 1999). The mole % of processing in lane 2 is 30% of the total. The DNA used here, pSV40E/L′, had the same promoter and poly(A) signal as in (A) but set in a different plasmid background that provided an intron to allow for expression in vivo. Lane 1 shows a control with cytoplasmic RNA isolated from transfected cells as previously described (Park et al., 2004). The probe used was a run-off transcript from a derivative of pSV40E/L′ containing an inserted T7 RNA polymerase promoter. Because only RNase T1 (specific for G residues) was used in the RNase protection, a single probe sufficed for both wt and mt RNAs. The intensities of lanes 6 and 7 were reduced in ImageQuant for purposes of comparison. (C) This assay was like that in (A) but using DNA with a different promoter and poly(A) signal, and at [citrate] and [MgCl2] of 5 and 6 mM, respectively. (D) Cleavage at the BGH poly(A) signal is accurate. Reactions were performed as in (C), with pCMV/BGH having either a wild-type or mutant poly(A) signal. For samples receiving 3′ dATP to block polyadenylation, α-amanitin was also added, simply because this had become part of a standard procedure (see Figure 2). Lane 4 was as for lane 3 except that oligo number 3 was added with the chase to direct RNaseH cutting to the BGH poly(A) site. (E) Transcription-polyadenylation as a function of time for different extracts, promoters, and poly(A) signals. The % polyadenylation is quantitated as for Figure 1A. The data for extract 1 are from an experiment that differed from the standard assay in having a 15 min preincubation in a volume of 6.9 μl, a chase of 2.6 μl containing PVA, and a final [ATP] of 500 μM. PVA was also used in the experiment for extract 2. The data for extract 3 are from the gels in Figures 1A and 1C and include some time points not shown in Figure 1A. Molecular Cell 2005 20, 733-745DOI: (10.1016/j.molcel.2005.09.026) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 2 Coupling Requires a Ternary Complex but Not Ongoing Transcription (A) Ongoing transcription and poly(A) tail growth are not required for coupling. The assay was done using pSV40E/L, extract 1, and PVA. (B) Quantitative comparison of processing efficiency with and without transcription. The data points are from Figure 2A, and the line is from Figure 1E. (C) Exogenous RNA added to a coupled reaction does not get processed. Pre-made 32P-labeled RNA was isolated from a 10-fold coupled processing reaction of pSV40E/L in extract 3. RNA running slower than poly(A)-cleaved RNA was purified from a 5% polyacrylamide gel, and 15,000 CPM of the RNA was added to a standard (i.e., containing no PVA) coupled processing reaction in extract 3 (along with α-amanitin to 34 ng/μl and 3′ dATP to 372 μM) that was pulsed with cold rather than hot CTP. The resulting gel was exposed 3 days to the phosphor screen. (D) Exogenous RNA is not inhibitory. A reaction was carried out in parallel with the above that differed only in that the coupled reaction to which the gel-purified RNA was added was pulsed with 32P as usual. This gel was exposed only for 8 hr to the phosphor screen, and the newly made RNA accounts for over 97% of the signal. (E) To demonstrate that the gel-purified RNA is capable of undergoing processing under special conditions, it was incubated at 37°C with PVA for 2 hr under standard uncoupled processing conditions (e.g., Wahle and Keller, 1994). Final amounts or concentrations in 30 μl were 2.5 μg tRNA, 0.67 mM 3′ dATP, 17 mM creatine phosphate, 1.9 mM DTT, 10 U anti-RNase (Ambion), 42 μM PMSF, 2.1% PVA, 8.3% glycerol, 8.3 mM HEPES (pH 7.9), 42 mM KCl, 83 μM EDTA, 12.5 μl extract 3, and 6.25 μl PBS. Molecular Cell 2005 20, 733-745DOI: (10.1016/j.molcel.2005.09.026) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 3 Severing the RNA Tether from the Poly(A) Signal to the Polymerase Disrupts Coupling (A) Cartoon of a ternary elongation complex in the process of assembling a cleavage and polyadenylation apparatus. A DNA oligonucleotide is shown hybridized to a target in the RNA, thereby directing RNaseH to cut the tether. (B) Severing the tether prevents coupled 3′ end processing. The oligonucleotide names refer to the distance from the principal poly(A) cleavage site to the predominant RNaseH cutting site (Wu et al., 1999). The control oligos for lanes 4–6 and 13–15 were the 77 oligo-complement and oligo number 7, respectively. (C) RNaseH-cut RNA is not intrinsically resistant to processing. RNaseH-cut RNA was generated as in (B) by using a 10-fold coupled processing reaction. The bands were gel purified and incubated under uncoupled processing conditions for 2 hr with PVA. The % processing given is mole % of the total. (D) Tether requirement for a weak poly(A) signal. This assay used PVA with [citrate] and [MgCl2] of 4 and 5 mM, respectively. Lane 4 was as for lane 3 except that oligo number 8 was added with the chase to direct RNaseH cutting to the poly(A) site. The 129 oligo here is the same as the 158 oligo in Figure 3B, but cloning has placed the identical cut site closer to the poly(A) site in this construct. Molecular Cell 2005 20, 733-745DOI: (10.1016/j.molcel.2005.09.026) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 4 The RNA Tether Mediates Coupling Wt and mt refer to a poly(A) signal with intact or mutated hexamer, respectively. Three independent experiments like that shown in part A were carried out (except that lanes 13–15 were lacking in one). The increases in poly(A) cleaved RNA (as a % of all RNA extending past the poly[A] site) between 5 min and either 10 or 30 min in the presence of the 77 and 397 oligos and the control oligo (77 oligo complement) are plotted as the standard deviations in the upper panel of part B. The decreases in RNaseH-cut RNA are plotted similarly in the lower panel. In each experiment, the % of decrease in the RNaseH-cut RNA was adjusted by subtracting the amount of decrease observed for the corresponding poly(A) signal mutant RNA. Molecular Cell 2005 20, 733-745DOI: (10.1016/j.molcel.2005.09.026) Copyright © 2005 Elsevier Inc. Terms and Conditions

Figure 5 Processed RNA Remains Associated with the Polymerase and the Processing Apparatus (A) Cartoon of an elongation complex. The template for all parts of this figure was pSV40E/L. (B) Processed RNA is preferentially retained with the template. Transcription was initiated in extract 2. At 30 s, 0.005 μg of the −181 (5′) oligo and 0.05 μg of either the control oligo (oligo number 7, lanes 1 and 2) or the 77 oligo (lanes 3 and 4) were added. PVA causes all cut RNA, short and long, to remain template-associated and was therefore not used. Bound RNA (pellet, lanes 2 and 4) was separated from released RNA (supernatant, lanes 1 and 3) by magnetic selection. The averages given are the mean ± the difference from the mean for two independent experiments using two different 5′ oligos (−181 or −147). (C) Processed RNA is preferentially retained by the polymerase (extract 2). The error shown is the standard deviation. (D) Processed RNA remains preferentially associated with CstF (extract 4). This experiment was carried out with 5 mM MgCl2, 4 mM citrate, and 0.013 μg of the −181 (5′) oligo. Molecular Cell 2005 20, 733-745DOI: (10.1016/j.molcel.2005.09.026) Copyright © 2005 Elsevier Inc. Terms and Conditions