Functional Coupling of Capping and Transcription of mRNA Shin Moteki, David Price  Molecular Cell  Volume 10, Issue 3, Pages 599-609 (September 2002) DOI: 10.1016/S1097-2765(02)00660-3

Figure 1 Use of Anti-2,2,7-Trimethylguanosine Antibody to Analyze Capped Transcripts (A) Assay for capped and methylated transcripts. Pulse-chase transcription reactions were carried out using a CMV template as described in Experimental Procedures using a 40 s pulse with [α-32P] CTP followed by a 5 min chase with addition of 50 μM DRB or S-adenosyl-methionine (SAM) as indicated. T, total; U, unbound; B, m2,2,7G antibody bead bound; M, 25 nt ladder; G, total transcripts as in lane 1 bound to antibody beads and eluted with 1 mM GTP; m7G, same as G but eluted with 1 mM m7GTP. End labeled tRNAs are indicated. (B) Assay for guanylylated transcripts. Pulse-chase transcription reactions were carried out as described in Experimental Procedures using a 20 s pulse with [α-32P] UTP (lanes 1–6) followed by a 5 min chase with 50 μM DRB added to inhibit P-TEFb (lanes 7–16). S-adenosyl-methionine was added (+) or not (-) during transcription (SAM(T)) or during a posttranscription methylation step (SAM(PT)) as indicated. nd, posttranscription methylation step not done; T, total transcripts; B, m2,2,7G antibody bead bound fraction; M, 25 nt ladder. (C) Kinetics of guanylylation and methylation during transcription. Pulse-chase transcription reactions were carried out as in (B) with DRB and SAM with the indicated chase times. T, total transcripts; G, guanylylated transcripts isolated using the method in (B); M, m7G capped transcripts isolated using the method in (A); M, 25 nt ladder. (D) Quantitation of guanylylation and methylation. A Packard InstantImager was used to quantitate all transcripts above 25 nt in length from each lane. The percentage of the total counts found in the guanylylation or methylation assay were calculated and plotted. The artifactual band at about 100 nt was not included in the quantitation. Molecular Cell 2002 10, 599-609DOI: (10.1016/S1097-2765(02)00660-3)

Figure 2 Kinetics of Guanylylation of Free RNA by HeLa Nuclear Extract Unguanylylated transcripts were generated as described in Experimental Procedures by isolating early elongation complexes, chasing for 3 min, and then phenol extracting the RNA. The RNA was added back to HeLa nuclear extract (HNE) or HNE plus 1 μg of Hce1. RNA was re-extracted at the indicated times (T) and analyzed for the presence of the G cap (G). Molecular Cell 2002 10, 599-609DOI: (10.1016/S1097-2765(02)00660-3)

Figure 3 HNE Add-Back to Isolated Elongation Complexes (A) Time course of transcription. Early elongation complexes containing a 15 nt uncapped transcript were isolated using a high salt Sarkosyl containing buffer containing (0 time point). HNE was added to each reaction and elongation allowed to take place for the indicated times. Transcripts were analyzed for guanylylation as in Figure 1B. T, total; G, guanylylated. (B) Quantitation of guanylylation. Transcripts were quantitated as in Figure 1D and the results plotted. (C) Hce1 add-back to isolated elongation complexes. Stringently washed complexes were isolated as in (A) and 1 ng of Hce1 added. After 10 min, NTPs were added to start elongation and time points were taken and analyzed for guanylylation. T, total; G, guanylylated. (D) Time course with 30 ng of Hce1 per reaction. Same as (C) except 30 ng of Hce1 added. (E) Quantitation of guanylylation. The results in (C) and (D) and three other similar experiments with different levels of Hce1 were quantitated as in Figure 1 and plotted. Molecular Cell 2002 10, 599-609DOI: (10.1016/S1097-2765(02)00660-3)

Figure 4 Kinetics of Guanylylation of Free RNA by HCE Unguanylylated transcripts were generated as described in Figure 2. The RNA was incubated with the either 100 or 1000 ng of Hce1 and GTP for the indicated times and the RNA was re-isolated and subjected to the guanylylation assay. T, total; G, guanylylated. Molecular Cell 2002 10, 599-609DOI: (10.1016/S1097-2765(02)00660-3)

Figure 5 Association of Capping Activity with Transcription Complexes (A) Washed elongation complexes. Early elongation complexes were formed on an immobilized template during a 20 s pulse and washed 3 times with a buffer containing 20 mM HEPES, 7 mM MgCl2, 0.2 mg/ml BSA, and either 60 or 200 mM KCl. Transcripts were elongated for the indicated times and then subjected to the guanylylation assay. T, total; G, guanylylated. The upper and lower arrowheads indicate the position of the 150 and 50 nt marker, respectively. (B) Diluted elongation complexes. Early elongation complexes were formed during a 20 s pulse and then diluted 1-, 3-, 6-, or 12-fold into chase buffer. After a 0.5 or 1 min chase, RNA was isolated and subjected to the guanylylation assay. T, total; G, guanylylated. (C) Diluted preinitiation complexes. Preinitiation complexes were formed during the standard 10 min preincubation. A 20 s pulse was carried out with either a 3- or 9-fold dilution so that early elongation complexes formed under more diluted conditions. The complexes were then chased for the indicated times and RNA was isolated and subjected to the guanylylation assay. T, total; G, guanylylated. Molecular Cell 2002 10, 599-609DOI: (10.1016/S1097-2765(02)00660-3)

Figure 6 Role of the CTD in Capping (A) Kinase assay. Unlabeled early elongation complexes formed during a 20 s pulse were isolated using a high salt and Sarkosyl containing buffer and treated with the indicated amount of chymotrypsin as described in Experimental Procedures. These complexes were re-isolated and then subjected to phosphorylation with P-TEFb using [γ-32P]ATP. The complexes were then analyzed by 6% SDS-PAGE and autoradiography. (B) Western blot. Complexes treated with the indicated amount of chymotrypsin were analyzed by western blot using an antibody that recognizes the CTD (H16). HeLa nuclear extract (HNE) was used as a control. (C) Add-back of HNE to chymotrypsin treated complexes. HNE was added back to early elongation complexes treated with 75 μg/ml chymotrypsin and elongation was allowed to take place for the indicated times. RNA was isolated and subjected to the guanylylation assay. T, total; G, guanylylated. (D) Add-back of Hce1 to chymotrypsin treated complexes. 10 ng Hce1 was added back to early elongation complexes treated with 75 μg/ml chymotrypsin and elongation was allowed to take place for the indicated times. RNA was isolated and subjected to the guanylylation assay. T, total; G, guanylylated. (E) Comparison of guanylylation rates. Guanylylation was quantitated from results in (C) and (D), and parallel experiments done with untreated complexes (not shown). Molecular Cell 2002 10, 599-609DOI: (10.1016/S1097-2765(02)00660-3)

Figure 7 Role of Cdk7 in Capping (A) Inhibition of Cdk7 by DRB. A kinase assay was carried out with purified TFIIH and Pol II as a substrate. DRB was added to the indicated final concentrations and the concentration of ATP was maintained at 100 μM to match that with the transcription done in (B). The labeled polymerase was analyzed by SDS PAGE and the autoradiograph of the dried gel is shown as an inset to the graph of the quantitated results. (B) Guanylylation assay for transcripts generated during transcription in the absence of Cdk7 activity. A pulse-chase transcription assay similar to that in Figure 2 was carried out except that 1.5 mM DRB was present to inhibit Cdk7 (and P-TEFb). The indicated time points were taken and the RNA subjected to the guanylylation assay. T, total; G, guanylylated. (C) Comparison of guanylylation with and without Cdk7 activity. The results from (B) were quantitated and compared with the guanylylation results for transcription in the presence of Cdk7 activity from Figure 2. The upper and lower arrowheads indicate the position of the 150 and 50 nt marker, respectively. Molecular Cell 2002 10, 599-609DOI: (10.1016/S1097-2765(02)00660-3)