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• W1972628058 abstract "Article17 February 2003free access The C-terminal domain of pol II and a DRB-sensitive kinase are required for 3′ processing of U2 snRNA Joanne E. Medlin Joanne E. Medlin Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, OX1 3RE UK Search for more papers by this author Patricia Uguen Patricia Uguen Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, OX1 3RE UK Search for more papers by this author Alice Taylor Alice Taylor Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, OX1 3RE UK Search for more papers by this author David L. Bentley David L. Bentley Department of Biochemistry and Molecular Genetics, University of Colorado Health Sciences Center, B121, 4200 East 9th Avenue, Denver, CO, 80262 USA Search for more papers by this author Shona Murphy Corresponding Author Shona Murphy Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, OX1 3RE UK Search for more papers by this author Joanne E. Medlin Joanne E. Medlin Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, OX1 3RE UK Search for more papers by this author Patricia Uguen Patricia Uguen Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, OX1 3RE UK Search for more papers by this author Alice Taylor Alice Taylor Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, OX1 3RE UK Search for more papers by this author David L. Bentley David L. Bentley Department of Biochemistry and Molecular Genetics, University of Colorado Health Sciences Center, B121, 4200 East 9th Avenue, Denver, CO, 80262 USA Search for more papers by this author Shona Murphy Corresponding Author Shona Murphy Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, OX1 3RE UK Search for more papers by this author Author Information Joanne E. Medlin1, Patricia Uguen1, Alice Taylor1, David L. Bentley2 and Shona Murphy 1 1Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, OX1 3RE UK 2Department of Biochemistry and Molecular Genetics, University of Colorado Health Sciences Center, B121, 4200 East 9th Avenue, Denver, CO, 80262 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2003)22:925-934https://doi.org/10.1093/emboj/cdg077 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The human snRNA genes transcribed by RNA polymerase II (e.g. U1 and U2) have a characteristic TATA-less promoter containing an essential proximal sequence element. Formation of the 3′ end of these non-polyadenylated RNAs requires a specialized 3′ box element whose function is promoter specific. Here we show that truncation of the C-terminal domain (CTD) of RNA polymerase II and treatment of cells with CTD kinase inhibitors, including DRB (5,6-dichloro-1-β-D-ribofuranosylbenzimidazole), causes a dramatic reduction in proper 3′ end formation of U2 transcripts. Activation of 3′ box recognition by the phosphorylated CTD would be consistent with the role of phospho-CTD in mRNA processing. CTD kinase inhibitors, however, have little effect on initiation or elongation of transcription of the U2 genes, whereas elongation of transcription of the β-actin gene is severely affected. This result highlights differences in transcription of snRNA and mRNA genes. Introduction In addition to protein-coding genes, RNA polymerase II (pol II) transcribes the genes for the non-coding snRNAs such as U1 and U2, which are required for processing of pre-mRNAs. These snRNA genes have specialized TATA-less promoters with a very simple structure comprising the basal proximal sequence element (PSE) and an upstream enhancer-like distal sequence element (DSE) (reviewed by Hernandez, 2001). Unlike pre-mRNAs, snRNAs are not spliced, and 3′ end formation is dependent on a conserved ‘3′ box’ rather than a polyadenylation signal or the 3′ processing signal of replication-activated histone genes (reviewed in Cuello et al., 1999). We have shown that transcription of the U2 snRNA genes continues beyond the 3′ box (Cuello et al., 1999), and this element is, therefore, likely to function directly as a 3′ end processing element in the RNA, rather than a transcription termination signal. However, the 3′ box is required for termination of transcription to occur downstream from the U2 promoter (Cuello et al., 1999). Intriguingly, the 3′ box only functions efficiently if transcription by pol II is initiated from a PSE-containing promoter (see Cuello et al., 1999), indicating that events at the snRNA gene promoter are somehow linked to downstream processing reactions. The conserved C-terminal domain (CTD) of pol II is important for efficient transcription of some mRNA genes in vitro (reviewed by Dahmus, 1996). In addition, efficient transcription of some transfected mRNA genes is CTD dependent (Gerber et al., 1995), and there appears to be a global defect in transcription of chromosomal protein-coding genes in the absence of the CTD (Meininghaus et al., 2000). In mammals, the CTD has an unusual structure of 52 tandem heptad repeats, which can be phosphorylated in a reversible manner. CTD kinases include the cyclin-dependent kinase, CDK7, subunit of the basal transcription factor TFIIH and the CDK9 subunit of the elongation factor P-TEFb. A conserved CTD phosphatase, called Fcp1, has also been identified. Dynamic site-specific phosphorylation and dephosphorylation appear to be critical for CTD function and correct mRNA gene expression (reviewed by Prelich, 2002). For example, the phosphorylated form of the CTD interacts with capping enzymes and enhances 5′ capping of pre-mRNA, splicing and 3′ cleavage (reviewed by Bentley, 2002). Dantonel et al. (1997) have also shown that the pol II basal transcription factor TFIID can mediate interaction between polyadenylation factors and the CTD. Thus, the CTD couples transcription and RNA processing in expression of protein-coding genes. We proposed that the CTD plays a similar role to connect initiation and 3′ end processing in expression of the U2 snRNA genes, and that specific promoter factors could help recruit the 3′ box-dependent processing machinery (Cuello et al., 1999). In agreement with this, we show here that correctly initiated transcripts are not processed properly at the 3′ end when U2 genes are transcribed by pol II lacking most of the CTD. Inhibitors of CTD kinases affect recognition of the 3′ box, causing readthrough of this signal. These inhibitors, however, have little effect on U2 transcription, although transcription of the β-actin gene is drastically affected. These results indicate that, in common with protein-coding genes, snRNA genes require the CTD of pol II for accurate and efficient RNA processing. In addition, a phosphorylated CTD may be required for processing of the transcripts. In contrast to the β-actin gene, however, transcription of U2 snRNA genes does not require hyperphosphorylation of the CTD, pointing to differences in the molecular events associated with transcription of these two gene types. Results The CTD of pol II is required for high levels of steady-state RNA from human U2 snRNA gene constructs and correct 3′ end formation of the transcripts To test the hypothesis that the CTD of pol II plays a role in 3′ end formation of transcripts from the human U2 snRNA genes, we have used the in vivo complementation system devised by Gerber et al. (1995). An α-amanitin-resistant pol II large subunit is supplied exogenously by transfection, and transcription by polymerase containing the endogenous pol II large subunit can be inhibited by addition of this drug. In this way, the effect of changes in the large subunit can be assessed. Previous experiments using this system have shown that, in mammals, the CTD of this subunit of pol II is required for transcription of some mRNA genes (Gerber et al., 1995) and for processing of pre-mRNA (McCracken et al., 1997b). For our analysis, we have used U2 gene constructs where the majority of the region encoding the mature RNA is replaced by β-globin gene sequences (U2G; Figure 1A). This allows differentiation between the products of transfected and endogenous genes. In addition, the 3′ processed RNAs from the transfected, marked, genes are exported to the cytoplasm but are neither further 3′ end processed nor re-imported into the nucleus since the necessary signals are lacking (reviewed in Huang and Pederson, 1999). Constructs with or without the 3′ box and with a mutated PSE were transfected (Figure 1A), and RNA was analysed at the 5′ end by S1 nuclease mapping (Figure 1B) and by RNase protection (Figure 1C and D). We have used the adenovirus pol III-transcribed VAI gene as a co-transfection control, and the quantitation determined relative to this is shown at the bottom of each figure. Since several plasmids must be present together in the same cell for these experiments to work, we have used 293 human embryonic kidney cells that transfect with high efficiency. Figure 1.The CTD of pol II is required for high steady-state levels of RNA from U2 snRNA gene constructs and correct 3′ end formation of the transcripts. (A) The structure of the U2–globin (U2G) constructs (see Materials and methods). P–is transcriptionally inactive due to a mutation in the PSE of the promoter (Cuello et al., 1999). The Δ3′ box has had the 3′ box deleted (Cuello et al., 1999). The relative positions of the S1 probe and riboprobes are shown below the diagram of the U2G construct. The size of the expected products of RNase protection analysis is also noted on the right of each construct. +1RT and −111RT are products that initiate at +1 and −111, respectively, and read through beyond the mismatch with the riboprobe. (B) The results of S1 analysis of RNA transcribed from the constructs described in (A). The U2G and CTD construct used and the addition of α-amanitin is noted above each lane. The positions of S1 products corresponding to VAI RNA (VAI) (see Materials and methods) and U2–globin RNA (U2) are noted on the right, and the position of the probes is noted on the left. The amount of properly initiated U2-specific S1 product relative to lane 1 is shown below each lane. (C) The results of RNase protection analysis of RNA transcribed from the constructs described in (A). The U2G and CTD construct used and the addition of α-amanitin is noted above each lane. The positions of the protected products are noted on the right. The amount of properly initiated U2G-specific protection products relative to lane 1 is shown beneath each lane. A breakdown of the relative amount of correct 3′ end and readthrough (RT) is also noted below each lane. (D) The result of RNase protection analysis of U2G RNA transcribed by the CTD constructs indicated above the lanes. The positions of the protected products are noted on the right. The amount of properly initiated U2G-specific protection products relative to lane 1 is shown beneath each lane. A breakdown of the relative amount of correct 3′ end and readthrough (RT) is also noted below each lane. (E) The results of 5′ cap analysis of U2G RNA transcribed by the CTD constructs indicated above the lanes. RNase protection analysis was carried out on RNA selected by GST–eIF4E (capped) or unselected (uncapped) after addition of a positive control RNA (PC). The percentage of capped RNA is noted below the lanes. Download figure Download PowerPoint Analysis of the 5′ end using S1 nuclease (Figure 1B) indicates that removal of the 3′ box has little effect on the steady-state level of RNA quantitated relative to VAI (compare U2G, lane 1 and Δ3′ box, lane 2). Mutation of the PSE abolishes all specific transcription, as expected (PSE–, lane 3). After incubation of the cells with α-amanitin for 48 h, no RNA is detected from the U2G construct, indicating that transcription has been inhibited effectively and that RNA turnover has occurred (lane 4). Co-transfection of the α-amanitin-resistant large subunit of pol II with either a full-length CTD (WT, lane 5) or 31 repeats (Δ31, lane 6) restores the steady-state level of RNA from the U2G construct (lane 1). However, deletion of all but the last five repeats in the CTD has a drastic effect on the steady-state level of correctly initiated RNA (Δ5, lane 7), which is reduced to 14% of the level in lane 1. Importantly, the low level of properly initiated transcription is dependent on the PSE (compare lanes 7 and 8). The intensity of the U2G probe band is increased when Δ31 (lane 6) or Δ5 (lane 7) are transfected compared with the WT CTD (lane 5). This may indicate that some transcription is initiating upstream from the natural start site or that transcription continues round the vector when the CTD is truncated. This transcription appears to be largely dependent on the PSE since it is reduced when the PSE–template is used (PSE–, lane 8). Analysis of the 3′ ends by RNase protection (Figure 1C) demonstrates that the majority of the 3′ ends are formed correctly in the presence of the 3′ box (U2G, lane 1) although some unprocessed or ‘readthrough’ transcripts are detected as a single band due to mismatch with the riboprobe (see Figure 1A). In the absence of the 3′ box (lane 2, Δ3′ BOX), the proportion of readthrough is significantly increased in accordance with previous results (Cuello et al., 1999). The readthrough is now present as a series of bands, perhaps due to inappropriate processing or non-specific degradation. The low level of residual, correct 3′ end formation suggests that sequences in the U2G construct other than the 3′ box can also direct this process, albeit inefficiently. Complementation with WT CTD results in the same proportion of correct 3′ end to readthrough (Figure 1C, lane 5) as for the U2G construct before addition of α-amanitin (lane 1). The proportion of readthrough is slightly higher when Δ31 is used (lane 6), suggesting that recognition of the 3′ box is slightly defective. However, no RNA with the correct 3′ end is detected when most of the repeats have been deleted (Δ5, lane 7). In this case, all of the correctly initiated RNA is detected as readthrough transcripts. As was noted for the 5′ end analysis (Figure 1B), the band corresponding to protection of the U2G riboprobe from the 5′ end at −111 to the site of the 3′ mismatch is stronger in lane 7 (Δ5 and U2G) than in lane 5 (WT CTD and U2G). This suggests, again, that some transcription is starting upstream from the natural initiation site or that the polymerase is reading right around the vector. The −111 readthrough band is weaker in lane 8 (Δ5 and PSE–), suggesting a role for the PSE in this ‘aberrant’ transcription. The amount of unprocessed RNA detected as starting at +1 and reading through when the Δ5 CTD construct is transfected is similar for the 5′ S1 (14%) and RNase protection (12%) analyses (Figure 1B and C, lanes 7), indicating that most of the correctly initiated transcripts are >232 nucleotides long. Treatment of the cells with cycloheximide for 48 h had little effect on the level of steady-state U2G RNA relative to VAI and had no effect on the relative proportion of correct 3′ end to readthrough (data not shown). Furthermore, all three CTD constructs were expressed efficiently in the transfected cells (data not shown). Our results therefore indicate that the CTD of pol II is required for high steady-state levels of U2 RNA and for correct formation of the 3′ end. In addition, pol II with a truncated CTD may be reading around the vector and back through the natural site of initiation due to failure to terminate transcription. The results of analysing the effect of additional, intermediate deletions of the CTD (Fong and Bentley, 2001) are shown in Figure 1D. The amount of steady-state RNA is reduced when only repeats 1–25 are present, and the proportion of readthrough is increased (compare WT, lane 3, 6%, and 1–25, lane 4, 17%). Repeats 27–52 support a slightly higher steady-state level of RNA but an increased proportion of readthrough (27–52, lane 5, 34%). The level of readthrough is increased further to 84% when only repeats 1–15 are present (lane 6). The stepwise truncation of the CTD appears to cause a progressive decrease in processing, suggesting a correlation between the efficiency of processing and the number of repeats. However, deletion of repeats 25–16 causes a more drastic loss of processing than deletion of repeats 52–26. In addition, repeats 27–52 activate processing less well than the N-terminal 25 repeats, indicating that the two halves of the CTD are not completely interchangeable. The CTD is required for the co-transcriptional capping of mRNAs (McCracken et al., 1997a), and the effect of CTD truncation may be the result of loss of the 5′ cap. We therefore have analysed the effect of CTD truncation on capping of U2G transcripts by binding to GST–eIF4E as previously described (McCracken et al., 1997a), followed by RNase protection analysis (Figure 1E). We have used a short synthetic capped U2G RNA, which gives a 160 nucleotide protection product, as a positive control, and the uncapped VAI transcripts serve as a negative control. Transcription by endogenous pol II (lanes 1 and 2) and pol II expressed from the WT CTD construct (WT, lanes 3 and 4) results in a similar level of capping when normalized to the positive control. Capping is reduced using either the 27–52 CTD construct (27–52, lanes 5 and 6, 70%) or the 1–15 construct (1–15, lanes 7 and 8, 61%) and reduced further but not abolished when only five repeats remain (Δ5, lanes 9 and 10, 30%). These results are in agreement with the CTD requirements for mRNA capping (Fong and Bentley, 2001). Thus, 3′ processing of U2 RNA is more severely affected by loss of the CTD than capping, indicating that the effect on processing is not due entirely to loss of the 5′ cap. This does not, however, rule out any role of the cap in the processing reaction. The −111 readthrough RNA is mainly unselected, suggesting that most of it is uncapped (see lanes 7 and 8). We have noted that this RNA is relatively unstable through the cap-binding procedure and is not always recovered quantitatively (see, for example, Δ5 in lanes 9 and 10). The CTD is required for transcription of the endogenous U2 genes To assess the effect of CTD truncation on nascent transcription rather than steady-state levels of RNA, we have carried out nuclear run-on analysis. Analysis of nascent RNA from transfected genes is complicated by the PSE-dependent transcription across the initiation site from upstream, derived from aberrant initiation or read-around (see Figure 1). We therefore have analysed the level of nascent transcription of the endogenous U2 genes when most of the CTD is deleted. We have shown that transcription continues beyond the 3′ box of the U2 genes (Cuello et al., 1999) and we have now mapped termination to ∼1 kb downstream from the site of initiation (see Figure 5). To ensure a detectable signal in this analysis, we used sets of three oligodeoxynucleotides (oligos) in each slot corresponding to contiguous regions of 143 (oligo 1) and 240 bp (oligos 2 and 3) within the transcription unit of the U2 genes (Figure 2A). Transcription of the endogenous genes is readily detected using these U2 gene probes. Addition of α-amanitin specifically abolishes the signals over these probes, while transcription of the pol III-dependent 7SK gene is unaffected. Transfection of the WT CTD restores transcription of the U2 genes, and the profile of transcription is unchanged. However, little transcription is detected when the Δ5 construct is transfected. This suggests that the drop in specific transcription seen in the steady-state analysis of the U2G constructs reflects reduced transcription in the absence of the CTD. It also suggests that the aberrant transcription across the site of initiation detected in the steady-state analysis (see, for example, Figure 1A and C, lanes 7) is either the result of a low level of transcription or does not occur on the endogenous genes. Taken together, the results of the steady-state and nuclear run-on analyses indicate that the CTD participates in both transcription of the human U2 genes and 3′ end formation of the transcripts. Figure 2.The CTD of pol II is required for transcription of U2 genes. A diagram of the structure of the U2 gene, with the relative positions of the probes marked below. The numbers noted next to the probes indicate the position of the probes relative to the site of initiation. The results of run-on analysis in the absence and presence of α-amanitin, and with co-transfected WT CTD or Δ5 CTD constructs, are shown below each probe. AS is a non-specific probe, and 7SK was used as an α-amanitin-resistant control in this and experiments shown in Figures 5 and 6. VA indicates that the probe is complementary to VAI transcripts. Download figure Download PowerPoint Inhibition of CTD kinases affects recognition of the 3′ box Pol II is known to exist in two alternative forms: pol II containing an unmodified form of the CTD is termed pol IIa, whereas pol II containing a hyperphosphorylated CTD is termed pol IIo. Several CDKs phosphorylate the CTD during transcription. CDK7 is a component of the pol II basal transcription factor TFIIH, CDK8 is a component of mediator that plays a role in transcriptional activation, and CDK9 is the kinase subunit of the positive transcription elongation factor, P-TEFb. Whereas CDK7 and CDK9 activate transcription (see Price, 2000), CDK8 is inhibitory (Akoulitchev et al., 2000). We have tested the effect of inhibitors of CTD kinases on transcription of U2 genes and formation of the 3′ end of the transcripts (Figure 3). HeLa cells transfected with the U2G construct (Figure 1A) were treated with 5,6-dichloro-1-β-D- ribofuranosylbenzimidazole (DRB), 1-(5-isoquino linylsulfonyl)-3-methylpiperazin (H-7) and 8-(methylthio) -4,5-dihydrothieno[3′,4′:5,6]benzoisoxazole-6-carboxamide (KM05283) (100 μM final concentration for 24 h) (see Materials and methods). All three inhibitors cause loss of hyperphosphorylation of the CTD used at this concentration in vivo (Figure 3A; Dubois et al., 1994; Lavoie et al., 2001) and inhibit CTD kinases in vitro (Serizawa et al., 1993; Mancebo et al., 1997). The steady-state level of U2 globin RNA is reduced in all cases, but is still readily detectable, and the accuracy of initiation is unaffected (Figure 3B). The effect of these inhibitors on 3′ end formation is, however, dramatic (Figure 3C). DRB causes a large proportion of transcription to read through the 3′ box, and H-7 and KM05283 cause practically complete readthrough. Since the riboprobes used have a region of complementarity to the endogenous U2 gene transcripts (see Figure 1A), we can also detect the endogenous pre-U2 (En pre-U2) and readthrough (En U2 RT) (Figure 3D). There is also a drastic change in the ratio of pre-U2 RNA to readthrough transcribed from the endogenous genes (Figure 3D). Figure 3.Inhibition of CTD kinases affects recognition of the 3′ box. (A) The results of western blot analysis of cells treated with 100 μM of the CTD kinase inhibitors (KI) shown above the lanes using an antibody specific for the large subunit of pol II (α-Pol II LS). The positions of the hyperphosphorylated CTD form (IIo) and the hypophosphorylated CTD form (IIa) are indicated on the right. (B) The results of S1 analysis of RNA transcribed from the U2G construct in the presence of 100 μM of the kinase inhibitors (KI) shown above the lanes. The positions of the S1 products are indicated on the right. The amount of properly initiated U2G-specific S1 product relative to the amount in untreated cells (%UT) is shown below each lane. (C) The results of RNase protection analysis of RNA transcribed from U2G in the presence of 100 μM of each kinase inhibitor (KI). The positions of the protected products are noted on the right. The amount of properly initiated U2G-specific protection products relative to the amount in untreated cells (%UT) is noted underneath each lane. A breakdown of the relative amount of correct 3′ end and readthrough (RT) is also noted below each lane. (D) The results of RNase protection analysis of endogenous U2 precursor RNA transcribed in the presence of 100 μM of each kinase inhibitor (KI). The positions of the protected products are noted on the right. A breakdown of the relative amount of correct pre-U2 3′ end and readthrough (RT) is also noted below each lane. Download figure Download PowerPoint A time course was carried out using KM05283 (100 μM) and an additional inhibitor of CTD kinases, N-(2-[methylamino] ethyl)-5-isoquinolinesulfonamide hydrochloride (H-8; 200 μM) (Dubois et al., 1994) (Figure 4) to assess how rapidly the effect of these kinase inhibitors on 3′ end formation could be detected. With both inhibitors, the change in ratio of correct 3′ end to readthrough transcribed from the U2G construct is evident after 1 h (Figure 4A). The proportion of readthrough continues to increase with time, possibly due to turnover of RNA made before addition of inhibitor, and most of the RNA is detected as readthrough after 4 h treatment (lanes 3 and 6). Again, the effect is mirrored in the change in ratio of U2 precursor to readthrough transcribed from the endogenous U2 genes (Figure 4B). Both KM05283 and H-8 efficiently inhibit hyperphosphorylation of the CTD of pol II during the time course (Figure 4C). Figure 4.Inhibition of 3′ end processing by CTD kinase inhibitors is detectable within 1 h. (A) The results of RNase protection analysis of RNA transcribed from U2G in the presence of 100 μM of each kinase inhibitor (KI). The positions of the protected products are noted on the right. The time of incubation of cells with the inhibitors is noted above the lanes. A breakdown of the relative amount of correct pre-U2 3′ end and readthrough (RT) is also noted below each lane. (B) The results of RNase protection analysis of endogenous U2 precursor RNA transcribed in the presence of 100 μM of the kinase inhibitors (KI) shown above the lanes. The positions of the protected products are indicated on the right. The time of incubation of cells with the inhibitors is noted above the lanes. A breakdown of the relative amount of correct pre-U2 3′ end and readthrough (RT) is also noted below each lane. (C) The results of western blot analysis of cells treated with 100 μM of the CTD kinase inhibitors KM05283 and H-8 using an antibody specific for the large subunit of pol II. The positions of the hyperphosphorylated CTD form (IIo) and the hypophosphorylated CTD form (IIa) are indicated on the right. The time of incubation of cells with the inhibitors is noted above the lanes. (D) The results of 5′ cap analysis of U2G RNA from untreated and KM05832-treated cells. RNase protection analysis was carried out on RNA selected by GST–eIF4E (capped) or unselected (uncapped). The percentage of capped RNA (see Materials and methods) is noted below the lanes. Download figure Download PowerPoint Figure 5.Transcription of the U2 gene terminates 1 kb downstream from the site of initiation and is unaffected by CTD kinase inhibitors. (A) A diagram of the structure of the U2 gene, with the relative positions of the run-on probes marked below. The numbers noted next to the probes indicate the end of the probes used relative to the site of initiation. The results of run-on analysis, in the absence or presence of α-amanitin, and hybridization of synthetically produced RNA to the probes are shown below each probe. (B) A graphic representation of the results of the run-on analysis in (A) as a percentage of the signal over probe 1 after subtraction of α-amanitin-resistant transcription and correction for the hybridization efficiency of each probe. Download figure Download PowerPoint Efficient capping of mRNAs requires that the CTD is phosphorylated (McCracken et al., 1997a). It is therefore possible that inhibition of CTD kinases indirectly affects processing by inhibiting capping. To address this, we have determined the proportion of RNA selected by GST–eIF4E after treatment of cells with KM05283 for 4 h (Figure 4D). RNA selection is reduced after treatment of the cells with KM05283 (lanes 3 and 4), but a significant proportion (58%) of the, mainly readthrough, RNA is still selected and therefore capped. Identical results were obtained using RNA from H-8-treated cells (data not shown). Thus, the effect of the CTD kinase inhibitors on 3′ processing is not entirely due to inhibition of capping. Capping may be less sensitive to these drugs than 3′ processing because relatively little phosphorylation of the CTD is required to activate capping (McCracken et al., 1997a). In an attempt to identify the CTD kinase(s) involved in the processing reaction, we have transfected constructs encoding kinase mutants of CDK7 (Makela et al., 1995), CDK8 (Akoulitchev et al., 2000) and CDK9 (Garriga et al., 1996), since CDK9 kinase mutants have been shown to have a dominant-negative effect on transcription of HIV and protein-coding genes (Mancebo et al., 1997; Kanazawa and Peterlin, 2001). Expression of these mutants had no significant effect on the ratio of readthrough to precursor of endogenous U2 gene transcripts (Supplementary data available at the EMBO Journal Online). Transcription of the U2 genes terminates 1 kb downstream from the site of initiation and is unaffected by CTD kinase inhibitors As noted above, CTD kinases play a role in initiation an" @default.
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• W1972628058 title "The C-terminal domain of pol II and a DRB-sensitive kinase are required for 3' processing of U2 snRNA" @default.
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