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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2009 Aug 7;106(34):14276–14280. doi: 10.1073/pnas.0903642106

Phosphorylation of the RNA polymerase II C-terminal domain by TFIIH kinase is not essential for transcription of Saccharomyces cerevisiae genome

Sun Woo Hong a,b, Seong Min Hong a, Jae Wook Yoo a,b, Young Chul Lee c, Soyoun Kim d, John T Lis e, Dong-ki Lee a,b,1
PMCID: PMC2732804  PMID: 19666497

Abstract

Ser-5 phosphorylation of the RNA polymerase II (Pol II) C-terminal domain by TFIIH kinase has been implicated in critical steps in mRNA synthesis, such as Pol II promoter escape and mRNA 5′-capping. However, the general requirement and precise role of TFIIH kinase in Pol II transcription still remain elusive. Here we use a chemical genetics approach to show that, for a majority of budding-yeast genes, specific inhibition of the yeast TFIIH kinase results in a dramatic reduction in both mRNA level and Ser-5 C-terminal domain phosphorylation. Surprisingly, inhibition of TFIIH kinase activity only partially affected both Pol II density and Ser-2 phosphorylation level. The discrepancy between mRNA level and Pol II density is attributed to the defective 5′-capping, which results in the destabilization of mRNAs. Therefore, contrary to the current belief, our study points strongly toward a minor role of TFIIH kinase in Pol II transcription, and a more significant role in mRNA capping in budding yeast.

Keywords: chemical genetics, Kin28, mRNA capping

The C-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) contains a series of YSPTSPS heptad repeats that are multiply-phosphorylated during the eukaryotic transcription cycle. Phosphorylation of the Ser-5 residue of the CTD heptad repeats has been implicated in multiple aspects of mRNA synthesis, such as promoter clearance (for transition from initiation to early elongation) and 5′-end capping of pre-mRNA (1). CTD Ser-2 phosphorylation has been implicated in productive elongation and the 3′-end processing of the transcript (2). However, it remains unclear whether CTD phosphorylation is required for transcription in general or functions in a promoter- and activator-specific manner. Moreover, the requirement of Ser-5 phosphorylation for subsequent Ser-2 phosphorylation and transcriptional elongation remains controversial. An earlier study suggested that the CTD Ser-5 phosphorylation by Kin28 does not affect the Ser-2 phosphorylation level (3). In contrast, a recent study suggests that the CTD Ser-5 phosphorylation stimulates Ser-2 phosphorylation by BUR1/BUR2 kinase (4). These issues must be resolved to gain a clear understanding of the role of CTD phosphorylation in Pol II transcription in vivo.

Temperature-sensitive mutants of CTD kinases have been used to study the functions of these enzymes in vivo (5). A genome-wide expression analysis using a temperature-sensitive mutant of KIN28, the Saccharomyces cerevisiae TFIIH kinase gene, showed that the loss of Kin28 function resulted in global shutdown of Pol II transcription (6). However, it has been demonstrated that the same mutation also disrupts other subunits in the TFIIH complex upon temperature shift, which makes the unambiguous functional analysis of this kinase difficult (7). Inhibition of CTD kinases with conventional pharmacological inhibitors is also problematic because these agents can nonspecifically inhibit other kinases (8). To overcome these obstacles, we used the “analog-sensitive” kinase-mutant strategy to dissect the unique roles of specific CTD kinases (9). In this strategy, a specific amino acid within the ATP binding pocket of the target kinase is mutated to a smaller one to enlarge the binding pocket. Thus, a bulky ATP analogue kinase inhibitor, such as NA-PP1, can fit only into the active site of the analog-sensitive kinase mutant, which results in quick and reversible inhibition of the mutant kinase with single-kinase specificity. Using an analog-sensitive KIN28 mutant yeast strain (KIN28as) (10), we aimed to decipher both the general requirement and the specific roles of TFIIH kinase in the transcription of the yeast genome by Pol II in vivo.

Results

Inhibition of Kin28p Resulted in Global Impairment of Gene Expression.

First, we treated both the KIN28as and corresponding wild-type strains with several different concentrations of NA-PP1. The mRNA levels of ACT1 and HXT1 genes were determined with the real-time quantitative RT-PCR (qRT-PCR). In the wild-type yeast strain, the mRNA levels of both genes were not affected by the NA-PP1 treatment, while they were significantly reduced in the KIN28as mutant strain under the same conditions (Fig. 1). Treatment of the KIN28as mutant with 1-μM NA-PP1 was sufficient to cause a significant reduction in ACT1 and HXT1 mRNA levels, and maximal reduction was achieved when the yeast was treated with 5-μM NA-PP1 (see Fig. 1A). A time-course experiment showed the more rapid reduction of HXT1 mRNA relative to ACT1 upon inhibitor treatment that likely reflects the different half-lives of these mRNAs (see Fig. 1B) (11). These results demonstrate that specific inhibition of TFIIH kinase activity by NA-PP1 affects the steady-state mRNA level of the genes tested.

Fig. 1.

Fig. 1.

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Inhibition of Kin28p kinase activity affects ACT1 and HXT1 mRNA level. mRNA level of ACT1 and HXT1 upon Kin28 kinase inhibition. NA-PP1 was treated with varying concentrations for 1 h (A) or for varying time periods (with 1 μM) (B) to either KIN28as (as) or isogenic wild-type (WT) strains, and mRNA levels of ACT1 (Left) and HXT1 (Right) were analyzed with qRT-PCR. Expression levels were normalized with the Pol I-driven-Rdn18 RNA levels, and the abundance of each mRNA relative to the no-treatment control is presented. At least 3 independent experiments were performed and average values with positive SD are presented.

To acquire a comprehensive view of the consequences of Kin28p inhibition, we performed a genome-wide gene expression microarray experiment using the Nimblegen microarray platform. The KIN28as strain was treated with varying concentrations of NA-PP1 in DMSO (0, 1, 2.5, and 5 μM), and the genome-wide gene expression responses were measured. Microarray data were subjected to quantile normalization (12), and each gene-expression value was generated using the Robust Multichip Average algorithm (13). Unexpectedly, analysis of the global gene-expression levels revealed an inconsistency between the microarray and qRT-PCR data: expression of ACT1 and HXT1 was only moderately reduced according to the microarray data [supporting information (SI) Fig. S1]. This result suggests that the typically used array-normalization algorithm is not suitable for samples with global defects in gene expression. This observation might also explain the discrepancy between our study and a recent DNA microarray study, which reported that inhibition of Kin28p by the same method used in the present study did not affect global mRNA levels (14). Therefore, we performed an additional normalization procedure on our genome-wide gene-expression data, which is based on the qRT-PCR result of individual mRNAs.

The typical microarray normalization is performed under the assumption that the global gene-expression level does not change. As we observed that the gene expression was broadly inhibited under our experimental conditions, we subtracted a normalization factor x from each gene-expression ratio. For further normalization of the gene-expression ratio, another normalization factor, y, was introduced. The resulting equation is a = (b–x)y, where a and b denote the gene-expression ratios obtained from qRT-PCR and array data, respectively. Each normalization factor was determined by using the gene-expression ratio values of genes that displayed reduced mRNA level (ACT1 and HXT1) and those with little changes in mRNA level (YGR109W-B, Ty element gene) upon NA-PP1 treatment. Whole-microarray data were normalized by the normalization factors, and we found that this normalization process resulted in good agreement between the quantitative PCR and microarray data (see Fig. S1 and Table S1).

The DNA microarray data revealed global impairment of gene expression upon chemical inhibition of Kin28p kinase activity. In the presence of 5-μM NA-PP1, about 58% of genes (out of 5,747 yeast genes tested) showed a more than 2-fold reduction in mRNA levels relative to the control, and only 9% of the genes showed mRNA levels unchanged or increased (32% and 14% for 1-μM and 2.5-μM NA-PP1, respectively) (Fig. 2A). A heat-map graph generated by clustering analysis also showed a global reduction in mRNA levels in an NA-PP1 dose-dependent manner (Fig. 2B).

Fig. 2.

Fig. 2.

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Inhibition of Kin28p kinase activity affects global mRNA level. Genome-wide gene-expression microarray data analysis. (A) Distribution of the gene-expression ratio upon treatment of the KIN28as strain with NA-PP1. The log2 of each gene-expression ratio (NA-PP1 treatment to control) is plotted as a function of the percentile rank of the gene-expression ratio. Black, gray, and blue line represents 1, 2.5, and 5 μM NA-PP1 array data, respectively. (B) TreeView representation of microarray data obtained with KIN28as strain grown in the presence of various concentrations of NA-PP1. The gene-expression ratio (NA-PP1 treatment condition to control) of each gene was calculated and processed with the Cluster program. Hierarchical clustering was performed, and results were graphically browsed through the TreeView program: (red) increased expression compared to control; (green) decreased expression; (black) constant expression. (C) Correlation between transcription rate (x-axis) and transcript sensitivity to Kin28p kinase inhibition (y-axis). Each gene was arranged in order of its transcription rate, and the percentile rank of each gene was plotted as a function of the expression-ratio average of 100 adjacent genes (the 5-μM NA-PP1 array data were used in this analysis).

Highly Transcribed Genes Are Sensitive to Kin28p Inhibition.

We then applied our microarray data to the transcription-rate database generated from a genome-wide run on studies in budding yeast (15). Genes were sorted by their transcription rate, and average expression ratios (i.e., expression in the presence of 5-μM NA-PP1/expression under control conditions, such as DMSO) were calculated for 100 adjacent genes. The resulting values were presented as a scatter-chart (Fig. 2C). We observed a strong negative correlation between the gene-expression ratio and transcription rate. These results suggest that the TFIIH kinase activity is essential for maintaining steady-state expression of genes with high transcription rates, but is less essential for maintaining the expression of genes with low transcription rates.

Repressor-activator protein 1 (Rap1) is a well-characterized DNA-binding protein that activates transcription of target genes, such as ribosomal protein-encoding genes (16). Because the transcription rate of Rap1-target genes is extremely high during exponential growth of yeast, they might be highly susceptible to Kin28p inhibition by NA-PP1 treatment (17). As expected, we found in our array data that NA-PP1 treatment caused significant repression of ribosomal protein-encoding genes (Fig. S2). In addition, we analyzed the expression of a group of Rap1 target genes identified by another study (16) and found that expression level of these genes was highly sensitive to the inhibition of Kin28p activity as well (see Fig. S2).

In contrast, further analysis of our array data revealed that some genes that encode proteins involved in the transcription process showed consistent gene expression upon NA-PP1 treatment. These genes include ones that encode a component of the general transcription factor complex (TAF7), a gene-specific transcriptional regulator (MET31), and a transcription initiation factor (RRN7) (Fig. 3A and see Table S1). Interestingly, we also found that the mRNA level of KIN28 gene itself was relatively insensitive to Kin28p kinase inhibition (see Fig. 3A and Table S2).

Fig. 3.

Fig. 3.

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mRNA level and ChIP analysis. (A) mRNA levels in KIN28as (as) and wild-type (WT) yeast strains in the absence and presence of NA-PP1. Yeast cells were treated with varying concentrations of NA-PP1 for 1 h, and mRNA levels of each gene were analyzed by qRT-PCR. mRNA levels of each gene were normalized with the Rdn18 RNA levels and relative mRNA level to 0-μM NA-PP1 treatment control was calculated. For each gene (listed across the x-axis), the relative mRNA levels in the KIN28as strain were divided by the relative mRNA levels in the respective wild-type strain. (B–E) ChIP analysis of DNA from wild-type (WT) and KIN28as (as) yeast grown in the absence (0 μM) and presence (5 μM) of NA-PP1. Cells were treated with 5-μM NA-PP1 for 1 h, and the DNA in the cell lysates was immunoprecipitated with one of several specific antibodies. The immunoprecipitated DNA was then quantified by qRT-PCR analysis. Antibodies specific for the Ser-5 phosphorylated version of the CTD (H14) (B), the Ser-2 phosphorylated version of CTD (H5) (C), Rpb3 (D), and PolII (8WG16) (E) were used separately in the immunoprecipitations. Immunoprecipitated DNA from a nontranscribed region in chromosome V was used for normalization. At least 3 independent experiments were performed and average values with positive SD are presented. (F) Summary of mRNA and ChIP data. From the KIN28as strain mRNA level and ChIP data, ratio of NA-PP1 treated sample to nontreated control was calculated and presented with positive SD.

Ser-5 CTD Phosphorylation upon Kin28p Inhibition.

To more fully understand the role of CTD phosphorylation by the TFIIH kinase in Pol II transcription, we performed a ChIP experiment. First, using an antibody specific for the Ser-5 phosphorylated version of the CTD (H14), we found that there was a dramatic reduction in the Ser-5 CTD phosphorylation level in the promoter regions of Kin28p-dependent genes (e.g., ACT1, RPL16A, RPL25, and TPI1) in the KIN28as strain upon NA-PP1 treatment (Fig. 3B). In contrast, the promoter regions of Kin28p-independent genes did not show a significant reduction in Ser-5 phosphorylation upon NA-PP1 treatment. The reduction in Ser-5 phosphorylation level closely correlated with the reduction in mRNA level (see Fig. 3A). For example, analysis of Kin28p-dependent gene showed similar degree of reduction in Ser-5 phosphorylation (65–82% reduction) level and mRNA (74–88% reduction) level (Fig. 3F).

Ser-2 CTD Phosphorylation and Pol II Occupancy upon Kin28p Inhibition.

Ser-5 CTD phosphorylation is thought to be the prerequisite for Ser-2 CTD phosphorylation by CDK9 (18). To test whether the reduced Ser-5 CTD phosphorylation by Kin28p inhibition affects Ser-2 CTD phosphorylation, we used a phosphorylated Ser-2 specific antibody (H5) to examine the level of Ser-2 CTD phosphorylation in the ORF region of Kin28p-dependent and -independent genes upon NA-PP1 treatment of the KIN28as strain. To our surprise, we found that, for both Kin28p-dependent and -independent genes, the Ser-2 CTD phosphorylation level was not significantly affected by Kin28p inhibition (Fig. 3C). In the ORF region of Kin28p-independent genes, Ser-2 phosphorylation levels after NA-PP1 treatment were 71 to 95% of those without NA-PP1 treatment; for Kin28p-dependent genes, we also observed the presence of a substantial level of Ser-2 phosphorylated CTD (59–83%) upon NA-PP1 treatment.

Next, we measured the Pol II density on Kin28p-dependent and -independent genes by using an antibody specific to Rpb3, a Pol II subunit. Again, total Pol II occupancy, as measured by Rpb3 level, was not dramatically affected for both Kin28p-dependent and -independent genes upon NA-PP1 treatment (Fig. 3D). Significant amounts of Rpb3 were still present in the ORF region of Kin28p-dependent genes (52–63%, relative to NA-PP1 nontreated control). This observation was confirmed by ChIP experiments carried out with the 8WG16 antibody, which recognizes a nonphosphodependent epitope on the CTD. It showed that 48 to 65% of Pol II still occupied the ORF region of Kin28p-dependent genes (Fig. 3E). The ratio between Ser-2 phosphorylation level and Pol II level also did not change upon NA-PP1 treatment (Fig. S3). Therefore, for Kin28p-dependent genes, the inhibition of the TFIIH kinase activity severely affects CTD Ser-5 phosphorylation but does not cause significant reduction of either total Pol II occupancy or CTD Ser-2 phosphorylation level (see Fig. 3F).

Restoration of mRNA Level in a XRN1-Deletion Strain.

The partial reduction of Pol II level on the body of Kin28p-dependent genes from a Kin28as strain treated with NA-PP1 did not correlate with the dramatic reduction of the mRNA level (see Fig. 3F). Thus, we hypothesized that the reduction in the mRNA levels of Kin28p-dependent genes could result from inefficient capping of the transcripts, which leads to the rapid degradation of uncapped mRNAs (19, 20).

To confirm this hypothesis, we first tested whether the recruitment of capping enzymes was compromised when the KIN28as strain were treated with NA-PP1. In accordance with previously published data (21–23), ChIP experiments confirmed that capping enzyme recruitment was severely reduced upon NA-PP1 treatment in the KIN28as strain, but not in the wild-type strain (Fig. 4A).

Fig. 4.

Fig. 4.

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Restoration of mRNA level in a XRN1-deletion strain. (A) ChIP of Ceg1 recruitment at the promoter of Kin28p-dependent genes. Wild-type (WT) and KIN28as (as) strains were treated with 5-μM NA-PP1 for 1 h, and the DNA in the cell lysates was immunoprecipitated with hemagglutinin (HA) antibody for HA-tagged Ceg1. The immunoprecipitated DNA was then quantified by qRT-PCR analysis. Immunoprecipitated DNA from a nontranscribed region in chromosome V was used for normalization. At least 3 independent experiments were performed and average values with SD are presented. (B) mRNA level analysis. KIN28as and KIN28as xrn1Δ strains were treated with 5-μM NA-PP1 for 1 h, and the expression levels of each gene were analyzed with qRT-PCR. Expression levels were normalized with Rdn18 RNA level, and the abundance of each mRNA level relative to control (DMSO treatment) is shown on the y-axis. At least 3 independent experiments were performed and average values with positive SD are presented.

Next, we deleted the XRN1 gene from the KIN28as strain. Xrn1p is a 5′ to 3′ exonuclease that is responsible for the degradation of uncapped mRNA transcripts, and deletion of XRN1 is known to lead to the partial stabilization of uncapped transcripts (24). We found that in the XRN1-deleted KIN28as strain, the reduction in mRNA level upon NA-PP1 treatment was indeed partially restored (Fig. 4B). However, XRN1 deletion did not completely restore mRNA concentrations to normal levels and the effects of XRN1 deletion differed for different genes, probably reflecting the fact that another exonuclease might also be involved in the degradation of uncapped transcripts (24). In addition, the partial reduction of Pol II level upon Kin28p inhibition might be responsible for the incomplete restoration of mRNA level. Nevertheless, these findings, along with the ChIP results, strongly support the idea that defective capping of mRNAs is the major cause of the reduced steady-state mRNA levels upon Kin28p inhibition.

Discussion

Our results demonstrate that the inhibition of the TFIIH kinase results in defective mRNA capping for a majority of yeast genes. This leads to the degradation of mRNAs by exonucleases, which results in the dramatic reduction in steady-state mRNA levels. However, inhibition of the TFIIH kinase did not significantly affect other transcriptional processes, such as overall Pol II density and Ser-2 CTD phosphorylation. Our observation is in line with previous results that showed that Cdk9p, the main CTD Ser-2 kinase, is critical for 3′-end processing of mRNAs, but less so for Pol II transcription (25, 26). In addition, we demonstrate that the expression of a small portion of the yeast genome is Kin28p-independent, which expands our previous observation (27, 28).

Our findings are also in agreement with a previous study using a kinase-crippled mutant version of Kin28p (22), which showed that for 2 genes tested, reduced Kin28p kinase activity inhibits capping-enzyme recruitment but does not affect Pol II distribution. Another previous study on Kin28p and Srb10p, 2 Ser-5 CTD kinases, suggests that Kin28p is responsible for high-level CTD phosphorylation, whereas Srb10p maintains low-level CTD phosphorylation (10). Combining these results with our genome-scale findings, we propose the following model. Low-level Ser-5 phosphorylation, presumably triggered by Srb10p or other CTD kinases, is sufficient for the 5′-capping of mRNAs of low transcription-rate genes; however, for high transcription-rate genes, full-level CTD phosphorylation by Kin28p is necessary for optimal mRNA capping. We found that the mRNA level of most Kin28p-independent genes were not affected in Srb10as or Srb10as/Kin28as mutants upon NA-PP1 treatment (data not shown), suggesting that kinases other than Srb10 might play an important role in maintaining the basal-level Ser-5 phosphorylation.

During the analysis of gene-expression microarray data, we found that the typical normalization method does not apply when the experimental condition induces global reduction of mRNA level. By introducing individual gene-expression data for further normalization, we were able to acquire accurate genome-wide gene-expression data upon Kin28 inhibition. Therefore, we suggest that extra caution is needed when genome-wide expression analysis is performed on transcription factors whose defect can lead to global changes in mRNA level.

Finally, we note that the precise transcription program in higher organisms is distinct from that in yeast, such as the wide existence of paused Pol II (29) and the presence of some early elongation factors, such as NELF (30). Thus, it would be intriguing to probe the functional consequences of TFIIH kinase inactivation with the analog-sensitive kinase mutant strategy in mammalian cells.

Materials and Methods

Yeast strain KIN28as and isogenic wild-type strains were grown in YPD media. For HA-tagged Ceg1p expression, each yeast strain was transformed with pRS315-HA3-CEG1. To create an XRN1-deleted KIN28as strain, the XRN1 ORF was replaced by the hygromycin-resistance gene. For NA-PP1 treatment, yeast cells were grown to OD600 of 0.5 to 1.0 at 30 °C, and the indicated concentrations of NA-PP1 were added to the cell cultures. For qRT-PCR analysis, RNA was isolated using the hot phenol method and reverse transcribed using the High Capacity RNA-to-cDNA Kit (Applied Biosystems). Quantitative real-time PCR was performed with the StepOne Real-Time PCR System (Applied Biosystems) using SYBR Premix Ex Taq (Takara). The gene-expression microarray experiments and analyses were performed by using the Nimblegen S. cerevisiae 4-Plex Array platform. For ChIP experiments, cross-linked chromatin from each condition was immunoprecipitated with indicated antibodies. DNA was eluted, extracted, precipitated, and subjected to quantitative real-time PCR analysis. Full description of the materials and methods used can be found in SI Text. Sequence information of all primers used in this study is listed in Table S3.

Supplementary Material

Supporting Information
supp_106_34_14276__index.html (851B, html)

Acknowledgments.

We thank Profs. Eun Jung Cho (Sungkyunkwan University, Suwon, Korea) and Steve Hahn (University of Pennyslvania School of Medicine, Philadelphia) for plasmids and yeast strains. This study was supported in part by Korean Research Foundation Grant KRF-2006–312-C00255 and Global Research Laboratory Grant 2008-00582 from the Korea Foundation for International Co-operation of Science and Technology (to D.-k. L.), a National Research Laboratory grant from the Korea Science and Engineering Foundation (to S.K.), and National Institutes of Health Grant GM25232 (to J.T.L.).

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/cgi/content/full/0903642106/DCSupplemental.

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