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. Author manuscript; available in PMC: 2017 Jan 21.
Published in final edited form as: Mol Cell. 2016 Jan 21;61(2):297–304. doi: 10.1016/j.molcel.2015.12.021

Direct analysis of phosphorylation sites on the Rpb1 C-terminal domain of RNA polymerase II

Hyunsuk Suh 1,2, Scott B Ficarro 1,2, Un-Beom Kang 1,2, Yujin Chun 1, Jarrod A Marto 1,2, Stephen Buratowski 1,*
PMCID: PMC4724063  NIHMSID: NIHMS747877  PMID: 26799764

Summary

Dynamic interactions between RNA polymerase II and various mRNA processing and chromatin modifying enzymes are mediated by the changing phosphorylation pattern on the C-terminal domain (CTD) of polymerase subunit Rpb1 during different stages of transcription. Phosphorylations within the repetitive heptamer sequence (YSPTSPS) of CTD have primarily been defined using antibodies, but these do not distinguish different repeats or allow comparative quantitation. Using a CTD modified for mass spectrometry (msCTD), we show that Ser5-P and Ser2-P occur throughout the length of CTD and are far more abundant than other phosphorylation sites. msCTD extracted from cells mutated in several CTD kinases or phosphatases showed the expected changes in phosphorylation. Furthermore, msCTD associated with capping enzyme was enriched for Ser5-P while that bound to the transcription termination factor Rtt103 had higher levels of Ser2-P. These results suggest a relatively sparse and simple "CTD code".

Introduction

RNA polymerase II (RNApII) transcribes all mRNAs and many noncoding RNAs. Rpb1, its largest subunit, has a C-terminal domain (CTD) consisting of roughly 25 repeats of the heptad sequence ‘Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7’. Five of these amino acids can be phosphorylated, and the two prolines can exist in cis or trans configurations (reviewed in Corden, 2013; Eick and Geyer, 2013). During the transcription cycle, dynamic CTD phosphorylation serves as a marker of RNApII progression along the gene, with specific phosphorylations creating binding sites for recruitment of chromatin regulators and RNA processing factors at specific stages of transcription (Buratowski, 2009; 2003). Chromatin immunoprecipitation (ChIP) experiments using antibodies that recognize phosphorylations at individual CTD residues showed that modification of Ser5 (Ser5-P) was strongest near the promoter while Ser2-P was stronger downstream (Komarnitsky et al., 2000). Later genome-wide experiments confirmed this pattern and mapped phosphorylations at Tyr1, Thr4, and Ser7 (Bataille et al., 2012; Mayer et al., 2010; Tietjen et al., 2010). However, these ChIP experiments have limitations: 1) because antibody affinities vary, it is impossible to compare phosphorylation levels at different positions in the heptad repeat, 2) epitopes typically span several amino acids, so nearby phosphorylations can interfere with antibody recognition and hinder proper detection (Chapman et al., 2007; Hintermair et al., 2012), 3) because antibodies are multivalent and CTD has many repeats, avidity effects are likely to produce non-linear responses between phosphorylation and reactivity, and 4) due to its repetitive nature, antibodies do not reveal the spatial distribution of phosphorylations along the CTD.

Results

CTD modified for mass spectrometry analysis functionally substitutes for wild-type CTD

Tandem mass spectrometry (MS/MS) provides much of the information unavailable from antibody studies. However, the CTD presents two challenges for MS/MS. First, it lacks a suitable distribution of protease cleavage sites that could generate unique peptides amenable to MS/MS analysis. Second, its repetitive structure makes it difficult to map phosphorylations to specific heptads. To overcome these hurdles for the yeast Rpb1 protein, selected Ser7 residues were mutated to Lys or Arg at two and three repeat intervals. Several studies have shown that two repeats represent the minimal functional unit of the CTD (reviewed in Corden, 2013; Eick and Geyer, 2013). Ser7 was chosen because Lys or Arg is found at position 7 in several heptads of metazoan CTDs, and all Ser7s can be mutated to alanine without affecting viability in several species (reviewed in Corden, 2013; Eick and Geyer, 2013). Di- and tri-heptads with unique masses were produced by taking advantage of several naturally occurring non-consensus residues at position 7 and by substituting four other Ser7s with Thr. In addition, to facilitate purification and detection, a Prescission protease cleavage site followed by 8xhistidine tag was inserted at the N-terminal end of CTD, and a 3xFLAG tag at the C-terminus. The primary structure of this msCTD is shown in Figure 1A.

Figure 1.

Figure 1

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msCTD supports cell growth and is functional for factor recruitment. (A) Amino acid sequences of wild-type CTD and msCTD shown with heptad repeats stacked vertically. msCTD has an additional N-terminal Prescission protease cleavage site (LEVLFQ/GP) followed by a 8xhistidine tag, and a C-terminal 3xFLAG tag. Mutated amino acids (K, R, or T incorporated at position 7 to generate tryptic peptides of unique mass) are highlighted in green. (B) Rpb1 with msCTD complements rpb1Δ. Plasmids carrying wild type RPB1 (WT) or RPB1-msCTD (MC) were introduced into a yeast strain lacking the chromosomal RPB1 by plasmid shuffling. Cell growth was assayed on rich media (YPD) or synthetic complete media (SC) plates. Three-fold serial dilutions of cultures were spotted onto plates and incubated at temperatures as labeled. (C) Immunoblotting of whole cell lysates from WT or MC strains. FLAG epitope is present only on Rpb1-msCTD. Rpb3 serves as a loading control for total RNApII. The asterisk (*) on the Ser7-P panel is a reminder that msCTD carries a reduced number of Ser7 residues due to S7K/R/T substitutions. (D) RNApII subunit Rpb3; CTD binding proteins Ceg1 and Rna15; and CTD Ser5, Ser7, and Ser2 phosphorylations were analyzed by chromatin immunoprecipitation (ChIP) along the PMA1 gene. These experiments all used the same chromatin preparations probed with specific antibodies. Schematic at the top shows PCR primer regions (numbered boxes) along the gene. Graphs underneath show the average of results from three independent ChIP experiments, with the y-axis indicating fold enrichment over a non-transcribed background signal and error bars showing standard error. See also Figure S1.

Complementation of an RPB1 deletion by plasmid shuffling was used to test whether msCTD is functional. Cells expressing only Rpb1-msCTD (this strain is hereafter referred to as MC) were viable, with a similar growth rate to wild-type cells (WT) at 30°C and 37°C (Figure 1B). Unlike many mutants with defective transcription elongation, MC grew normally on media containing 6-azauracil or mycophenolic acid (Figure S1A). MC had slightly slower growth at 14°C, particularly on minimal media (Figure 1B), so all subsequent experiments were carried out at 30°C. Based on Northern blot analysis, MC did not show defects in snoRNA 3' end processing or mRNA splicing (Figure S1B). Immunoblotting of whole cell extracts for CTD phosphorylations showed lower levels of Ser7-P in MC, as expected due to the Ser7 substitutions. However, total Ser5-P and Ser2-P levels on msCTD were similar to WT (Figure 1C). ChIP assays showed comparable occupancy levels and patterns of total RNApII (the Rpb3 subunit) and Ser5-P, but slightly reduced Ser2-P in MC versus WT. Notably, essentially identical occupancy patterns of the CTD binding factors Ceg1 (the capping enzyme guanylyltransferase) and Rna15 (an mRNA cleavage and polyadenylation factor) further demonstrated that msCTD can functionally replace wild-type CTD.

Analysis of msCTD phosphorylation on total yeast RNApII

To validate the efficacy of MS/MS for analyzing CTD phosphorylation, pilot experiments were carried out using purified recombinant GST-msCTD. Peptides were analyzed by shotgun sequencing on a hybrid linear ion trap/Orbitrap mass spectrometer. With unphosphorylated GST-msCTD, all expected tryptic peptides were easily observed (Figure S2C, D and data not shown). To create controls with known phosphorylation sites, GST-msCTD was phosphorylated with TAP-tagged Kin28 (the CTD Ser5 kinase of TFIIH, Figure S2A) purified from yeast or recombinant Abl tyrosine kinase (Figure S2B). Mono-, di-, and triphosphorylated peptides were detected, with up to one phosphorylation per heptad. Importantly, Mascot phosphorylation site assignments were reevaluated by manual inspection, with final assignments based on accurate mass measurements (<10 ppm mass error) of site-differentiating product ions. Using this approach, we found that Abl and Kin28 kinases exhibited the expected strong preferences for Tyr1 and Ser5, respectively (Figure S2E, F). Time course experiments indicated that counts of Peptide Spectrum Matches (PSMs) could be used for relative quantitation (Figure S2).

We next proceeded to analyze msCTD isolated from yeast cells (Figure 2A). RNApII was isolated from whole cell extract via Rpb3-TAP binding to IgG-agarose, and then msCTD was released by Prescission protease cleavage and further purified by Ni-NTA resin via the 8xhistidine tag. Following elution with imidazole, msCTD was digested with trypsin and analyzed as described above. All predicted peptides covering the entire CTD were readily detected (Figure 2B). For each peptide, a mix of non-, mono-, and di-phosphorylated forms was detected. Tri-phosphorylation was observed but rare, so to maximize spectral quality and avoid the stochastic sampling of phosphopeptides used in shotgun MS/MS experiments, we employed a targeted, high resolution MS2 approach based on a database of all predicted non-, mono-, and di-phosphorylated peptides from msCTD. Phosphorylations were distributed throughout the CTD, including the two non-consensus repeats closest to the RNApII core (peptide 1, repeats -2 and -1). An average density of 0.3–0.4 phosphates per repeat was seen uniformly throughout the CTD.

Figure 2.

Figure 2

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Analysis of msCTD phosphorylation on total RNApII extracted from yeast. (A) Schematic of msCTD purification for MS/MS analysis. RNApII is isolated from cell extracts using IgG-Sepharose binding to the TAP tag on Rpb3. Prescission protease releases msCTD, which is then further purified via binding of the 8x histidine tag to Ni-NTA agarose. Following elution, msCTD is digested by trypsin, subjected to desalting and analyzed by tandem mass spectrometry. (B) Box at left shows predicted tryptic peptides from msCTD. The three bar graphs in the center show the number of Peptide-Spectrum-Matches detected within peak reverse phase chromatographic fractions for each peptide in the un-, mono-, or diphosphorylated form. The number of phosphorylations divided by total PSMs was then normalized to the number of repeats in that peptide to estimate the average density of phosphorylations per heptad as shown in the right bar graph. (C) For each potential phosphorylation site in the heptad, the number of phosphorylations detected (middle line) was divided by the total occurrences of that amino acid (bottom line) to calculate the percentage of phosphorylation (top line). (D) The percentage of phosphorylation at each residue along the length of msCTD was calculated and graphed using a color scale as shown at bottom. The tryptic peptide and heptad numbers are shown at left shown from N-terminus to C-terminus. (E) Multiple phosphorylations detected on tryptic peptides. Only those that were confirmed by manual validation of MS2 spectra and detected in more than two different tryptic peptides are listed. See also Figure S3.

Relative levels of phosphorylation (% phosphorylation) at each heptad position were estimated by dividing the number of phosphorylated occurrences by the total detections. Each position within the heptad repeat was identified thousands of times. Highest levels of phosphorylation were seen on Ser5, followed by Ser2 (Figure 2C, D). Both of these marks were distributed throughout the length of msCTD, although some reduction at the Ser2 position following trypsin cleavage sites was observed. The relatively uniform levels of phosphorylations throughout the CTD are consistent with experiments showing that Ser2 or Ser5 mutations are equally tolerated in either the N-terminal or C-terminal half of the CTD (reviewed in Corden, 2013; Eick and Geyer, 2013). Multiply phosphorylated peptides most often carried Ser5-P or Ser2-P modified in adjacent or alternating repeats (Figures 2E and S3). The appearance of Ser5-P and Ser2-P together was less frequent. Interestingly, when these two phosphorylations did occur within a single seven amino acid span, Ser5-P always appeared N-terminal to Ser2-P.

Surprisingly, while phosphorylations at Tyr1, Thr4, and Ser7 (or the substituted Thr7) were detected, their levels were almost two orders of magnitude lower than Ser2-P and Ser5-P. This was unanticipated from immunoblotting and ChIP experiments, but illustrates why different antibody reactivities cannot be directly compared. Given the number of position seven mutations in msCTD, we cannot rule out that phosphorylation at this position is more extensive in WT CTD. However, the low frequency of Tyr1-P makes it difficult to imagine how this modification could sterically mask recognition of Ser2-P in vivo (Mayer et al., 2012). Low detection of Tyr1-P was not a technical issue, as this modification was easily seen on recombinant CTD phosphorylated with Abl kinase (Figure S2).

Next, msCTD was analyzed in some CTD kinase or phosphatase mutants. Fcp1 is a CTD phosphatase thought to act primarily at Ser2-P (Cho et al., 2001), but perhaps also at other phosphorylated residues (reviewed in Corden, 2013; Eick and Geyer, 2013). Immunoblotting confirmed a marked increase of Ser2-P reactivity on msCTD purified from an fcp1-1 strain compared to the isogenic FCP1 control (Figures 3A and S4). MS/MS detected far fewer nonphosphorylated peptides in fcp1-1, and a four-fold increase of triply phosphorylated peptides. The mutant showed a striking increase of Ser2-P throughout the length of the CTD, but also a smaller increase in Ser5-P (Figures 3B and C, S4). No effect at Tyr1, Thr4, or Ser7 was seen.

Figure 3.

Figure 3

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Effect of CTD phosphatase mutant fcp1-1 on msCTD phosphorylation. Rpb1-msCTD was introduced by plasmid shuffling into FCP1 and fcp1-1 strains. (A) Prior to trypsin digestion, a fraction of each sample was separated by SDS-PAGE and blots were probed with the indicated antibodies (Chapman et al., 2007). Protein size estimates (kDa) to the left are based on protein size markers. (B) The percentage of phosphorylation at each heptad residue was calculated as in Figure 2, with the number listed below the bar graph. Fold change was calculated by dividing %phosphorylation in the mutant by that in the isogenic wild-type strain. (C) The left panel shows percent phosphorylation for individual residues along the length of msCTD, with color code shown at the bottom. Fold change was calculated by log2(%phospho[mutant]/%phospho[wild-type]). As indicated by the color code at the bottom, purple signifies an increase and green a decrease. See also Figure S4.

msCTD was similarly analyzed from cells lacking the major CTD Ser2 kinase Ctk1 (Cho et al., 2001; Bartkowiak et al., 2010). As expected, both immunoblotting and MS/MS showed a strong drop in Ser2-P, as well as an unexpected slight increase of Ser5-P compared to an isogenic wild-type control. BUR2 encodes the cyclin subunit of Bur1 kinase, the CDK9 homolog. While BUR1 is essential for cell growth, cells lacking BUR2 are viable but have lower Ser2-P level in whole cell lysates (Qiu et al., 2009). Immunoblotting and MS/MS showed that msCTD from bur2Δ cells showed a smaller decrease in Ser2-P, but a similar increase in Ser5-P, compared to the ctk1Δ strain (Figure 4A–C). Altogether, MS/MS analyses of msCTD confirmed the previously reported effects of FCP1, BUR2, and CTK1 mutants based on antibody reactivity, and showed that these effects occur throughout the CTD.

Figure 4.

Figure 4

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Effect of CTD kinase mutants on msCTD phosphorylation. Rpb1-msCTD was introduced by plasmid shuffling into BY4741, bur2Δ, ctk1Δ strains. (A) Prior to trypsin digestion, a fraction of each sample was separated by SDS-PAGE and blots were probed with the indicated antibodies (Chapman et al., 2007). Protein size estimates (kDa) to the left are based on protein size markers. (B) The percentage of phosphorylation at each heptad residue was calculated as in Figure 2, with the number listed below the bar graph. Fold change was calculated by dividing %phosphorylation in the mutant by that in the isogenic wild-type strain. (C) The left panel shows percent phosphorylation for individual residues along the length of msCTD, with color code shown at the bottom. Fold change was calculated by log2(%phospho[mutant]/%phospho[wild-type]). As indicated by the color code at the bottom, purple signifies an increase and green a decrease.

Analysis of phosphorylations on msCTD during early and late stages of transcription

The experiments above analyzed total RNApII, which is a mixture of species from different stages of the transcription cycle as well as non-transcribing polymerase. To test the ability of msCTD to monitor phosphorylation at specific stages of transcription, we isolated RNApII associated with capping enzyme guanylyltransferase Ceg1 or the termination factor Rtt103, two well-characterized CTD binding proteins bound to RNApII at early or late stages of transcription, respectively. ChIP assays of TAP-tagged Ceg1 or Rtt103 showed comparable occupancy for both of these proteins in WT and MC, indicating recruitment was unaffected by msCTD (Figure 5A and B). TAP purification from cell extracts of these two CTD binders was carried out alongside Rpb3-TAP as a control for total RNApII. Although the overall yield of Ceg1- and Rtt103-associated msCTD was only 10–20% of that with Rpb3-TAP, shotgun MS/MS nonetheless detected hundreds of PSMs spanning the full msCTD.

Figure 5.

Figure 5

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Phosphorylations on msCTD associated with CTD binding proteins Ceg1 or Rtt103. (A) PMA1, and (B) ADH1 genes were analyzed for ChIP crosslinking of the TAP-tagged proteins indicated on top in strains carrying wild-type RPB1 (WT) or RPB1-msCTD (MC). Schematic at top shows the PCR primer regions (numbered boxes) along the gene. Graphs underneath show the average of quantified results from three independent ChIP experiments, with the y-axis representing fold enrichment over a nontranscribed background signal and error bars showing standard error. (C) Percent phosphorylation detected on each residue of the heptad was calculated as in Figure 2C. (D) Percent phosphorylation for each residue along the length of msCTD is graphed as in Figure 2D. (E) Listing of peptides carrying two phosphorylations, with phosphorylated sites marked in blue color. Only those that were detected in more than two different tryptic peptides are shown. See also Figure S5.

Ceg1-associated msCTD carried much higher levels of Ser5-P and very little Ser2-P compared to total RNApII (Figure 5C). The Ser5-P increase was seen along the entire msCTD, showing that early Ser5-P is not localized to a specific region of CTD. Low levels of Ser2-P, Thr4-P, and Ser7-P were seen in proximal heptads, although the low number of PSMs makes it difficult to know if this asymmetry is meaningful (Figure 5D). Peptides with multiple phosphorylations were more frequent on Ceg1-associated msCTD, revealing a pattern of neighboring Ser5-P pairs throughout msCTD (Figures 5E and S5A). This finding agrees well with crystal structures showing other yeast guanylyltransferases contacting Ser5-Ps in two different CTD repeats (Doamekpor et al., 2014; Fabrega et al., 2003).

The transcription termination factor Rtt103 was isolated as a Ser2-P binding protein (Kim et al., 2004). In agreement, msCTD associated with Rtt103 was highly enriched for Ser2-P, which was evenly distributed throughout the whole CTD (Figure 5C and D). Structural and biochemical studies show the phosphorylated Ser2 contacting an arginine in the Rtt103 CID, and there may be some additional contribution of the preceding Ser2-P to affinity (Lunde et al., 2010). Also, multiple Rtt103 monomers may bind cooperatively to the CTD (Lunde et al., 2010). These models are consistent with observation of some neighboring Ser2-P pairs (Figures 5E and S5B). Ser5-P was lower than in total RNApII but still present throughout the CTD, and a small number of peptides with neighboring Ser2-P and Ser5-P marks were also detected, consistent with some Ser5-P persisting even late into transcription (Bataille et al., 2012; Komarnitsky et al., 2000; Mayer et al., 2010; Tietjen et al., 2010).

Discussion

Using a modified CTD engineered for mass spectrometry, we have been able to determine the location and relative levels of phosphorylations along the CTD. Furthermore, by isolating CTD bound to factors that interact with RNApII we can monitor phosphorylation status at specific stages of transcription. Our results provide strong validation for the antibody-based model originally proposed by Komarnitsky et al. (2000), with high levels of Ser5-P at early stages and stronger Ser2-P at late stages of transcription. There was no evidence for differential phosphorylation between proximal and distal regions of CTD, consistent with general findings of redundancy between repeats (reviewed in Corden, 2013; Eick and Geyer, 2013).

Several surprises emerged from the MS data. First, phosphorylations at Tyr1 and Thr4 were roughly two orders of magnitude less frequent than at Ser2 and Ser5. Ser7 phosphorylation was similarly rare, but this low level may also reflect the multiple mutations made at this position. While we cannot rule out that these other residues are highly phosphorylated in a very restricted population of RNApII, genome-wide ChIP experiments did not reveal highly localized crosslinking at narrow peaks along genes or at a small number of specific genomic loci (Bataille et al., 2012; Mayer et al., 2010; Tietjen et al., 2010). Rather, the patterns for these marks in yeast generally resemble either Ser2-P or Ser5-P. The antibodies used for these ChIP experiments ostensibly recognize specific phosphorylated residues, but given a >100-fold higher frequency of Ser2-P and Ser5-P, the antibody selectivity against those abundant sites must exceed that amount to rule out any cross-contamination of signals.

A second surprise was that multiple phosphorylations within a single repeat length are relatively rare, with only Ser5-P followed by Ser2-P detected at appreciable levels. Therefore, the CTD code is unlikely to involve adjacent modifications within one heptad, and instead any combinatorial phosphorylation site recognition probably occurs over multiple repeat units. In the future, msCTD will be a valuable tool for characterizing phosphorylation patterns of CTD bound to other factors and in different mutant backgrounds. More complex patterns involving other residues may emerge. However, our current results suggest the CTD code in yeast may be relatively simple, primarily involving Ser2-P and Ser5-P.

Experimental Procedures

Yeast strains and Plasmids

Yeast strains used in this study are listed in Table S1. Strains WT (YSB2980) and MC (YSB2982) are isogenic derivatives of BY4741. In order to make available the TRP1 marker for tryptophan auxotrophy for use in BY4741, TRP1 was deleted by integration of the trp1Δ:: hisG-URA3-hisG cassette (Alani et al., 1987). The URA3 marker was subsequently removed by 5-FOA selection, resulting in replacement of TRP1 with the hisG sequence. This strain was transformed with a plasmid carrying wild-type RPB1 and a URA3 marker gene, and then with a rpb1Δ::NATMX cassette in order to delete the genomic copy of RPB1. Plasmid shuffling was carried out with a LEU2-marked plasmid carrying either "wild-type" RPB1 (pCK859, a gift from Craig Kaplan, Texas A&M University) or RPB1-msCTD gene (pRS315-RPB1-msCTD), generating WT or MC, respectively. MC strain was further transformed with RPB3-TAP::HIS3MX (YSB3007), CEG1-TAP::HIS3MX (YSB2990), or RTT103-TAP::HIS3MX (YSB2996) cassette for ChIPs and co-precipitation experiments. CTK1 or BUR2 gene was further deleted using ctk1Δ::KanMX (YSB3102) or bur2Δ::KanMX (YSB3195) cassette. FCP1 (YSB3135) and fcp1-1 (YSB3137) are paired isogenic strains engineered to carry a plasmid harboring either wild-type RPB1 or RPB1-msCTD, with the genomic copy of RPB1 having been replaced by rpb1Δ::NATMX, and then was transformed with RPB3-TAP::HIS3MX cassette.

Plasmids used in this study are listed in Table S2. Oligos used to amplify KanMX cassettes for CTK1 or BUR2 deletion, to amplify TAP::HIS3MX cassettes for TAP-tagging, and to swap KanMX of rpb1Δ::KanMX to NATMX cassette are listed in Table S3. Yeast transformations and plasmid shuffling was performed by standard methods (Guthrie and Fink, 1991).

DNA encoding msCTD (873 base pair long) was synthesized using the Gene Synthesis service of GenScript USA Inc., and was provided on a plasmid, pU57-msCTD (DNA sequence information is available upon request). In order to construct pRS315-RPB1-msCTD, first pRS315-RPB1-NheI was generated by inverse PCR using oligos listed in Table S3 and pRS315-RPB1 (pCK859, kind gift from Craig Kaplan, Texas A&M University) as template, followed by NheI digestion and self-ligation. This procedure replaced coding region 4471–5202 of RPB1 gene with a NheI site. msCTD fragment was released from pUC57-msCTD through digestion by NheI and was subcloned into the NheI site of pRS315-RPB1-NheI. For GST-tagging, msCTD was subcloned from pUC57-msCTD into pGEX4T3 using BamHI and SalI sites. Sequences were confirmed through sequencing.

Phenotype analysis

Cells were grown overnight at 30°C in selective media. The overnight culture was diluted to OD[600]=0.3, which was subjected to 3-fold serial dilutions. 4µl of each dilution were spotted onto plates as indicated, and the plates were incubated at 14°C, 30°C, or 37°C for 3–6 days, until being photographed.

Northern blot analysis

Cells were grown at 30°C until OD[600] reached 0.5, after which pcf11-9 cells were subjected to a temperature shift to 37°C for 30min. Total RNA was isolated using hot acidic phenol extraction. 20µg of each sample were separated by electrophoresis on a 1.5% agarose gel with 6.7% formaldehyde and 1× MOPS, transferred onto a nylon membrane (Nytran SPC, GE healthcare) by capillary transfer, and crosslinked by UV irradiation using UV Stratalinker 1800 (Stratagene). Probe preparation and hybridization were carried out as previously described (Marquardt et al., 2011). Oligos used for probe preparation are listed in Table S3. Detection was performed using phosphorimager BAS-2500 (Fujifilm).

Preparation of whole cell lysate and immunoblot analysis

Whole cell lysate was prepared as previously described in Suh et al. (2013). In brief, cells were grown overnight in 5 ml of YPD media at 30°C. 50 ml of fresh selective media was inoculated with 3 ml of overnight culture and cells were grown at 30°C until OD[600] reached 0.5. Cells were harvested by centrifugation and resuspended in 500 µl lysis buffer (50 mM Tris-Cl (pH 8.0), 150 mM NaCl, 0.1% NP-40, 2 µg/ml Aprotinin, 2 µg/ml Leupeptin, 2 µg/ml Pepstatin A, 2 µg/ml Antipain, 1 mM PMSF, 1 mM sodium fluoride, 1 mM sodium orthovanadate). 500 µl of glass beads were added and cells were lysed by repeating 20 seconds of vortexing and 20 seconds of cooling on ice for a total of 6 cycles. Lysate was transferred to a 1.5 ml microfuge tube, and insoluble material was precipitated by centrifugation. Supernatant was transferred to a fresh tube, and protein concentration was measured using Coomassie Protein Assay Reagent (Thermo Scientific) following manufacturer’s protocol.

For immunoblot analysis, 20 µg of each sample were loaded per well. Samples were resolved on an 8% or 12% SDS-polyacrylamide gel, transferred to an Optitran BA-S 83 reinforced nitrocellulose membrane (Whatman), and analyzed by immunoblotting. Primary antibodies that were used include mouse anti-phosphoSer2 (H5, 1:500), rat anti-phosphoSer2 (3E10, 1:1000), rat anti-phosphoSer5 (3E8, 1:1000), rat anti-phosphoSer7 (4E12, 1:200), mouse anti-FLAG (Sigma M2, 1:5000), and mouse anti-RPB3 (NeoClone 1Y26, 1:1000) antibodies. Peroxidase-conjugated secondary antibodies were anti-rat IgG (Sigma A9037, 1:10,000), anti-mouse IgG (Sigma A2304, 1:10,000), or anti-mouse IgM (1:10,000). Signals were developed with the SuperSignal West Pico chemiluminescent substrate (Thermo Scientific) and the SuperSignal West Femto maximum sensitivity substrate (Thermo Scientific), depending on signal intensity.

Chromatin immunoprecipitation

Chromatin samples were prepared as previously described in Komarnitsky et al. (2000). For immunoprecipitation, 3 µl of anti-RPB3, 3 µl of anti-phosphoSer5 (3E8), 25 µl of anti-phosphoSer7 (4E12), 3 µl of anti-Ceg1, or 3 µl of anti-Rna15 antibody (a gift from Claire Moore, Tufts Medical School) was added along with 20 µl of ProteinG-sepharose mixture (50% slurry in 10mM Tris-Cl (pH 8.0), 1mM EDTA (pH 8.0)) to 500 µg chromatin in 500 µl of binding buffer: 50mM HEPES-KOH (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 1 mM PMSF. For phosphoSer2 ChIP, 3µl of H5 antibody along with 20µl of anti-IgM-sepharose mixture was added to 500 µg chromatin dissolved in 500 µl binding buffer. For TAP-tagged proteins, 20µl of IgG-sepharose mixture was added to 500 µg chromatin dissolved in 500 µl binding buffer. Subsequent washes, decrosslinking, DNA precipitation, PCR reaction, and quantification were carried out as previously described in Kim et al. (2004). Oligos used for PCR are listed in Table S3.

In vitro kinase assay

Kinase reactions were performed as previously described (Keogh et al., 2003). In brief, reactions were carried out at 30°C in 100 µl of 20 mM HEPES-KOH (pH 7.5), 7.5 mM magnesium acetate, 2 mM dithiothreitol, 100 mM potassium acetate, 2% glycerol, 200 µM ATP, 5 µg of purified recombinant GST-msCTD, and approximately 100 ng of purified Abl tyrosine kinase (kind gift from Michael Eck) or 10 µl of Kin28-TAP-bound IgG sepharose. Kin28-TAP protein was pulled down by incubating 2 mg/ml of whole cell extract from KIN28-TAP strain with 10ul IgG sepharose at 4°C for overnight with end-to-end inversions, followed by three washes with 1.5 ml of lysis buffer (50 mM Tris-Cl (pH8.0), 150 mM NaCl, 0.1% NP-40, 2 µg/ml Aprotinin, 2 µg/ml Leupeptin, 2 µg/ml Pepstatin A, 2 µg/ml Antipain, 1 mM PMSF, 1 mM sodium fluoride, 1 mM sodium orthovanadate).

Purification of msCTD from yeast

Cells were grown in 2 L of YPD at 30°C until OD[600] reached 1.0 and then harvested by centrifugation. Collected cells were frozen by dispensing as drops into liquid nitrogen. Frozen cells were ground into powder using an MM301 ball mill (Retsch) following manufacturer’s instructions. Frozen powder was rehydrated in 40 ml of lysis buffer (50 mM Tris-Cl (pH8.0), 50 mM NaCl, 0.1% NP-40, 2 µg/ml Aprotinin, 2 µg/ml Leupeptin, 2 µg/ml Pepstatin A, 2 µg/ml Antipain, 1 mM PMSF, 1 mM sodium fluoride, 1 mM sodium orthovanadate). Lysate was centrifuged at 5,000 rpm in a Sorvall SS-34 rotor for 10 min at 4°C, and the supernatant was cleared once more by centrifugation at 9,000 rpm in a Sorvall SS-34 rotor for 10 min at 4°C. Cleared supernatant was then incubated with IgG-sepharose (bed volume 200 µl) for 3hrs at 4°C to bind the TAP tag, followed by three washes with 10 ml lysis buffer and then one wash with 10 ml cleavage buffer (50 mM Tris-Cl (pH8.0), 50 mM NaCl, 0.1% NP-40, 1 mM sodium fluoride, 1 mM sodium orthovanadate, 7 mM β-mercaptoethanol). Resins were incubated in 500µl of cleavage buffer containing 10 units of Prescission protease (GE Healthcare Lifesciences) at 4°C overnight with gentle end-to-end inversions. Supernatant was supplemented with imidazole to a final concentration of 10 mM, passed through a column of glutathione-agarose (Sigma) to remove His-GST-tagged Prescission protease, and then was incubated with 50 µl of Ni-NTA resin (QIAGEN) at 4°C for 1hr with gentle end-to-end inversions to bind msCTD. Resins were washed with 6 column volumes of 50 mM Tris-Cl (pH8.0), 50 mM NaCl, 0.1% NP-40, 10 mM imidazole, and then 6 column volumes of 50mM Tris-Cl (pH8.0), 50 mM NaCl, 10 mM imidazole in order to remove NP-40. Bound proteins were eluted in 50 mM Tris-HCl (pH8.0), 50 mM NaCl, 200 mM imidazole.

Tandem mass spectrometry (nano LC/MS)

Proteins were digested with trypsin (37°C, overnight) and analyzed by nanoLC-MS (Ficarro et al., 2009). In each analysis, one-fifth of the total sample was injected onto the precolumn (4 cm POROS 10R2, Applied Biosystems, Framingham, MA) and eluted with an HPLC gradient (NanoAcquity UPLC system, Waters, Milford, MA; 2–35% B in 120 minutes; A=0.1 M acetic acid in water, B=0.1 M acetic acid in acetonitrile). Peptides were resolved on a self-packed analytical column (50 cm Monitor C18, Column Engineering, Ontario, CA) and introduced to the mass spectrometer (LTQ Orbitrap XL) at a flow rate of ~30 nL/min (ESI spray voltage = 3.2 kV). The mass spectrometer was programmed to perform data-dependent "shotgun" MS/MS on the 10 most abundant precursors (35% collision energy) in each MS1 scan (image current detection, 60K resolution, m/z 300–2000). MS/MS spectra were matched to peptide sequences using Mascot (version 2.2.1) after conversion of raw data to .mgf using multiplierz scripts (Askenazi et al., 2009). Search parameters specified trypsin digestion with up to 2 missed cleavages, as well as variable oxidation of methionine and phosphorylation of serine, threonine, or tyrosine residues. Precursor and peptide ion mass tolerances were 10 ppm and 0.6 Da, respectively. For higher accuracy, positions of phosphorylated sites were confirmed and corrected by manual validation of spectra. Targeted scan experiments were performed in a similar fashion using an LTQ Orbitrap Velos. HCD-MS2 (image current detection, resolution = 7500 @ m/z 400) was performed on up to 14 precursors per analysis to afford ~4–5 points across each peak. Sequence of targeted peptides and corresponding m/z values are listed in Table S4.

Supplementary Material

Acknowledgements

This work was supported by DFCI/Blais Proteomics Center support to J.M. and NIH grant GM56663 to S.B. We thank Mike Eck (HMS), Eun-Young Park (HMS), Craig Kaplan (Texas A&M), Claire Moore (Tufts University) for materials, Byung-hoon Lee (HMS), Nahid Iglesias (HMS), Dane Hazelbaker (HMS) for technical advice, So-Young Hwang (HMS), Marto lab members, and Buratowski lab members for helpful discussions, and Dirk Eick for antibodies and discussion of unpublished results.

Footnotes

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Author Contributions

S.B. and H.S. conceived and designed MS-compatible CTD. H.S. carried out the experiments. S.B.F. and U.B.K. supervised mass spectrometry experiments. Y.C. assisted generating kinase and phosphatase mutant strains. Data analysis and manuscript preparation were done by H.S., S.B.F., J.A.M. and S.B.

References

  1. Alani E, Cao L, Kleckner N. A method for gene disruption that allows repeated use of URA3 selection in the construction of multiply disrupted yeast strains. Genetics. 1987;116:541–545. doi: 10.1534/genetics.112.541.test. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Askenazi M, Parikh JR, Marto JA. mzAPI: a new strategy for efficiently sharing mass spectrometry data. Nat Meth. 2009;6:240–241. doi: 10.1038/nmeth0409-240. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bartkowiak B, Liu P, Phatnani HP, Fuda NJ, Cooper JJ, Price DH, Adelman K, Lis JT, Greenleaf AL. CDK12 is a transcription elongation-associated CTD kinase, the metazoan ortholog of yeast Ctk1. Genes Dev. 2010;24:2303–2316. doi: 10.1101/gad.1968210. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bataille AR, Jeronimo C, Jacques P-E, Laramée L, Fortin M-È, Forest A, Bergeron M, Hanes SD, Robert F. A universal RNA polymerase II CTD cycle is orchestrated by complex interplays between kinase, phosphatase, and isomerase enzymes along genes. Mol Cell. 2012;45:158–170. doi: 10.1016/j.molcel.2011.11.024. [DOI] [PubMed] [Google Scholar]
  5. Buratowski S. Progression through the RNA Polymerase II CTD Cycle. Mol Cell. 2009;36:541–546. doi: 10.1016/j.molcel.2009.10.019. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Buratowski S. The CTD code. Nat Struct Biol. 2003;10:679–680. doi: 10.1038/nsb0903-679. [DOI] [PubMed] [Google Scholar]
  7. Chapman RD, Heidemann M, Albert TK, Mailhammer R, Flatley A, Meisterernst M, Kremmer E, Eick D. Transcribing RNA polymerase II is phosphorylated at CTD residue serine-7. Science. 2007;318:1780–1782. doi: 10.1126/science.1145977. [DOI] [PubMed] [Google Scholar]
  8. Cho EJ, Kobor MS, Kim M, Greenblatt J, Buratowski S. Opposing effects of Ctk1 kinase and Fcp1 phosphatase at Ser 2 of the RNA polymerase II C-terminal domain. Genes Dev. 2001;15:3319–3329. doi: 10.1101/gad.935901. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Corden JL. RNA polymerase II C-terminal domain: Tethering transcription to transcript and template. Chem. Rev. 2013;113:8423–8455. doi: 10.1021/cr400158h. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Doamekpor SK, Sanchez AM, Schwer B, Shuman S, Lima CD. How an mRNA capping enzyme reads distinct RNA polymerase II and Spt5 CTD phosphorylation codes. Genes Dev. 2014;28:1323–1336. doi: 10.1101/gad.242768.114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Eick D, Geyer M. The RNA polymerase II carboxy-terminal domain (CTD) code. Chem. Rev. 2013;113:8456–8490. doi: 10.1021/cr400071f. [DOI] [PubMed] [Google Scholar]
  12. Fabrega C, Shen V, Shuman S, Lima CD. Structure of an mRNA capping enzyme bound to the phosphorylated carboxy-terminal domain of RNA polymerase II. Mol Cell. 2003;11:1549–1561. doi: 10.1016/s1097-2765(03)00187-4. [DOI] [PubMed] [Google Scholar]
  13. Ficarro SB, Zhang Y, Lu Y, Moghimi AR, Askenazi M, Hyatt E, Smith ED, Boyer L, Schlaeger TM, Luckey CJ, Marto JA. Improved electrospray ionization efficiency compensates for diminished chromatographic resolution and enables proteomics analysis of tyrosine signaling in embryonic stem cells. Anal Chem. 2009;81:3440–3447. doi: 10.1021/ac802720e. [DOI] [PubMed] [Google Scholar]
  14. Guthrie C, Fink GR, editors. Methods in Enzymology. New York, NY: Academic Press; 1991. Guide to yeast genetics and molecular biology. [PubMed] [Google Scholar]
  15. Hintermair C, Heidemann M, Koch F, Descostes N, Gut M, Gut I, Fenouil R, Ferrier P, Flatley A, Kremmer E, Chapman RD, Andrau J-C, Eick D. Threonine-4 of mammalian RNA polymerase II CTD is targeted by Polo-like kinase 3 and required for transcriptional elongation. EMBO J. 2012;31:2784–2797. doi: 10.1038/emboj.2012.123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Keogh MC, Podolny V, Buratowski S. Bur1 kinase is required for efficient transcription elongation by RNA polymerase II. Mol Cell Biol. 2003;23:7005–7018. doi: 10.1128/MCB.23.19.7005-7018.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kim M, Krogan NJ, Vasiljeva L, Rando OJ, Nedea E, Greenblatt JF, Buratowski S. The yeast Rat1 exonuclease promotes transcription termination by RNA polymerase II. Nature. 2004;432:517–522. doi: 10.1038/nature03041. [DOI] [PubMed] [Google Scholar]
  18. Komarnitsky P, Cho EJ, Buratowski S. Different phosphorylated forms of RNA polymerase II and associated mRNA processing factors during transcription. Genes Dev. 2000;14:2452–2460. doi: 10.1101/gad.824700. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lunde BM, Reichow SL, Kim M, Suh H, Leeper TC, Yang F, Mutschler H, Buratowski S, Meinhart A, Varani G. Cooperative interaction of transcription termination factors with the RNA polymerase II C-terminal domain. Nat Struct Mol Biol. 2010;17:1195–1201. doi: 10.1038/nsmb.1893. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Marquardt S, Hazelbaker DZ, Buratowski S. Distinct RNA degradation pathways and 3' extensions of yeast non-coding RNA species. Transcription. 2011;2:145–154. doi: 10.4161/trns.2.3.16298. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Mayer A, Heidemann M, Lidschreiber M, Schreieck A, Sun M, Hintermair C, Kremmer E, Eick D, Cramer P. CTD tyrosine phosphorylation impairs termination factor recruitment to RNA polymerase II. Science. 2012;336:1723–1725. doi: 10.1126/science.1219651. [DOI] [PubMed] [Google Scholar]
  22. Mayer A, Lidschreiber M, Siebert M, Leike K, Söding J, Cramer P. Uniform transitions of the general RNA polymerase II transcription complex. Nat Struct Mol Biol. 2010;17:1272–1278. doi: 10.1038/nsmb.1903. [DOI] [PubMed] [Google Scholar]
  23. Qiu H, Hu C, Hinnebusch AG. Phosphorylation of the Pol II CTD by KIN28 enhances BUR1/BUR2 recruitment and Ser2 CTD phosphorylation near promoters. Mol Cell. 2009;33:752–762. doi: 10.1016/j.molcel.2009.02.018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Suh H, Hazelbaker DZ, Soares LM, Buratowski S. The C-terminal domain of Rpb1 functions on other RNA polymerase II subunits. Mol Cell. 2013;51:850–858. doi: 10.1016/j.molcel.2013.08.015. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Tietjen JR, Zhang DW, Rodríguez-Molina JB, White BE, Akhtar MS, Heidemann M, Li X, Chapman RD, Shokat K, Keles S, Eick D, Ansari AZ. Chemical-genomic dissection of the CTD code. Nat Struct Mol Biol. 2010;17:1154–1161. doi: 10.1038/nsmb.1900. [DOI] [PMC free article] [PubMed] [Google Scholar]

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Supplementary Materials

NIHMS747877-supplement.pdf (1.1MB, pdf)

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