Yeast strains and plasmids

Yeast strains used in this study are listed in Table S2. Plasmids were described previously [29], [30]. Construction of strains containing CTK1 or BUR1 under the control of the GAL1 promoter was performed as described [31].

Northern blot analysis

RNA was isolated by phenol extraction from cultures grown to early mid-log phase. Northern blot analysis was performed using standard protocols. Hybridization was performed either in 6× SSC, 5× Denhardt's solution, 0.1% SDS and 100 µg/nl fragmented haring sperm DNA buffer or in UltraHyb buffer (Ambion). Quantification of the signals was carried out using a Storm 820 phosphoimager. Oligonucleotide probes and primers used to amplify the PCR probes are listed in Table S3.

Protein analyses and immunoprecipitations

For immunoprecipitations, cells were grown to OD 1.0–1.6 and harvested. The cell pellet was resuspended in 4 ml of Lysis buffer (6 mM Na₂HPO₄, 4 mM NaH₂PO₄, 200 mM sodium acetate, 0.25% NP-40, 2 mM EDTA, 1 mM EGTA, 5% glycerol, 20 mM PMSF, 1 mM benzamidine, EDTA-free protease inhibitor cocktail from Roche) and flash frozen. Cells were disrupted using a ball mill (MM400 Retsch; 5 times 10 Hz, 3 min) and the extract cleared at 13krpm for 25 min. Immunoprecipitation assays were performed using IgG Fast Flow Sepharose beads (GE Healthcare). Proteins were eluted for 15 min at 37°C with 60 µl of lysis buffer and 80 µl 4× Laemmli buffer containing 50 mM DTT. RNAseA treatment was performed after IP on beads washed once with 1 ml Lysis buffer (10 µg/ml RNaseA for 20 min in 25°C).

To analyze the phosphorylation status of RNA Pol II CTD after Ctk1p and Bur1p depletion, 4OD units of exponentially growing cells were harvested after growth in the appropriate medium and resuspended in 150 µl of 8 M urea. Cells were broken in a Magnalyser (Roche) and the lysate was cleared by centrifugation for 2 min at 4000 rpm. Alternatively, exponentially growing ctk1Δ and wt cells were harvested and disrupted in FA lysis buffer (50 mM Hepes KOH pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Deoxycholate, 0.1% SDS), containing protease and phosphatase inhibitors using a genie-disruptor (Scientific Industries). Western blot analysis was performed using antibodies 8WG16 (gift from H.T.M. Timmers), 3E8, 3E10, 4E12 (Millipore) [32], Tub1p (Immunologicals), H5, H14 (Covance), Y-80 (Santa Cruz), 12ca5 (Roche), αSpt5 (gift from G. Hartzog), αNab3p (gift from M. S. Swanson), αRbp3p (Neoclone), α-Nrd1p (gift from D. Brow).

Expression profiling

Each mutant strain was profiled four times from two independently inoculated cultures and harvested in early mid-log phase in SC medium with 2% glucose. An exception is Pcf11, for which two different alleles (pcf11-2 and pcf11-ts2) were profiled two times from single inoculated cultures. Sets of mutants were grown alongside corresponding wt cultures and processed in parallel. Growth of ctk1Δ and rpb1-S2A for Figure 5 was performed in YPD, because the rpb1-S2A strain grew very poorly in SC. Dual-channel 70-mer oligonucleotide arrays were employed with a common reference wt RNA. All steps after RNA isolation were automated using robotic liquid handlers. These procedures were first optimized for accuracy (correct fold-change) and precision (reproducible result), using spiked-in RNA for calibration [33]. After quality control, normalization, and dye-bias correction [34], statistical analysis for mid-log cultures was performed for each mutant versus the collection of 200 wt cultures as described previously [35]. The temperature sensitive mutants of SEN1 and PCF11 were compared to wt Mat a strains. The reported fold-change is an average of the replicate mutant profiles versus the average of all wildtypes. Microarray data have been submitted to the ArrayExpress database (http://www.ebi.ac.uk/microarray) under accession number E-MTAB-1261 and to the GEO database (http://www.ncbi.nlm.nih.gov/geo/) under accession number GSE40254

Genome-wide analyses

Tiling arrays were performed for two independent replicates of wt and ctk1Δ as described previously [36], [37]. The occurrence of readthrough at ORFs and snoRNAs was assessed as follows: the probe intensities from the two replicates were averaged over 25 bp regions and aligned to the 3′ end of ORFs and snoRNAs. Readthrough was scored positive if the expression difference between ctk1Δ and wt was more than 0.6 in the 150 bp regions downstream of the 3′ end (at least 4 out of 6 25 bp regions). Transcripts for which the 3′ end was less than 150 bp from the following transcript were excluded. Metagene analysis was performed by averaging the intensity over 25 bp regions for all ORFs and snoRNAs. For visualization in Figure 2, the tiling data was segmented separately for wt and ctk1Δ using the library tilingArray in R [36], [37]. The optimal number of segments was selected based on the maximum log likelihood (BIC). The data can be visualized for individual genes at http://steinmetzlab.embl.de/holstegeLabArray/.

Chromatin Immunoprecipitation

500 ml of cultures in early mid-log phase were crosslinked with 1% formaldehyde for 20 min at 25°C. The reaction was stopped with 300 mM glycine. Cells were washed with ice-cold TBS and FA lysis buffer (50 mM Hepes KOH pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Deoxycholate, 0.1% SDS). Cells were disrupted in FA-lysis containing protease and phosphatase inhibitors using a gene-disruptor. Chromatin was fragmented by sonication (Bioruptor, Diagenode: 7 cycles, 30 sec on/off, medium setting), resulting in fragment sizes of 300–500 bp. Chromatin extracts were incubated overnight at 4°C with antibody (1 µl α-Nrd1, 1 µl α-rpb3 (Neoclone), 2 µl 3E8, 2 µl 3E10, 10 µl 4E12). 20 µl of dry protein A (for α-Nrd1) or protein A+G beads (for all other antibodies) were added and incubated for 1.5 hr at RT. For TAP-Pcf11, 20 µl of IgG sepharose beads were used. The beads were washed 2× with FA-lysis buffer, 2× with FA lysis buffer containing 500 mM NaCl, 2× with 10 mM Tris pH 8, 0.25 M LiCl, 0.5% Nonidet P-40, 0.5% sodium deoxycholate, 1 mM EDTA and 1× with TE pH 8.0. Samples were eluted with TE-SDS 1% at 65°C. Crosslinking was reversed at 65°C for ∼16 hr with 5 µg RNAse. Samples were treated with Proteinase K for 2 hr at 37°C. DNA was recovered with phenol extraction and a PCR purification kit (Qiagen). Samples were analyzed by qPCR with the primer pairs in Table S3. Every experiment is based on at least 2, in most cases 4 biological replicates and the signals represent enrichment over input. Mock ChIPs contain no antibody. For mock ChIP of Pcf11, untagged strains were used.

The expression signature of ctk1Δ is most similar to loss of an NNS complex component

Expression profiles that describe the genome-wide changes in gene expression upon mutation of different regulatory factors can provide detailed phenotypical information. For example similarities between the genome-wide expression signatures derived from mutation of different factors can indicate different types of interactions, including same protein complex or regulatory pathway membership [35], [38]. To assess the role of the CTD-Ser2 kinase Ctk1p in early and late termination pathways, genome-wide expression changes of a ctk1Δ mutant were compared with expression changes of mutants of different CTD-interacting factors (Figure 1A). For each factor, we determined the expression profile in quadruplicate (two technical replicates from two independent cultures), averaged and statistically analyzed versus many wild type strains grown and processed identically (Materials and Methods). Most temperature-sensitive (ts) strains already show partial loss of function at the permissive temperature [39]. To ensure data comparability, all strains, including the ts strains for the essential genes SEN1 and PCF11, were grown under identical conditions including temperature (30°C).

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Figure 1. Loss of CTK1 results in readthrough at snoRNAs.

(A) Unsupervised hierarchical cluster diagram (cosine correlation) of expression changes in mutants of Ctk1p and several CTD-binding factors. All genes with significant changes (p<0.05, FC>1.7) in any of the mutants individually are shown, yellow indicating upregulation, blue indicating downregulation, and black indicating no change compared to wt. The dendrogram indicates the similarity between the mutants. The relationship between Ctk1p and Sen1p is highlighted in red. Overlapping expression changes between ctk1Δ and other mutants are underlined and marked by I (ctk1Δ, sen1-1), II (ctk1Δ, set2Δ, rco1Δ, eaf3Δ), III (ctk1Δ, pcf11-2, pcf11-ts2), IV (ctk1Δ, rtt103Δ). (B) Expression changes for the first gene that is positioned within 2 kB of a snoRNA termination site. Upregulation suggests defective termination at the snoRNA. (C, D and E) Northern blot analysis at snR5, snR13, and snR33. The positions of the probes are indicated on the scheme above the Northern blots. TUB1 mRNA is used as a loading control. Precursors and readthrough transcripts (RT) are indicated. (F) Wt and ctk1Δ cells were assessed for their ability to express the HIS3 genes, which was positioned on a plasmid containing no terminator, or the terminators of snR13 or snR47 upstream of the HIS3 gene. Expression of HIS3 is reflected by the ability to grow on plates containing the histidine analogue 3-AT, and indicates readthrough. Serial dilutions (1∶5) are shown starting at 1*10⁴ cells after growth for 5 days on SC -URA -HIS and SC -URA -HIS+ 2.5 mM 3-AT plates.

https://doi.org/10.1371/journal.pone.0080495.g001

Figure 1A shows all genes with a significant change in expression in any mutant as compared to wild type (p<0.05, fold change (FC)>1.7). In keeping with previous observations that similar expression profiles may reflect interactions, loss of Ctk1p results in mRNA expression changes that overlap with changes observed upon mutation of several different binding partners of CTD-Ser2P. For example, the expression changes in ctk1Δ overlap with changes observed upon deletion of the Ser2P binder Set2p (Figure 1A, II, Table S1). Upon binding to Ser2P, Set2 trimethylates histone 3 on lysine 36 in the coding region of actively transcribed genes [17], [19], which leads to activation of Rpd3S complex and deacetylation of coding regions, preventing initiation of cryptic transcription in gene bodies [40]–[43]. Removal of Ctk1p, Set2p and the downstream components of Rpd3S complex, Eaf3p and Rco1p, all result in elevated expression levels of a similar set of genes (Figure 1A, II). As expected, this set includes genes with internal transcription initiation sites, such as STE11 [40] (Table S1). Besides reflecting this previously known interaction between Ctk1p and Set2p, loss of Ctk1p also results in changes that overlap with mutants of the Ser2P binding factors Rtt103p and Pcf11p (Figure 1A, III and IV, Table S1). Gene set III is significantly enriched for binding sites of the gene specific transcription factors Msn2p, Msn4p and Gcn4p, which regulate stress and amino acid biosynthesis genes. Gene set IV, shared primarily between ctk1Δ and rtt103Δ, is enriched for genes with weak polyA sites (hypergeometric test, p<2.82*10⁻⁴) [11], suggesting that their decrease might be caused by instability after readthrough. Both mutants result in defects in the late termination pathway that acts on mRNA genes [20], [22].

The dendrogram on the right side of figure 1A reflects overall similarities between expression signatures and is automatically generated using hierarchical clustering with no input of prior knowledge. Surprisingly, the overall expression profile of ctk1Δ is more similar to sen1-1, a member of the Ser5P binding NNS-complex, than to any of the Ser2P binding factors (Figure 1A, red part of tree). The expression changes of ctk1Δ and sen1-1 cells show a large overlap (Figure 1A, I, Table S1), of which some transcripts are also affected in mutants of the nuclear exosome, rrp6Δ and lrp1Δ, involved in degradation and trimming of NNS-dependent transcripts. The large overlap between sen1-1 and ctk1Δ suggests a prominent role of Ctk1p in early termination.

Defective termination at snoRNAs in ctk1Δ cells

One class of transcripts terminated by the early termination pathway consists of snoRNAs [22], [27]. Detailed analysis of the genes with altered expression in both ctk1Δ and sen1-1 reveals that many are positioned downstream of snoRNAs, suggesting that transcriptional readthrough from the upstream snoRNA might artificially increase the signal downstream. Previous studies have indeed reported readthrough at a few model snoRNAs in ctk1Δ [27], [28], although whether this reflects a direct or indirect effect mediated by Ser2 phosphorylation of the CTD was unclear.

Deletion of CTK1 appears to lead to the upregulation of the first gene following a snoRNA terminator in approximately one third of cases. All of these genes are also upregulated in sen1-1 (Figure 1B). To confirm that these transcripts originate from defective snoRNA termination, RNA from ctk1Δ cells was analyzed by Northern blot. A double mutant of ctk1Δ and rrp6Δ was included to avoid transcripts being missed due to nuclear degradation. In ctk1Δ, ctk1Δ/rrp6Δ, and sen1-1, a long snR5 transcript is observed that also hybridizes to a probe for the downstream gene HEM4 (Figure 1C), indicating the presence of a fusion transcript between snR5 and HEM4. Similar fusion transcripts are also observed for snR13, snR33, snR43, snR60 and snR82 (Figure 1D, 1E, Figure S1). The same fusion transcripts are observed in sen1-1, showing that these transcripts are caused by defective termination at the snoRNA termination sites. Although the termination defect is not as strong as for the sen1-1 mutant (note that this analysis is performed at permissive temperature and that only a partial termination effect occurs in sen1-1 cells), it is clear that integrity of Ctk1p is nevertheless important for appropriate termination of snoRNAs.

In all these cases we also noticed that the snoRNA precursor (only visible in double ctk1Δ/rrp6Δ mutants when its conversion to the mature form is less efficient) migrates anomalously, with longer forms that extend in some cases for several hundred bases. These transcripts likely derive from delayed termination events that in most cases depend on the NNS pathway, since they are degraded in the wild type strain.

Ctk1p has been shown to influence the co-transcriptional assembly of snoRNAs, which, in turn, might affect termination [28]. It is also possible that the lack of Ser2P alters the local chromatin context, which could impact termination. Therefore we assessed the efficiency of termination using a reporter system in which a snoRNA terminator sequence is inserted upstream of the HIS3 gene and prevents its expression unless transcriptional readthrough occurs. This construct misses sequences directing the co-transcriptional assembly of the core particle and has previously been used to analyze cis and trans elements that direct termination of snoRNAs [29], [30]. As expected, wild type cells are inhibited in growth in the absence of histidine if either snR13 or snR47 termination sequences are placed upstream of the HIS3 marker. In contrast, CTK1 deletion mutants show near-normal growth with the snR47 reporter and only partial inhibition of growth with the snR13 reporter, indicating readthrough at both sites (Figure 1F). These results indicate that the readthrough phenotype observed in ctk1Δ cells is linked to inefficient recognition and/or use of snoRNA termination signals, independent of the genomic context. The generality and the mechanism of Ctk1p dependency were therefore further investigated.

Widespread termination defects at NNS-dependent genes and minor defects at mRNA coding genes in ctk1Δ mutants

The NNS-dependent early termination pathway also terminates other non-coding transcripts including CUTs [1], [2] and several SUTs [44]. The microarrays used in the previous analysis mainly carry probes for detection of mRNAs. To assess how general the role of Ctk1p is in NNS-termination, RNA from ctk1Δ and wild type cells were analyzed on whole-genome tiling arrays [36], [37] (Figure 2). Although reliable detection of cryptic transcripts requires the use of degradation defective strains [3], we reasoned that some extended transcripts would be easily revealed when terminated at downstream CPF/CF sites and therefore stabilized as for snoRNA precursors [45]. Metagene analysis of hybridization signals observed around snoRNAs clearly showed increased average intensity in ctk1Δ cells downstream of the 3′ end (Figure 2A). In addition to snoRNAs, ctk1Δ also shows readthrough of several other sites (Figure 2 C–I). These readthrough sites include CUTs and SUTs, and also non-coding transcripts that lie upstream of coding genes and have been shown to have regulatory potential. For example, the upstream regulatory RNA of SER3, SRG1, shows readthrough into SER3 in ctk1Δ (Figure 2C) [46]. Similar readthrough in ctk1Δ is observed at CUT882 (or one of the more upstream CUTs/SUTs), CUT152 and SUT277, upstream of the GRE1, ASK10, and ERF2 genes, respectively (Figure 2D, E, F). Early termination at the NRD1 gene, which is known to be dependent on the NNS complex [1], is also affected in ctk1Δ cells, as indicated by the increase in full length NRD1 mRNA abundance (Figure 2G). Termination defects were confirmed by Northern analysis at SER3 and CUT882 (Figure S2) although in the latter case the readthrough appears to be unstable and only detected in the ctk1Δ/rrp6Δ double mutant (Figure S2C). Note that, as for snoRNA precursors, longer forms are observed for CUT882 in ctk1Δ/rrp6Δ cells, again suggesting the occurrence of delayed termination.

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Figure 2. Loss of CTK1 also results in termination defects at several upstream regulatory ORFs, CUTs and coding genes.

Expression analysis using tiling arrays in two replicate cultures of wt and ctk1Δ. (A–B) Metagene analyses displaying the average probe intensity over 25 bp regions in wt and ctk1Δ, aligned by the 3′ end of the mature snoRNAs (A) or ORFs (B). Insets show the difference in expression between ctk1Δ and wt. (C–I) Examples of readthrough at non-coding and coding genes: (C) SRG1-SER3, (D) CUT882-GRE1, (E) CUT152-ASK10, (F) SUT277-ERF2 (G) NRD1 (H) BIO4-BIO5, (I) MF(ALPHA)2. Note also the readthrough at snR10 in (I). Expression data are displayed along the chromosome (x axis) for the Watson (W, upper half) and the Crick (C, lower half) strands. Normalized signal intensities (higher in darker blue) are shown for 2 replicates of wt and ctk1Δ (y axis). Grey lines indicate segment boundaries as described in materials and methods. Genome annotations are shown in the center: annotated ORFs (blue boxes), SUTs (orange boxes), CUTs/non-coding RNAs (green boxes), and snoRNAs (brown). Note that CUTs and snoRNA precursors are normally not detectable in wt cells, but the occurrence of a readthrough allows detection because the longer RNAs escape degradation. Readthrough is confirmed by Northern blot for SRG1, CUT882 and BIO5 (Figure S2).

https://doi.org/10.1371/journal.pone.0080495.g002

Ser2P phosphorylation is thought to be important for the recruitment of the CPF/CF complex, because of the interaction of Rtt103p and Pcf11p with the CTD-Ser2P. Surprisingly, we did not observe a global defect in mRNA termination upon loss of the major Ser2P kinase Ctk1p. Indeed some readthrough is observed at a set of mRNAs, but termination by the CPF/CF complex is not affected in general (Figure 2B). When the expression difference between ctk1Δ and wt was calculated downstream of ORFs, only 2.8% of all ORFs show clear indications of readthrough, while similar analysis for snoRNAs reveals readthrough at 32 out of 77 sites (42%). This was confirmed by northern analysis for a few model genes in a ctk1Δ strain (Figure S3) and by metagene analyses of the hybridization signal for ORFs and snoRNAs (Figure 2A–B and S2E–F). This result is seemingly at odds with the finding that mutation of CPF/CF components (the recruitment of which is strongly dependent on Ctk1p [47]) has a generally dramatic effect on termination of mRNA genes ([48], [49]; and data not shown). Examples of mRNA coding genes with readthrough are BIO4/5 and MF(ALPHA)2 (Figure 2H, I). Readthrough was not specifically observed at short (<600 nt) or long (>3 kB) mRNA coding genes (Figure S2E, F). Moreover, it cannot be excluded that the NNS-pathway contributes to termination at some mRNA coding genes as it was shown for BIO4/5 [1].

Together these experiments reveal that the absence of Ctk1p has a general impact in termination of snoRNAs and likely other NNS-dependent transcripts but only affects termination of a subset of mRNA coding genes.

Readthrough is kinase-dependent and partially explained by Ser2 phosphorylation

We first assessed whether defective termination in ctk1Δ was dependent on its kinase activity. A kinase independent role for Ctk1p in the release of basal initiation factors from elongating polymerase has previously been described [50]. To this end, two Ctk1p mutants were generated by introducing a T338A change in the T-loop and a D324N change in the active site. The first mutation results in reduced kinase activity because Ctk1p cannot be phosphorylated and activated anymore by Cak1p. The second mutation abolishes all kinase activity of Ctk1p in vitro [51]. Northern analysis of SNR13 transcripts revealed readthrough defects to similar levels as in ctk1Δ cells only when the kinase activity of Ctk1p is fully impaired in the Ctk1-D324N mutant (Figure 3A). We note that in agreement with a kinase-independent role for Ctk1p in transcription [50], the Ctk1-D324N only partially rescues the growth defect of ctk1Δ (Figure 3B).

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Figure 3. Readthrough is kinase-dependent.

(A) Northern blot analysis of ctk1Δ with a genomically integrated copy of CTK1-wt, CTK1-T338A, or CTK1-D324N. Ctk1-D324N is kinase-dead (data not shown) and cannot rescue the readthrough. (B) Growth of ctk1Δ with a genomically integrated copy of CTK1-wt, CTK1-T338A, or CTK1-D324N. Ctk1-D324N is kinase-dead (data not shown) and can partially rescue the growth defect of ctk1Δ. Serial dilutions (1∶5) are shown starting at 1*10⁴ cells after growth for 2 days on YPD plates at 30°C.

https://doi.org/10.1371/journal.pone.0080495.g003

These results strongly suggest that the readthrough observed at NNS targets is due to an altered CTD phosphorylation pattern in ctk1Δ cells. Although Ctk1p has been shown to affect Ser2 phosphorylation in vivo [52], subtle effects on the other phosphoisoforms might occur that impact termination. Indeed it has been proposed that recruitment of Nrd1 depends on the interaction with Ser5 phosphorylated Pol II CTD [21], [23]. However, western blot analysis of whole cell extracts show a major decrease of Ser2P levels, possibly elevated Ser5P levels and unchanged Ser7P levels (Figure S4A, Figure S6).

CTD phosphorylation patterns were also examined at individual snoRNAs by ChIP. Ser2P is low at snoRNA sites in wt, as previously observed in genome-wide experiments (Figure 4D–F) [10], and strongly reduced in ctk1Δ cells. The 3′ end of PMA1 is used as a positive control and shows high Ser2P enrichment that is lost upon deletion of CTK1. Levels of Ser5P are slightly elevated downstream of the snoRNA genes in ctk1Δ (Figure 4G–I), which can be explained by increased Pol II density, possibly due to readthrough. It is therefore unlikely that changes in the distribution of Ser5 phosphorylation explain the readthrough observed in ctk1Δ. Levels of Ser7P are slightly decreased (Figure 4J–L), but a role for Ser7P in transcription termination has not been identified.

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Figure 4. CTD phosphorylation patterns at snoRNAs are not substantially changed in ctk1Δ cells.

(A, B, and C) Positions of the genomic regions analyzed by ChIP-qPCR. (D, E, and F) ChIP analysis of CTD-Ser2P in wt and ctk1Δ at snR5, snR13 and snR82. The 3′ end of PMA1 is used as a positive control. (G, H, and I) ChIP analysis of CTD-Ser5P in wt and ctk1Δ at snR5, snR13 and snR82. (J, K and I) ChIP analysis of CTD-Ser7P in wt and ctk1Δ at snR5, snR13 and snR82. All ChIP values (average of 3 biological replicates) represent the fold enrichment, expressed as percent fraction of input. Error bars represent standard error. A telomeric region from chromosome V (TEL V) is used as a negative control.

https://doi.org/10.1371/journal.pone.0080495.g004

Upon deletion of Ctk1, the Ser2P levels are strongly reduced, but are not completely absent. To assess whether the low remaining Ser2P levels can be explained by the redundant action of Bur1p, another CTD kinase that has been implicated in Ser2 phosphorylation [13], we constructed strains expressing the CTK1 and BUR1 genes under control of the glucose repressible GAL1 promoter (P_GALCTK1 and P_GALBUR1) and combined the two in a double P_GALCTK1/P_GALBUR1 strain. Depletion of Ctk1p for 3 hours was effective in preventing Ser2P to similar levels as in a ctk1Δ strain (Figure S4B). Co-depletion of both Ctk1p and Bur1p only marginally affected the Ser2P signal with respect to Ctk1p depletion alone (Figure S4B), which indicates a minor (if any) role of Bur1p in global CTD-Ser2P.

We also assessed the effect of depletion of Ctk1p on NNS-dependent termination, as well as the effect of double Ctk1p and Bur1p depletion to consider the occurrence of partial redundancy in Ser2P, either under normal conditions or specifically in the absence of Ctk1p. Transcriptional readthrough leading to the production of fusion transcripts terminating at a downstream CPF/CF site were clearly observed upon Ctk1p depletion (Figure S2D). Longer unstable transcripts, likely terminating at downstream sites by the NNS pathways were also observed, the levels of which were not further increased by Bur1p co-depletion, suggesting a minor role for Bur1p in NNS-dependent termination (Figure S2D). These results also verify that readthrough is not an indirect effect of the strong growth defect of ctk1Δ.

NNS-dependent termination was only partially impaired in ctk1Δ cells. This could occur because residual levels of CTD-Ser2P in ctk1Δ, possibly generated by another pathway, are sufficient for basal termination levels. Therefore we analyzed the efficiency of the NNS pathways in a rbp1-S2A mutant, in which Ser2 in every repeat was mutated to alanine [53]. rpb1-S2A cells show clear readthrough at SNR13, to similar levels (or even lower) than in ctk1Δ cells (Figure 5A). A double ctk1Δ/rpb1-S2A mutant behaved similarly to the single ctk1Δ mutant. This indicates that Ser2P impacts the efficiency of NNS-dependent termination but is not absolutely required. Finally, we also considered the possibility that low CTD-Ser2P levels might be the reason why mRNA termination is not globally affected in ctk1Δ cells. However, the correlation between the expression profile of ctk1Δ and rpb1-S2A is high (0.86, Figure 5B), showing that the overall expression effects are highly similar and that, similarly to deletion of Ctk1p, the rpb1-S2A mutant does not have a global termination defect at mRNAs genes.

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Figure 5. Readthrough is partially explained by Ser2 phosphorylation.

(A) Northern blot analysis of an rpb1-S2A mutant in which every Ser2 in the CTD is mutated to an alanine. Readthrough transcripts (RT) are indicated. (B) Correlation plot of expression changes (M, log2 fold change) in ctk1Δ and rpb1-S2A versus wt, as measured by microarray analysis.

https://doi.org/10.1371/journal.pone.0080495.g005

The above data strongly suggest that CTD-Ser2 phosphorylation is the primary target of Ctk1p in promoting termination at NNS-dependent genes. They also confirm that CTD-Ser2P impacts the efficiency of NNS-dependent termination but is generally not required for termination of mRNA coding genes.

Nrd1p recruitment is not affected in ctk1Δ

Termination defects have previously been associated with aberrant recruitment of termination factors [54]. In order to understand the mechanism by which Ctk1p contributes to the early termination pathway, binding of Pol II, Nrd1p and Pcf11p was determined by chromatin immunoprecipitation (ChIP) at SNR5, SNR13 and SNR82 (Figure 6 and S5). Pol II levels are increased downstream of all three snoRNAs in ctk1Δ, consistent with readthrough into adjacent genes (Fig. S5D–F). As expected, Nrd1p is recruited to the 3′ end of all three snoRNAs in wild type (Fig. S5G–I). Upon deletion of CTK1, Nrd1p occupancy closely follows polymerase levels, being unchanged in the body of the genes and elevated downstream (Figure S5G–I; better visible when Nrd1p signals are normalized to Rpb3p signals (Figure 6D–F). Interestingly, we also detected an increased association of Nrd1p with Ser5P Rbp1p in ctk1Δ cells by co-immunoprecipitation (Figure S6), which could either be a consequence or a cause of the termination defect (see discussion).

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Figure 6. Recruitment of Nrd1p and Pcf11p, relative to Rpb3p.

(A, B, and C) Scheme of the genomic regions analyzed by ChIP-qPCR. (D, E, and F) Analysis of Nrd1p occupancy relative to Rpb3p occupancy as determined by ChIP in wild type and ctk1Δ strains at the snR5, snR13 and snR82 loci. Relative ChIP values are normalized to the TEL-V signal in wt. (G, H, and I) Analysis of Pcf11p occupancy relative to Rpb3p occupancy in wt and ctk1Δ at snR5, snR13 and snR82. Relative ChIP values are normalized to the TEL-V signal in wt. Error bars represent standard errors. Raw Rpb3p, Nrd1p and Pcf11p levels are shown in Figure S5.

https://doi.org/10.1371/journal.pone.0080495.g006

A different pattern is observed for Pcf11p. We observed recruitment of Pcf11p to snoRNA genes that was highest in the termination region. The Pcf11p ChIP signal was seemingly similar in wild type and ctk1Δ cells (Figure S5J–L). However, when the signal was considered relative to the level of transcription in each region, i.e. when normalized to Rpb3p signal (that is higher in ctk1Δ cells due to the readthrough, Figure S6G–I), Pcf11p occupancy was clearly found to be negatively affected by deletion of Ctk1p, which mirrors Ctk1p dependency at mRNA genes [26]. From these experiments we conclude that defective recruitment of Nrd1p cannot account for the termination defect observed in ctk1Δ strain. The results also indicate that Pcf11p (and presumably the CPF/CF complex) is recruited at snoRNA genes in a Ctk1p-dependent manner.

Partial suppression of the ctk1Δ phenotype by a transcription elongation mutant

The observation that longer transcripts, presumably representing primary termination products, can be observed at NNS-dependent transcription units in ctk1Δ cells, suggests that transcription termination is delayed in the absence of functional Ctk1p. It has recently been shown that termination is sensitive to the elongation properties of RNA pol II, with “faster” polymerase mutants delaying termination and which is suggested to depend on the existence of kinetic competition between transcription and termination [55]. This suggests that in ctk1Δ cells, presumably because of the low or absent Ser2P levels, RNA polymerase might behave as a fast mutant with regard to termination. We therefore investigated whether altering the elongation properties of RNA Pol II might impact the delayed termination phenotype of ctk1Δ cells. Spt5p is an elongation factor that has an important role in regulating the processivity of RNA Pol II [56]. Its C-terminal region has been implicated to be involved in the recruitment of the PAF complex, another elongation complex. Deletion of the Spt5p C-terminal region (spt5ΔCTR) is lethal in a ctk1Δ strain. We therefore introduced the spt5ΔCTR mutation in a P_GalCTK1 strain and analyzed the effects of Ctk1p depletion in an spt5ΔCTR context. We performed these experiments in an rrp6Δ background to visualize CUTs or snoRNA precursors as before. The spt5ΔCTR mutation alone produced slightly shorter precursors at most NNS-dependent genes (Figure 7), consistent with the notion of kinetic competition between transcription elongation and termination. Interestingly, the spt5ΔCTR mutation suppressed the delayed termination phenotype observed at NEL025C and SNR47 genes upon Ctk1p depletion. Indeed, the upshift of the transcript smear observed upon Ctk1p depletion (compare lanes 4 to 9) is not observed in the spt5ΔCTR context.

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Figure 7. Partial suppression of ctk1Δ termination phenotype by a spt5ΔCTR mutant.

Northern blot showing the effect of the spt5ΔCTR mutation on termination at snR47 and the NEL025c CUT in strains metabolically depleted for Ctk1p and Bur1p. CTK1 and BUR1 were expressed under control of the GAL1 promoter and depletion was carried for the times indicated. Note the appearance of longer RNA species upon depletion of CTK1 that are suppressed in an spt5ΔCTR background. An rrp6Δ background was used to allow detection of unstable RNA species. Transcripts were revealed with double stranded probes that span the entire snR47, NEL025c genes or the 3′ end of ACT1.

https://doi.org/10.1371/journal.pone.0080495.g007