Molecular Cell
Article
Rtr1 Is a CTD Phosphatase that Regulates RNA
Polymerase II during the Transition from Serine 5
to Serine 2 Phosphorylation
Amber L. Mosley,1 Samantha G. Pattenden,1 Michael Carey,1,2 Swaminathan Venkatesh,1 Joshua M. Gilmore,1
Laurence Florens,1 Jerry L. Workman,1 and Michael P. Washburn1,*
1Stowers
Institute for Medical Research, Kansas City, MO 64110, USA
of Biological Chemistry, David Geffen School of Medicine, University of California, Los Angeles, 10833 LeConte Avenue,
Los Angeles, CA 90095, USA
*Correspondence: [email protected]
DOI 10.1016/j.molcel.2009.02.025
2Department
SUMMARY
Messenger RNA processing is coupled to RNA
polymerase II (RNAPII) transcription through coordinated recruitment of accessory proteins to the Rpb1
C-terminal domain (CTD). Dynamic changes in CTD
phosphorylation during transcription elongation are
responsible for their recruitment, with serine 5 phosphorylation (S5-P) occurring toward the 50 end of
genes and serine 2 phosphorylation (S2-P) occurring
toward the 30 end. The proteins responsible for regulation of the transition state between S5-P and S2-P
CTD remain elusive. We show that a conserved
protein of unknown function, Rtr1, localizes within
coding regions, with maximum levels of enrichment
occurring between the peaks of S5-P and S2-P
RNAPII. Upon deletion of Rtr1, the S5-P form of
RNAPII accumulates in both whole-cell extracts and
throughout coding regions; additionally, RNAPII transcription is decreased, and termination defects are
observed. Functional characterization of Rtr1 reveals
its role as a CTD phosphatase essential for the S5to-S2-P transition.
INTRODUCTION
From yeast to mammals, there are three highly conserved RNA
polymerase complexes that are responsible for the transcription
of all classes of cellular RNAs. RNA processing is closely tied to
transcription in order to ensure the fate of nascent RNA. One
unique mechanism for proper RNA processing involves the
recruitment of a wide variety of accessory proteins to the
C-terminal domain (CTD) of the largest subunit of RNAPII, Rpb1
(for review, see Phatnani and Greenleaf, 2006). The CTD consists
of 27 repeats of the sequence Y1S2P3T4S5P6S7 in yeast and is not
conserved within the Rpb1 counterparts found in RNAP I and
RNAPIII, thereby serving as a unique signaling platform for
RNAPII. In order to form a competent initiation complex at the
promoter of a target gene, the CTD must exist in a hypophosphorylated state. Following assembly of the initiation complex,
168 Molecular Cell 34, 168–178, April 24, 2009 ª2009 Elsevier Inc.
the CTD exhibits increased phosphorylation on serine 5 (S5-P),
carried out by the cyclin-dependent kinase Kin28, a subunit of
the general transcription factor TFIIH (Komarnitsky et al., 2000;
Schroeder et al., 2000). This phosphorylation event is responsible
for the recruitment of the capping machinery, which begin processing of the nascent mRNA during early transcription (Cho
et al., 1997; Fabrega et al., 2003; Komarnitsky et al., 2000;
Schroeder et al., 2000). As transcription elongation progresses,
there is a change in the modification state of the CTD as serine
2 phosphorylation (S2-P) increases through the action of the
CTDK-I complex (Cho et al., 2001). Chromatin immunoprecipitation (ChIP) experiments have demonstrated that the increase
in S2-P occurs as transcription progresses through the open
reading frame (ORF) (Komarnitsky et al., 2000). As transcription
approaches the 30 end of the ORF, the termination and polyadenylation machinery are recruited, some of which interact with the
S2-P CTD (Licatalosi et al., 2002; Meinhart and Cramer, 2004;
Kim et al., 2004). Although this transition state from S5-P to
S2-P during the transcription cycle is thought to distinguish
different phases of RNAPII elongation, the proteins involved in
the decrease of S5-P during elongation have yet to be identified.
In addition to the aforementioned CTD kinases, the actions of
CTD phosphatases are also required to manage the different
CTD modification states. Two CTD phosphatases, Fcp1 and
Ssu72, have been characterized in yeast (for review, see Meinhart
et al., 2005). Fcp1 has a preference for the S2-P modification and
has been shown by ChIP analysis to colocalize with RNAPII
throughout coding regions (Cho et al., 2001). In addition, Fcp1
mutants show an increase in the level of S2-P in the coding region
of genes, indicating that the phosphatase plays a role in dephosphorylation of S2-P during the transcription cycle (Cho et al.,
2001). Fcp1 is also thought to play a major role in RNAPII recycling
after the complex has dissociated from the coding region (Cho
et al., 1999; Kong et al., 2005; Archambault et al., 1997; Chambers
et al., 1995; Aygun et al., 2008). Ssu72, conversely, is a S5-Pspecific CTD phosphatase and a component of the yeast cleavage
and polyadenylation factor (CPF), which is involved in mRNA processing at the 30 ends of genes (Krishnamurthy et al., 2004; ReyesReyes and Hampsey, 2007). ChIP assays have revealed that
Ssu72 is predominately enriched at the 30 ends of genes, with little
to no enrichment found at the promoter (Nedea et al., 2003; Ansari
and Hampsey, 2005). Although Fcp1 and Ssu72 have both been
Molecular Cell
Rtr1 Regulates RNAPII CTD Phosphorylation
Figure 1. Rtr1 Interacts with the Intact RNAPII Complex
(A) Summary of five different MudPIT analyses of the baits given at the top. The sequence coverage and total number of unique peptides for each subunit of
interest are shown to the left.
(B) Western blot analysis of anion-exchange fractions obtained following separation of Rpb3-TAP complexes using antibodies directed against the unmodified
CTD (UM CTD), S5-P CTD, Anti-CBP (Rpb3), or Anti-FLAG (Rtr1).
(C) Heat map representation of SAF values obtained from MudPIT analysis of each fraction (top, corresponding to Figure 1B). The highest SAF values are indicated in bright yellow, and the absent values are indicated in black, as shown in the intensity scale.
implicated in dephosphorylation of the RNAPII CTD, neither phosphatase has been shown to regulate the S5-P-to-S2-P transition
during transcription elongation. Therefore, it is likely that an additional regulatory protein(s) is required to direct the S5-P-to-S2-P
transition dephosphorylation event.
In this study, we have characterized the interaction of a
conserved protein of unknown function, Rtr1 (regulator of transcription) (Gibney et al., 2008), with RNAPII. Recent studies on
Rtr1 revealed genetic interactions implicating the protein in the
regulation of RNAPII transcription (Gibney et al., 2008). Our
current study reveals that Rtr1 is a bona fide RNAPII-associated
protein that copurifies with a transcriptionally competent form of
the enzyme and can interact with CTD peptides in vitro. ChIP
assays show that Rtr1 localizes to the coding regions of PMA1
and PYK1 (also known as CDC19) and that the highest level of
association was seen between the peaks of S5-P and S2-P
RNAPII. Deletion of Rtr1 results in the accumulation of S5-P
RNAPII in whole-cell extracts, as well as across the ORFs of
PMA1 and PYK1 as shown by ChIP. In addition, we show that
Rtr1 is able to dephosphorylate RNAPII that is present in a ternary
complex, supporting our hypothesis that Rtr1 is a CTD phosphatase that targets RNAPII during transcription elongation.
RESULTS
Identification of Rtr1 as a Bona Fide RNAPII
CTD-Associated Protein
Our initial analysis focused on the identification of RNAPII-interacting proteins through multidimensional protein identification
technology (MudPIT) analysis (Florens et al., 2006) of RNAPII
complexes. Using RNAPII subunits as bait, we were able to identify all 12 known subunits of the complex (Figure 1A). In addition,
Molecular Cell 34, 168–178, April 24, 2009 ª2009 Elsevier Inc. 169
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Rtr1 Regulates RNAPII CTD Phosphorylation
Figure 2. Rtr1 Associates with Transcriptionally Active RNAPII
(A) In vitro transcription reactions were performed
using increasing concentrations of Rpb3-TAP or
Rtr1-TAP as shown. The full-length (FL) RNA transcript is indicated to the right. The volume and
intensity of the bands were measured using ImageQuant, and the percent intensity compared to the
maximum level is given at the bottom of the figure.
(B) Western blot analysis using monoclonal antibodies directed against the different forms of the
RNAPII CTD as shown. Approximately 30 mg of
whole-cell yeast extract (WCE) was analyzed for
comparison to Rtr1-TAP purifications.
(C) Western blot analysis on CTD peptide-binding
assays visualized using antibodies against HA.
The CTD peptides used were either unmodified
(UM) or S5-P, S2-P, or S2,5-P.
(D) Quantitation of western blots represented in (C)
was performed by ImageQuant on four biological
replicates and is expressed as the average binding
intensity normalized to the UM CTD peptide ± SD.
we identified a protein of unknown function, Rtr1. Purification of
Rtr1 confirmed its interaction with RNAPII, in agreement with
data from large-scale proteomics studies in yeast (Gavin et al.,
2002). Interestingly, the human homolog of Rtr1, RPAP-2, has
also been shown to be an RNAPII-associated protein, indicating
that the interaction is conserved in higher eukaryotes (Jeronimo
et al., 2007). In addition to the identification of all 12 RNAPII
subunits in the Rtr1-TAP purification, we were able to identify
Rtr1 in four purifications using TAP-tagged RNAPII subunits as
a bait, which has not been reported previously to our knowledge
(Figure 1A). This information indicates that Rtr1 is associated with
a significant fraction of RNAPII and prompted us to further characterize the interaction. In order to determine whether Rtr1 was
a bona fide RNA polymerase II-associated protein, RNAPII was
purified via the TAP-tagged subunit Rpb3 in a strain that also contained Rtr1-His6-FLAG3-HA3 (Rtr1-HFH). The resulting RNAPII
population was then fractionated by anion exchange chromatography performed as previously described (Hu et al., 2006). Rtr1
cofractionates with Rpb3 and Rpb1 (using antibodies against
either the S5-P or unmodified [UM] form of the CTD; Figure 1B).
The western blots were confirmed by MudPIT analyses on the
peak fractions as shown, which is visualized through a heat
map displaying the spectral abundance factors (SAF) of the
proteins (Figure 1C and Table S3 available online). These data
show that Rtr1 does indeed cofractionate with all 12 subunits of
RNAPII, indicating that they form a stable complex.
To determine whether Rtr1 associates with functional RNAPII,
electromobility shift assays (EMSA) and in vitro transcription
experiments were performed. We used equal amounts of RNAPII
purified through either Rpb3-TAP or an Rtr1-TAP strain as previ-
170 Molecular Cell 34, 168–178, April 24, 2009 ª2009 Elsevier Inc.
ously described and performed experiments using a C-tailed DNA template,
which allowed for RNAPII binding in
the absence of the general transcription
factors (Carey et al., 2006). We found
that the DNA-binding activity of RNAPII
to the C-tailed template was similar for both the Rpb3-TAP and
Rtr1-TAP complexes (Figure S1). The amount of RNAPII used
in the in vitro transcription experiments was normalized to this
DNA-binding activity. Both Rtr1-TAP- and Rpb3-TAP-purified
RNAPII were able to produce full-length (FL) transcripts, indicating that Rtr1 interacts with a transcriptionally competent
RNAPII (Figure 2A). However, we did observe a slight decrease
in the total amount of FL transcript when comparing the highest
concentrations of Rpb3-TAP to Rtr1-TAP (Figure 2A, bottom).
These data indicate that the association of Rtr1 with RNAPII
may be inhibitory to transcription elongation.
We next sought to determine whether Rtr1 interacts with a
specific form of RNAPII that may direct its function during the
transcription cycle. We performed western blot analysis on
whole-cell yeast extracts (WCE) and Rtr1-TAP purifications using
antibodies directed against the different forms of the RNAPII CTD.
Rtr1 interacts with both the unmodified (UM) and S5-P forms of
the RNAPII CTD in vivo, which was also recently shown by Gibney
et al. (Figure 2B; Gibney et al., 2008). Because Rtr1 displays
a binding preference for a particular form of the modified CTD,
we next asked whether Rtr1 was able to directly interact with
the RNAPII CTD repeat sequence. CTD peptide-binding assays
were performed using recombinant Rtr1 (rRtr1) purified from
bacteria and biotinylated CTD peptides containing four repeats
of the Y1S2P3T4S5P6S7 sequence, which were unmodified or
phosphorylated at S5, S2, or S2 and S5 (S2,5). These experiments
show that rRtr1 is able to directly interact with the CTD peptides
in vitro (Figure 2C). Although able to bind all four peptides, Rtr1
shows a preference for the S5-P form of the CTD and has the
lowest affinity for the S2,5 modified peptide (Figure 2D).
Molecular Cell
Rtr1 Regulates RNAPII CTD Phosphorylation
Figure 3. Rtr1 Localizes to Open Reading
Frames
(A) ChIP assays were performed using antibodies
directed against different forms of RNAPII and
Rtr1 as indicated. Quantitation of the S5-P (black
circles) and S2-P (blue squares) occupancy
compared to the occupancy of Rtr1 (red triangles)
across the PMA1 genomic loci.
(B) Quantitation of protein occupancy across the
PYK1 genomic loci as performed above. These
data are shown as the average percent maximum
IP ± SD for comparison purposes between the
different antibodies (n = 3). The midpoint of the
PCR amplicon was used as the distance from
the ATG. A schematic representation of the
genomic loci for each gene is shown below the
graphs to illustrate the approximate location of
the promoter and polyadenylation regions.
(Figure S2). This localization, in combination with our other results, could indicate
a role for Rtr1 in the transition from S5-P
CTD to S2-P CTD in vivo.
Rtr1 Is Found in the Open Reading Frames
of PMA1 and PYK1
To determine whether Rtr1 colocalizes with elongating, initiating,
or terminating RNAPII in vivo, chromatin immunoprecipitation
(ChIP) assays were performed with an Rtr1-HFH strain using
antibodies directed against HA. The genomic loci of PMA1 and
PYK1 were analyzed for Rtr1, S5-P, and S2-P RNAPII occupancy
using qPCR (Figures 3A and 3B). Both of these genes are highly
expressed and have previously been used to study the localization of different phosphorylated forms of RNAPII (Komarnitsky
et al., 2000). As shown in Figures 3A and 3B, a peak in S5-phosphorylated RNAPII is observed at the 50 end of both PMA1 and
PYK1 and decreases prior to an observed increase of S2-P, in
agreement with previous results (Komarnitsky et al., 2000).
Surprisingly, we found a strong and distinct peak of Rtr1 that
localized to a region of both genes between the enriched regions
of S5-P and S2-P. This peak was also observed after normalization of the levels of Rtr1 to the level of RNAPII occupancy
Rtr1 Is Required for S5-P
Dephosphorylation during Early
RNAPII Elongation
The Rtr1 deletion strain was analyzed for
defects in CTD phosphorylation in vivo
by western blot analysis of whole-cell
extracts. Loss of Rtr1 resulted in increased
levels of S5-P CTD in vivo (Figure 4A,
upper panel) and corresponded with a
slight decrease in the cellular level of
unmodified RNAPII (Figure 4B, third
panel). The level of S2-P was not affected
in rtr1D extracts, nor was the level of Rpb3,
which was used as a control for protein
loading (Figure 4A, bottom two panels).
Quantitation was performed on triplicate
experiments and is shown in Figure 4B. These data indicate that
Rtr1 is involved in the regulation of S5-P in vivo and support the
hypothesis that Rtr1 is involved in decreasing S5-P in wild-type
cells, given that there is an accumulation of the S5-P form in
rtr1D cells.
In order to determine the effects of Rtr1 deletion on the levels of
DNA-associated RNAPII, we performed ChIP analyses in the
rtr1D strain and compared the levels of S5-P and S2-P RNAPII
to those found in wild-type. The levels of S5-P RNAPII dramatically increase in the rtr1D cells when compared to the levels
observed in wild-type cells at both the PMA1 and PYK1 ORFs
(Figures 4C and 4D). This finding is especially true toward the
30 ends of these coding regions, where S5-P on the CTD is
normally very low. The level of S5-P RNAPII decreases near the
polyadenylation sites of both genes to near wild-type levels.
This decline is likely due to decreased RNAPII occupancy in these
regions, which was observed for Rpb3 in both the rtr1D and
wild-type cells (Figure 5A). The levels of S2-P RNAPII were also
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Rtr1 Regulates RNAPII CTD Phosphorylation
Figure 4. Rtr1 Deletion Causes Increased RNAPII CTD S5-P In Vivo
(A) Western blot analysis of whole-cell extracts of wild-type (WT) compared to rtr1D.
(B) Quantitation of western blots represented in (A). The data are shown as average fold enrichment over WT ± SD (n = 3).
(C and D) Quantitation of the fold enrichment of S5-P RNAPII occupancy across the PMA1 (C) and PYK1 (D) open reading frames using the H14 monoclonal
antibody. The midpoint of the PCR amplicon was used as the distance from the PMA1/PYK1 ATG. Data are expressed as average fold enrichment ± SD
(n = 3), which was calculated by dividing the percent IP of each region over the percent IP of a control region on chromosome 5, where RNAPII does not bind.
analyzed at the PMA1- and PYK1-coding regions (Figure S3). In
the Rtr1 deletion, there was a slight increase in the amount of
S2-P RNAPII at both PMA1 and PYK1 throughout their ORFs,
suggesting that there may be local increases in S2-P, although
the total cellular level of S2-P remains unaffected in rtr1D cells.
Loss of Rtr1 Causes Defects in RNAPII Transcription
We next addressed the effects of Rtr1 deletion on the RNAPII
transcription cycle by measuring the occupancy of RNAPII
across the PMA1 and PYK1 open reading frames. For these
experiments, we used antibodies directed against the RNAPIIspecific subunit Rpb3 in both WT and rtr1D cells for ChIP analysis. As shown in Figures 5A and 5B, the total amount of Rpb3
occupancy at PMA1 and PYK1 decreases an average of 40%
in the absence of Rtr1. A similar loss of RNAPII occupancy has
been observed when analyzing Rpb3 occupancy in strains containing conditional mutants of the CTD phosphatase Fcp1 (Cho
et al., 2001).
To address whether or not loss of Rtr1 may result in other transcriptional defects such as transcription readthrough, northern
blot analysis was performed on total RNA isolated from either
WT or rtr1D cells using a probe directed against the 30 end of
PMA1. Although no readthrough transcript was observed, we
172 Molecular Cell 34, 168–178, April 24, 2009 ª2009 Elsevier Inc.
could detect a decrease in the total amount of PMA1 in the
rtr1D cells when compared to the levels of an RNAPIII transcript,
SCR1, or when compared to the RNAPI transcript for the 18S
rRNA (Figure 5C). Quantitation was performed on triplicate
experiments and is shown in Figure 5D, confirming the decrease
in the level of the PMA1 transcript. This observation was further
verified using qRT-PCR analysis of PMA1, as well as PYK1 and
ACT1, compared to the levels of SCR1 (Figure 5E). These data
support that the loss of Rtr1 results in both loss of RNAPII occupancy and reduced transcription.
Because no readthrough transcripts were observed at the
PMA1 gene, we performed additional northern blot experiments
with WT or rtr1D polyA+ mRNA using probes directed against the
NRD1 and MRPL17 loci. We chose these loci for analysis
because they had previously been shown to display a readthrough phenotype in cells containing a thermosensitive allele
of Ssu72, whose inactivation also causes accumulation of S5-P
RNAPII in vivo (Ganem et al., 2003; Krishnamurthy et al., 2004).
The loss of Rtr1 results in a readthrough transcript at NRD1 that
can also be detected with probes directed against a downstream
gene, MRPL17, confirming that it was a readthrough product
(Figure 5F). The observation of this phenotype in rtr1D cells
suggests that the activity of both Ssu72 and Rtr1 is required for
Molecular Cell
Rtr1 Regulates RNAPII CTD Phosphorylation
Figure 5. Deletion of Rtr1 Causes Transcription Defects In Vivo
(A) The level of RNAPII occupancy decreases in rtr1D. Quantitation of the total RNAPII (measured by Rpb3) occupancy PMA1 genomic loci in both WT (black
diamonds) and rtr1D (open diamonds) deletion strains.
(B) Quantitation of Rpb3 occupancy across the PYK1 genomic loci as performed above. Data are expressed as average fold over background ± SD (n = 3).
(C) Northern blot analysis of total RNA isolated from either WT or rtr1D cells as indicated. Northern blots (left panel) were visualized with probes directed against
the RNAPII transcript PMA1 followed by probes directed against the RNAPIII transcript SCR. The 18S and 25S rRNA were visualized through ethidium bromide
staining.
(D) Quantitation of three replicate northern blot analyses were performed by ImageQuant and are expressed as the average intensity of PMA1 normalized to the
level of SCR1 or 18S rRNA ± SD.
(E) Quantitative RT-PCR analysis from either WT or rtr1D total RNA for PMA1, PYK1, and ACT. Transcript levels are expressed as the average total ng of transcript
normalized to the total ng of the RNAPIII transcript SCR1 ± SD.
(F) Northern blot analysis of polyA mRNA isolated from either WT or rtr1D strains using probes directed against the 30 end of NRD1 or the entire coding region
of MRPL17. The location of the different transcripts is shown to the left of each panel, and a schematic of the NRD1/MRPL17 loci is shown for visualization at
the bottom.
proper RNAPII termination at some loci in vivo. Their requirement
for proper termination at some loci may be related to the affinity of
termination factors such as Pcf11 and Rtt103 for S2-P rather than
S5-P CTD (Licatalosi et al., 2002; Meinhart and Cramer, 2004;
Kim et al., 2004).
Rtr1 Is a CTD Phosphatase
The effects of Rtr1 on RNAPII transcription mirror effects previously seen with thermosensitive alleles of Fcp1 and Ssu72, the
two known CTD phosphatases in yeast. In light of these parallels,
we sought to determine whether Rtr1 could also function as a
CTD phosphatase in vitro. For these experiments, Rtr1 was purified from bacteria using a two-step affinity purification to limit
contaminating bacterial proteins (Figure S4). Recombinant Rtr1
was used for in vitro phosphatase reactions using TFIIK 32Plabeled CTD peptide. As shown in Figure 6A, Rtr1 is able to
dephosphorylate the CTD peptide in vitro, indicating that it is
capable of acting as a CTD phosphatase. To test whether Rtr1
has a preference for either S2-P or S5-P, we performed phosphatase reactions with GST-CTD that had been modified in vitro by
either CTDK-I or MAPK2 and visualized the different forms of
GST-CTD using western blotting with antibodies that detect
either S2-P or S5-P. The kinases CTDK-I and MAPK2 were
used for these experiments because they both exhibit robust
activity in vitro and target both S2 and S5 for phosphorylation
(Trigon et al., 1998; Jones et al., 2004). Rtr1 displays phosphatase
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Rtr1 Regulates RNAPII CTD Phosphorylation
Figure 6. Rtr1 Is a CTD Phosphatase
(A) In vitro phosphatase reactions using 1 ug of
TFIIK 32P-labeled biotinylated CTD peptide (containing four YSPTSPS repeats) as substrate and
increasing concentrations of purified recombinant
Rtr1. The CTD peptide was resolved on a 16%
Tricine gel that was then dried and exposed to
a phosphorimaging screen.
(B) Western blot analysis of Rtr1 phosphatase
reactions performed using CTDK-I-labeled GSTCTD as a substrate. Reactions were visualized
using antibodies directed against S5-P, S2-P, or
HA. Approximately 5 pmol of GST-CTD was used
as a substrate in each reaction using increasing
concentrations of Rtr1.
(C) Western blot analysis of Rtr1 phosphatase
reactions performed as in (B), except that the
GST-CTD substrate was modified by MAPK2 prior
to the phosphatase experiments. *Quantitation for
the MAPK2 experiments was performed on the
slower-migrating bands as illustrated.
(D) Quantitation of three replicate phosphatase
reactions performed on either CTDK-I- or MAPK2modified GST-CTD. Quantitation was performed
using ImageQuant and is expressed as the average
percent maximum intensity ± SD.
(E) Western blot analysis of whole-cell extracts
from wild-type and Rtr1 mutant strains. Cells
were grown in SD. Ura minus media and total
cellular protein were extracted with 10, 50,
and 100 mg of total protein loaded. The BY4741
and rtr1D strains were transformed with the
URA3-containing plasmid pRS416. Proteins were
visualized using antibodies directed against S5-P,
Pgk1, Rpb3 (monoclonal), or Rtr1-TAP (TAP tag
detected using Peroxidase antiperoxidase).
(F) Quantitation of three replicate western blots as
shown in (E) using ImageQuant software. Data are
expressed as the average percent of wild-type
(BY4741) S5-P intensity normalized to the level
of Pgk1 as a loading control ± SD.
activity on both CTDK-I- and MAPK2-modified GST-CTD (as
shown in Figures 6B and 6C) and appears to have a preference
for the S5-P form of the protein (Figure 6D). Some dephosphorylation of a high-mobility form of S2-P GST-CTD was seen in the
CTDK-I-modified reactions, which are likely hyperphosphorylated (Figure S5). The dephosphorylation of S2-P may be due to
the fact that the H5 monoclonal antibody has some cross-reactivity with the S5 modification, or it could indicate that Rtr1 also
has some affinity toward S2-P (Jones et al., 2004).
Although Rtr1 does not contain a previously described phosphatase motif, it does have a number of conserved residues
that could contribute to its catalytic activity. In fact, studies by
Gibney et al. revealed that C73 is required to compensate for
loss of Rtr1 in yeast (Gibney et al., 2008). In order to address
whether the defects seen with C73 mutation correlate with
changes in the total levels of RNAPII phosphorylation, we performed western blot analysis on yeast strains containing either
WT or C73A TAP-tagged Rtr1 integrated into its chromosomal
locus. Using increasing concentrations of extract, we observed
increased levels of S5-P in the Rtr1-C73A-TAP strain as
compared to Rtr1-TAP (Figures 6E and 6F). The level of S5-P in
174 Molecular Cell 34, 168–178, April 24, 2009 ª2009 Elsevier Inc.
the C73A-TAP strain was similar to that observed in rtr1D cells,
indicating that the mutant does not efficiently regulate S5-P levels
in vivo. In addition, copper induction experiments were performed in rtr1D cells that had been transformed with a CUP1
promoter-driven plasmid containing either wild-type or C73S
RTR1. Upon copper induction, there was a decrease in the total
level of S5-P RNAPII in rtr1D cells containing the RTR1 wildtype plasmid. However, induction of the C73S-Rtr1 mutant did
not result in decreases in the total level of S5-P in the cells, which
again suggests that C73 is required for Rtr1 function in vivo
(Figures S6A and S6B).
Our experiments suggest that Rtr1 is a CTD phosphatase that
functions during the transcription cycle to target S5-P for
dephosphorylation. For this to occur, Rtr1 would have to display
phosphatase activity on an intact RNAPII complex that was
engaged on DNA. In order to test whether Rtr1 was able to
dephosphorylate RNAPII present in an elongation complex, we
prepared ternary complexes in vitro, as previously described,
on a biotinylated DNA template with a 30 overhang (Figure 7A;
Kong et al., 2005). For these experiments, we used Rpb3-TAPpurified RNAPII, which contains all 12 subunits (Figure 1A), and
Molecular Cell
Rtr1 Regulates RNAPII CTD Phosphorylation
Figure 7. Rtr1 Can Dephosphorylate RNAPII
that Is Present in a Ternary Complex In Vitro
(A) Schematic representation of the ternary complex
substrate assembled on a doubled-stranded oligonucleotide with a 30 overhang using Rpb3-TAP-purified RNAPII.
(B) Rtr1 phosphatase reactions performed on
5 pmol of TFIIK 32P-labeled ternary complex.
The concentration of Rtr1 in each reaction is indicated at the top of the panel.
(C) Rtr1 phosphatase reactions performed on
5 pmol of MAPK2 32P-labeled ternary complex.
The reactions in (B) and (C) were stopped by the
addition of 63 SDS-loading buffer and were separated by SDS-PAGE. Bands were visualized by
autoradiography.
(D) Quantitation of replicate phosphatase reactions as shown in (B) and (C) were performed using
ImageQuant. Values are expressed as average
intensity normalized to the input sample ± SD.
phosphorylated the complex in the presence of [g-32P]-ATP with
either TFIIK or MAPK2. As shown in Figures 7B and 7C, Rtr1 is
able to dephosphorylate RNAPII that is present in an elongation
complex with similar efficiency as seen with the free GST-CTD
protein (Figures 6D and 7D). These data support our hypothesis
that Rtr1 is a CTD phosphatase that targets RNAPII during the
transition in the CTD modification state.
DISCUSSION
We have shown that Rtr1 is a phosphatase that preferentially
dephosphorylates the S5 modification on the RNAPII CTD
in vivo. In addition to Rtr1, the phosphatases Ssu72 and Fcp1
also play an important role in the regulation of CTD phosphorylation. The depletion of Fcp1 in vivo has been shown to result in
the specific accumulation of S2-P RNAPII, which is enriched at
the 30 end of genes (Cho et al., 2001). The depletion of Ssu72,
conversely, has been shown to lead to an accumulation in the
S5-P form of RNAPII by western blot analysis (Krishnamurthy
et al., 2004). However, it has been unclear whether Ssu72 is the
transition phosphatase, given that, as a member of the CPF
complex, its major site of localization is toward the 30 end of the
gene (Nedea et al., 2003) and inactivation of Ssu72 has not
been shown to increase S5-P across open reading frames. The
localization of Rtr1 by ChIP indicates that the protein is not
enriched at the promoter but is highly enriched at a region
preceding the phosphorylation of serine 2, which is consistent
with Rtr1 being the transition phosphatase. This hypothesis is
also supported by the observation that Rtr1-TAP purifications
are enriched in RNAPII complexes that contain some combination
of nonphosphorylated and S5 phosphorylated repeats. These
data provide evidence of a phosphatase specifically involved in
the regulation of the S5-P-to-S2-P transition and indicate that
Rtr1 is involved in S5-P dephosphorylation during elongation.
We have shown that mutation of C73 in Rtr1 leads to increased
S5-P of the RNAPII CTD in vivo to levels that rival the rtr1D full
deletion strain. This cysteine residue is completely conserved
in Rtr1 homologs in higher eukaryotes, and our data indicate
that it is required for proper Rtr1 function. Rtr1 does not contain
a characterized phosphatase motif, but there is precedence for
a low level of sequence homology between known phosphatases and the other identified CTD phosphatases, Fcp1 and
Ssu72. Fcp1 was originally isolated through its ability to dephosphorylate the CTD, not by sequence homology (Chambers and
Dahmus, 1994; Chambers et al., 1995; Archambault et al., 1997).
When Fcp1 was characterized, it was described to contain an
‘‘unusual’’ phosphatase domain that was unique for eukaryotic
protein phosphatases (Kobor et al., 1999). By contrast, Ssu72
was found to contain a known phosphatase motif, CX5R, but
had no other significant homology to known phosphatases
(Meinhart et al., 2003).
What are the consequences of misregulated CTD modification
during the transcription cycle? In addition to the hyperphosphorylation of RNAPII, loss of Rtr1 leads to diminished transcription of
a number of RNAPII target genes and causes transcription readthrough at some loci, indicating a termination defect. A decrease
in RNAPII occupancy has previously been reported in studies that
made use of different thermosensitive alleles of Fcp1 (Cho et al.,
2001). The loss of RNAPII occupancy may be a result of RNAPII
hyperphosphorylation, either at S5-P as seen with deletion of
Rtr1 or at S2-P as seen with mutant alleles of Fcp1. It has been
suggested that CTD hyperphosphorylation may inhibit RNAPII
reinitiation, leading to decreased levels of RNAPII in the ORF
(Lux et al., 2005; Max et al., 2007; Payne et al., 1989). In addition
to the reduced transcription observed at the RNAPII loci tested in
our study, Gibney et al. observed that induction of GAL1 and
GAL7 was not detectable in rtr1D cells grown with galactose as
the sole carbon source (Gibney et al., 2008). Similar reductions
in transcription have also been observed using a thermosensitive
allele of Ssu72, ssu72-2. Although RNAPI transcripts were not
affected at the restrictive temperature, the levels of several
RNAPII transcripts were found to decrease in ssu72-2 cells (Dichtl
et al., 2002). The loci tested included ACT1, which decreased
52%—very similar to what we observed for the same loci in
rtr1D cells (Figure 5E). The transcription defects of Fcp1 thermosensitive alleles have been more extensively studied and have
been shown to cause a global defect in transcription. Using
expression arrays, 77% of all yeast genes were found to have
Molecular Cell 34, 168–178, April 24, 2009 ª2009 Elsevier Inc. 175
Molecular Cell
Rtr1 Regulates RNAPII CTD Phosphorylation
more than a 2-fold reduction in their expression levels when
mRNA was extracted from fcp1-1 cells grown at the restrictive
temperature (Kobor et al., 1999). These data show that the regulation of the RNAPII phosphorylation level is essential for the
maintenance of proper transcription in vivo.
In conclusion, our experiments have uncovered a previously
uncharacterized player in the RNAPII transcription cycle, Rtr1,
a phosphatase required for the regulation of the CTD modification
state during early RNAPII elongation events. In addition to the
recruitment of mRNA-processing factors, the different combinations of S5-P and S2-P CTD have been shown to be involved in
the recruitment of a number of accessory factors that are involved
in histone modification and chromatin-remodeling processes
during transcription (Li et al., 2007a). It remains to be determined
whether the correct arrangement of S5-P RNAPII across the open
reading frame is involved in the management of the chromatin
state during the transcription cycle and how alterations in the
regulation of this process may affect mRNA-processing events
other than termination.
EXPERIMENTAL PROCEDURES
Yeast Strains
The yeast strains used in this study are given in Table S1. All strains were
derived from BY4741.
Antibodies
Antibodies used in these studies are listed in Table S2.
Complex Isolation through Tandem Affinity Purification followed
by MudPIT
All TAP-tagged strains were obtained from Open Biosystems (Ghaemmaghami et al., 2003) and were purified as previously described with slight modification (Puig et al., 2001). In order to isolate complexes from chromatin-solubilized extracts, extracts were treated with 120 ug of heparin (sodium salt,
Sigma) and 100 units of DNase I (amplification grade, Sigma) for 10 min at
room temperature after cell lysis. Detailed methods are provided in the Supplemental Experimental Procedures.
Column Chromatography
Rpb3-TAP-purified RNAPII was isolated from the Rpb3-TAP Rtr1-His6-FLAG3HA3 strain via TAP purification and was then subjected to fractionation on
a Uno-Q1 anion exchange column (Biorad). Proteins were eluted with a gradient
of column buffer (50 mM TrisdHCl (pH 7.8)/10% glycerol/1 mM EDTA/10 mM
ZnCl2) starting at 150 mM (NH4)2SO4 and ending at 500 mM (NH4)2SO4 as previously described (Banks et al., 2007).
EMSA and In Vitro Transcription
EMSAs and in vitro transcription experiments were performed as previously
described (Carey et al., 2006) using equal concentrations of Rpb3-TAP or
Rtr1-TAP as illustrated. EMSAs were performed using the 32P-labeled C-tailed
nucleosomal template as described. The products were separated on 4%
native polyacrylamide gels (37.5:1 acrylamide-bis acrylamide) electrophoresed
in 0.5 3 TBE at 4 C. For the in vitro transcription reactions, the 32P-RNAs were
separated on an 8% polyacrylamide/urea gel. The resulting gels were dried,
exposed to a Phosphorimager or film.
CTD Peptide-Binding Assays
The Rtr1-His6-HA1-Protein A bacterial expression vector was constructed and
protein purified as described in the Supplemental Experimental Procedures.
The UM, S2-P, and S5-P peptides were synthesized, and the S2,5-P peptide
was prepared as previously described (Phatnani and Greenleaf, 2004; Phatnani
et al., 2004; Kizer et al., 2005). For CTD-binding assays, biotinylated CTD
176 Molecular Cell 34, 168–178, April 24, 2009 ª2009 Elsevier Inc.
peptides (2.5 mg) were incubated with 0.5 mg of streptavidin-coated Dynabeads M280 (Invitrogen) in binding buffer (50 mM Tris-HCl (pH 6.5), 300 mM
NaCl, 1 mM dithiothreitol, 0.5% Nonidet P-40, 1 mM PMSF) at 4 C for 2 hr.
Approximately 1 mg recombinant Rtr1-His6-HA1 and 100 mg of BSA were added
in binding buffer and allowed to interact with the peptides for 3 hr at 4 C. The
beads were washed four times in binding buffer containing 10 mg/ml BSA using
a Dynal MPC (Invitrogen) prior to addition of 43 SDS-loading buffer for elution
of bound proteins.
Chromatin Immunoprecipitation Assays
Chromatin immunoprecipitation (ChIP) assays were performed as previously
described (Hecht and Grunstein, 1999; Li et al., 2007c). Detailed methods
are available in the Supplemental Experimental Procedures.
Northern Blot Analysis
Northern blots were performed as previously described using either total or
polyA+ mRNA as indicated and were isolated from WT or rtr1D as given in
the text (Carrozza et al., 2005; Li et al., 2007b). Probes were generated by
PCR with primers listed in Table S4.
Quantitative RT-PCR
Quantitative RT-PCR was performed on total RNA isolated from WT or rtr1D
using the RNeasy Mini Kit (QIAGEN) according to the manufacturer’s protocol.
First-strand synthesis was performed using Superscript II Reverse Transcriptase (RT, Invitrogen) and random hexamers with 2.5 mg total RNA. Real-time
PCR was performed with samples generated in the presence and absence
of RT using a Biorad iCycler and SYBR Green master mix (Stratagene) and
primers directed against the gene of interest (Table S4). Total nanograms of
transcript was determined by comparison to a standard curve generated
with the same primers and five 10-fold serial dilutions of yeast genomic DNA.
Kinase Reactions
TFIIK was generated by purification of the complex from yeast through Kin28TAP. CTDK-I was purified through the Ctk1-TAP subunit. Active MAPK2/Erk2
was obtained from Millipore. For kinase reactions, 400 ng of RNAPII, 200 ng
of GST-CTD, or 5 mg of biotinylated CTD peptide was incubated with the indicated kinase for 1 hr at 30 C in kinase buffer (40 mM HEPES [pH 7.5], 10 mM
MgCl2, 5 mM dithiothreitol, and either 10 mCi of [g-32P]-ATP [6000 Ci/mmol;
Perkin Elmer] or 500 mM ATP [Roche]). Kinase reactions were stopped by
removal of the unincorporated ATP through an Illustra MicroSpin Column
(GE Healthcare).
Rtr1 Phosphatase Reactions
Reactions were performed with 5 pmol of phosphorylated RNAPII or GSTCTD (Thompson et al., 1993) as indicated or 1 ug of CTD peptide in phosphatase buffer (50 mM Tris-HCl [pH 6.5], 10 mM MgCl2, 20 mM KCl, and 5 mM
dithiothreitol). Reactions were quenched by the addition of SDS-loading buffer
and incubation at 98 C for 5 min prior to loading on a gel. Reactions performed
with RNAPII ternary complex or GST-CTD were separated on a 10% or 12%
SDS-PAGE gel. Reactions performed with the CTD peptides were separated
on a 16% Tricine gel. After separation, 32P-labeled protein gels were dried
and exposed to either a PhosphoImager screen or BioMax film with an intensifying screen. Reactions performed in the absence of 32P were subjected to
western blot analysis as described.
Preparation of RNAPII Ternary Complexes
Ternary complexes were prepared as described in (Kong et al., 2005). Approximately 5 pmol of RNAPII present in a ternary complex was used for phosphatase assays.
SUPPLEMENTAL DATA
Supplemental Data include Supplemental Experimental Procedures, six
figures, and five tables and can be found with this article online at http://
www.cell.com/molecular-cell/supplemental/S1097-2765(09)00141-5.
Molecular Cell
Rtr1 Regulates RNAPII CTD Phosphorylation
ACKNOWLEDGMENTS
complexes using shotgun proteomics and normalized spectral abundance
factors. Methods 40, 303–311.
We are grateful to all of the members of the Washburn and Workman laboratories for useful discussions and technical suggestions and would also like to
thank Drs. Joan and Ron Conaway for critical reading of our manuscript and
useful suggestions. We would like to thank Dr. Richard Young for the gift of
the GST-CTD plasmid, Dr. Kevin Morano for the CUP1-Rtr1 plasmids, and
William McDowell of the Stowers Institute for the production of the RTR1
C73A plasmid. This work was supported by a postdoctoral fellowship from
the NIGMS to A.L.M. (F32 GM075541) and funding from the Stowers Institute.
Ganem, C., Devaux, F., Torchet, C., Jacq, C., Quevillon-Cheruel, S., Labesse, G.,
Facca, C., and Faye, G. (2003). Ssu72 is a phosphatase essential for transcription
termination of snoRNAs and specific mRNAs in yeast. EMBO J. 22, 1588–1598.
Received: April 18, 2008
Revised: February 3, 2009
Accepted: February 23, 2009
Published: April 23, 2009
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