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J. Biol. Chem., Vol. 277, Issue 30, 27423-27432, July 26, 2002
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From the
Received for publication, February 6, 2002, and in revised form, May 6, 2002
Cycloheximide inhibits ribosomal DNA (rDNA)
transcription in vivo. The mouse homologue of yeast Rrn3, a
polymerase-associated transcription initiation factor, can complement
extracts from cycloheximide-treated mammalian cells. Cycloheximide
inhibits the phosphorylation of Rrn3 and causes its dissociation from
RNA polymerase I. Rrn3 interacts with the rpa43 subunit of RNA
polymerase I, and treatment with cycloheximide inhibits the formation
of a Rrn3·rpa43 complex in vivo. Rrn3 produced in
Sf9 cells but not in bacteria interacts with rpa43 in
vitro, and such interaction is dependent upon the phosphorylation
state of Rrn3. Significantly, neither dephosphorylated Rrn3 nor Rrn3
produced in Escherichia coli can restore transcription by
extracts from cycloheximide-treated cells. These results suggest that
the phosphorylation state of Rrn3 regulates rDNA transcription by
determining the steady-state concentration of the Rrn3·RNA polymerase
I complex within the nucleolus.
In the early 1970s Feigelson and colleagues (1-3) reported that
cycloheximide caused a rapid cessation of nucleolar RNA synthesis (ribosomal DNA transcription) and concluded that a rapidly turning over
protein was required for RNA polymerase I (pol
I)1 activity in
vivo. Subsequent studies have demonstrated that transcription by
RNA polymerase I is subject to regulation at many levels (4, 5). At
least three, and possibly more, polymerase-associated proteins, TIF-IA,
Factor C*, and TFIC (6-8), have been demonstrated to contribute to the
regulation of rDNA transcription. TIF-IA and Factor C* were identified
as factors that were required for the complementation of extracts of
quiescent or cycloheximide-treated cells. TFIC was identified as that
activity required to reconstitute transcription by extracts of
glucocorticoid-treated P1798 cells. This lymphosarcoma cell line exits
the cell cycle in response to the synthetic glucocorticoid
dexamethasone (DEX) (6). Interestingly, TIF-IA, Factor C*, and TFIC
shared several properties, including a tight association with the core
polymerase (8-10). TIF-IA and TFIC were purified and consisted of
different polypeptides (10, 11). However, the lack of immunological and
molecular tools precluded a definitive statement that TIF-IA and TFIC
were the same or different proteins (reviewed in Refs. 4 and 5).
The formation of the stable preinitiation complex in yeast requires an
interaction between the upstream activating factor bound to the
upstream promoter element and core factor, bound to the core promoter
element. This complex then recruits transcriptionally competent RNA
polymerase I to the transcription initiation site (Ref. 12 and
references therein). Mechanistically, Rrn3 appears to "bridge" the
polymerase and transcription initiation complexes (13-15). Thus, only
pol I molecules in complex with Rrn3 are able to recognize the
preinitiation complex and initiate transcription.
Studies comparing the state of RNA polymerase I in growing and
stationary yeast cells demonstrated that ~2% of the pol I in whole
cell extracts was capable of initiating transcription in vitro (16). This correlated with the observation that the
association of Rrn3 with pol I corresponded to the growth state of the
cells and was confirmed by the observation that transcriptionally
active pol I was associated with Rrn3 (16, 17). Milkereit and
Tschochner (16) demonstrated that the association of Rrn3 with pol I
was independent of either the total pol I or total Rrn3 content but varied with the growth rate of the cells. This could reflect
alterations in the state of Rrn3 and/or pol I that would shift the
equilibrium between free Rrn3 and Rrn3 associated with pol I. Fath
et al. (18) have recently reported that this equilibrium is
modulated by growth-related phosphorylation of specific sites of yeast
RNA polymerase I.
Moorefield et al. (19) demonstrated that hRrn3 could
complement yeast Rrn3 mutants, and there is strong evidence
that mammalian Rrn3 is the equivalent of TIF-IA (20). Recombinant
mammalian Rrn3 has been shown to complement extracts from
cycloheximide-treated cells (20), suggesting that Rrn3 was the rapidly
turning over activity originally identified by Feigelson's laboratory
(3). In addition, mammalian Rrn3 has been shown to interact with SL1 and would then serve as the bridge between RNA polymerase I and the
preinitiation complex on the rDNA promoter (21). However, the mechanism
by which Rrn3 activity is inhibited by CHX has not been identified.
Moreover, earlier studies on TIF-IA suggested that it did not
dissociate from RNA polymerase I (10). Thus, the role of Rrn3 in
mammalian rDNA transcription and its mode of regulation needed to be examined.
We report here that the mammalian homologue of Rrn3 plays a crucial
role in rDNA transcription. We have found that Rrn3 can complement
extracts of cells treated with CHX but not extracts of DEX-treated
P1798 cells, demonstrating that TIF-IA/Rrn3 and TFIC are not the same
activities. We have confirmed that hRrn3 can interact with both the
core subunits of RNA polymerase I and SL1. Although the Rrn3-SL1
interaction is not affected by CHX, the interaction between Rrn3 and
pol I is inhibited. Moreover, we demonstrate that the Rrn3·pol I
interaction is, at least in part, mediated by the mouse homologue of
rpa43 as it is in Saccharomyces cerevisiae (15). However,
the Rrn3·pol I interaction is regulated differently in mammals than
in yeast. Treatment with cycloheximide inhibits Rrn3 phosphorylation
and is associated with the dissociation of Rrn3 from its complex with
RNA polymerase I and the inhibition of the formation of an Rrn3·rpa43
complex in vivo. The interaction between Rrn3 and rpa43 was
confirmed using purified recombinant proteins in vitro.
Using this in vitro model, we found that dephosphorylation of Rrn3 weakened the interaction between Rrn3 and rpa43. The importance of the role of Rrn3 phosphorylation was confirmed by the observation that dephosphorylated Rrn3 could not reconstitute transcription when
added to extracts from cycloheximide-treated cells.
Cell Culture and Treatment with Cycloheximide and
Dexamethasone--
3T3 and N1S1 cells were grown as described
previously (22). P1798 cells were cultured and treated with
dexamethasone as described previously (6). Monkey CMT3 cells were grown
in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with
10% fetal calf serum (Hybaid or Invitrogen), 1% glutamine,
penicillin, and streptomycin. Where indicated, cells were treated with
cycloheximide, 2 µg/ml (Sigma Chemical Co.).
Transfection--
3T3 cells were plated at a density of 5 × 105 cells per 60-mm plate. Transfections were performed
with LipofectAMINE 2000 (Invitrogen) according to the manufacturer's
instructions. Full-length Rrn3 and rpa43 were cloned into pcDNA3.1
(Invitrogen). Various epitope tags were added to the coding regions by
PCR, and the constructs were confirmed by DNA sequencing. Six hours
after plating, cells were transfected with a total of 2 µg of DNA.
Cells cotransfected with Rrn3 and rpa43 received 1 µg of each
plasmid. Transfections were carried out for 36 h. Cells were
scraped into lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, containing protease inhibitors (Complete, Roche Molecular
Biochemicals)) and either used immediately or stored at
Metabolic Labeling--
Twenty-four hours after NIH3T3 cells
were transfected with pCDNA3.1FLAG-Rrn3, the medium was replaced
with phosphate-free Dulbecco's modified Eagle's medium (Invitrogen)
containing 10% fetal bovine serum, and
[32P]orthophosphate (PerkinElmer Life Sciences, 286 Ci/mg) was added at a concentration of 1 mCi/ml. Cells were grown
overnight in the radioactive medium, and cycloheximide (2 µg/ml) was
added to the medium prior to harvesting the cells. The cells were
harvested as described previously (22), and Rrn3 was immunoprecipitated using anti-FLAG M-2-agarose (Sigma). The immunoprecipitates were fractionated by SDS-PAGE, transferred to Immobilon P, and subjected to
autoradiography. The amount of protein immunoprecipitated was verified
by Western blots of the same filters used for autoradiography.
Phosphopeptide Mapping--
After autoradiography, the
radiolabeled protein bands from control and cycloheximide-treated cells
were cut from the filter, digested with
L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated trypsin and subjected to two-dimensional TLC using a Hunter apparatus (CBS Scientific) (23). The phosphotryptic peptides were then detected
by autoradiography.
Immunocytochemistry and FISH--
A full-length, 1953-bp clone
of human Rrn3 (provided by Drs. Moorefield and Reeder) was inserted
into the pEGFP-C1 vector (CLONTECH Laboratories,
Inc.). After electroporation with a BTX electroporator ECM 830 (150 V,
four 1-ms pulses at 0.5-s intervals, 2-mm gap cuvette) with
pEGFP-Rrn3C1, CMT3 cells were grown on 22- × 22-mm coverslips. 20 h after transfection, cells were washed in PBS, fixed with 4%
paraformaldehyde in PBS (15 min, 25 °C), rinsed in PBS,
permeabilized with 0.5% Triton X-100 in PBS (5 min, 4 °C), washed
with PBS, and washed with 2 × SSC. The hybridization probe,
corresponding to a portion of the 5'-ETS of human pre-rRNA, was
synthesized by linearizing a SacI-KpnI fragment
of human pre-rRNA, nt +934 to +1444, in pBluescript SK( In Vitro Transcription and Complementation Assays--
S100
extracts (5-8 mg of protein/ml) were isolated as described from either
control or cycloheximide-treated N1S1 cells (25) or from P1798 cells
treated with dexamethasone for 24 h (6). The heat-treated extract
( Production of Recombinant Proteins in Sf9 Cells and
Protein Purification--
Sf9 cells were infected with
baculovirus expressing FLAG epitope-tagged hRrn3-His6 or
His6-hTAFI68 (a gift of Dr. L. Comai), mouse rpa43 (V5, S, and
His6-tagged),2 or
human Rrn3 tagged with both the FLAG and polyoma epitopes (originally
provided by Dr. Beth Moorefield). After 3 days of infection, the
Sf9 cells were pelleted by centrifugation and washed with 5 ml
of ice-cold PBS. FLAG-hTAFI68 and FLAG-Rrn3 were purified as described previously (27). The quality of the purified proteins was
monitored by SDS-PAGE (Fig. 1B, inset).
Copurification of Rrn3/TAFI68 in Vitro--
Aliquots
of purified Rrn3 (4 µg) and TAFI68 (4 µg) were mixed
and incubated overnight at 4 °C in 200 µl of FLAG wash buffer (50 mM Tris-HCl, pH 8.0, 238 mM NaCl, 27 mM KCl, 20% glycerol). Ni-NTA-agarose (Qiagen, 100-µl
packed bead volume) was preincubated overnight with 1 ml of PBS
containing 2 mg/ml BSA. The beads were washed once with 1 ml of PBS
before use. Fifty microliters of a 50% slurry of BSA-coated
Ni-NTA-agarose was added to each reaction, and the mixture was tumbled
for 2 h at 4 °C. The beads were washed three times with 1 ml of
50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole. Bound protein was
eluted in 100 µl of the same buffer containing 250 mM
imidazole (two 50-µl elutions). The eluates were fractionated by
SDS-PAGE, transferred to Immobilon P, and probed with monoclonal
anti-FLAG antibodies.
Coimmunoprecipitation of Rrn3/TAFI68 from
Cycloheximide-treated Cells--
3T3 cells were transfected with
FLAG-Rrn3 and V5-tagged TAFI68 as described above.
Thirty-six hours after transfection, some of the transfected cells were
treated with cycloheximide for 3 h. All plates were harvested at
the same time, and whole cell lysates were prepared using lysis buffer
(100 µl/plate). Aliquots (180 µl) of the lysates were incubated
(4 °C) with anti-FLAG-agarose beads (25 µl), and the mixtures were
tumbled for 2 h at 4 °C. After the beads were washed with lysis
buffer, the bound protein was eluted (2 × 50 µl) with FLAG
peptide (0.5 mg/ml in 50 mM Tris-HCl, pH 8.0, 238 mM NaCl, 27 mM KCl, 20% glycerol),
fractionated by SDS-PAGE, and analyzed by Western blotting with the
indicated antibodies.
Association of Rrn3 and RNA Polymerase I--
NIH3T3 cells were
transfected with pCDNA3.1FLAG-Rrn3. After 36 h, the cells were
harvested, washed, and lysed, and the whole cell lysate was tumbled
with anti-FLAG-agarose for 2 h at 4 °C as described above. The
beads were washed three times with FLAG wash buffer (50 mM
Tris-HCl, pH 8.0, 238 mM NaCl, 27 mM KCl, 20% glycerol). Bound protein was eluted with FLAG peptide in the same buffer, fractionated by SDS-PAGE, and transferred to Immobilon P. The
blots were probed with monoclonal anti-FLAG antibody (to detect Rrn3)
or polyclonal antibodies to either the 194- or 127-kDa subunits of RNA
polymerase I (22).
Coimmunoprecipitation of Rrn3 and rpa43--
Exponentially
growing NIH3T3 cells were cotransfected with 1 µg of FLAG-Rrn3 and 1 µg of V5-tagged RPA43. Thirty-six hours after transfection, whole
cell lysates were prepared and the lysates were tumbled with
anti-FLAG-agarose for 2 h at 4 °C as described above. The beads
were boiled in SDS sample buffer, and the eluted proteins were analyzed
by SDS-PAGE and Western blotting using anti-FLAG (to detect Rrn3) and
anti-V5 (to detect rpa43) monoclonal antibodies.
Treatment with Phosphatase--
Where indicated, Rrn3 (4 µg)
immobilized on anti-FLAG-agarose was treated with either bacterial
alkaline phosphatase (BAP, Sigma) or calf intestinal alkaline
phosphatase (CIAP, Sigma). In those experiments, 50-µl aliquots of
Rrn3-FLAG beads were washed in buffer C/20 (28) containing proteinase
inhibitors and suspended to a final volume of 100 µl, and the
indicated amounts of the appropriate phosphatase were added to the
reaction. After 30 min at 4 °C, the Rrn3-FLAG beads were washed
three times with 200 µl of FLAG wash buffer and then used as an
affinity matrix. For in vitro transcription experiments,
affinity-purified Rrn3 (2 µg) was treated with the indicated amounts
of bacterial alkaline phosphatase (0.04, 0.4, and 4.0 units in buffer
C/20) at 15 °C for 30 min in a total volume of 20 µl. The reaction
was stopped by the addition of 1 µl of phosphatase inhibitor mixture
II (Sigma).
Western Blot Analysis--
SDS-PAGE and electroblotting
were carried out as described previously (22). Monoclonal antibodies to
FLAG (Sigma), polyoma (Research Diagnostics, Inc., Flanders, NJ),
and V5 peptide (Invitrogen) and S peptide conjugated to horseradish
peroxidase (S-HRP conjugate, Novagen) were used as recommended by the
suppliers. Polyclonal rabbit antisera to the subunits of RNA polymerase
I and UBF were as described previously (29). The antigens were
visualized by the enhanced chemiluminescent (ECL) method (Amersham Biosciences).
Rrn3/TIF-IA and TFIC Are Not the Same Factor--
The
complementation assay originally used to define TFIC (6) demonstrates
that it is a heat-stable activity (Fig.
1A), i.e.
heat-treated S100 extracts (N1S1
Thus, we sought to determine if Rrn3 was the heat-stable activity
present in the heat-treated S100 extract that can complement extracts
from both CHX- and DEX-treated cells. As shown in Fig. 1B
(lanes 2-4), the addition of purified, recombinant Rrn3 to an inactive S100 from cycloheximide-treated cells (N1S1 CHX)
resulted in the reconstitution of transcription in confirmation of the results reported by Bodem et al. (20). Rrn3 had no
transcriptional activity by itself (lane 5). As a control
for the addition of protein and because Rrn3 interacts with
TAFI68 (21), we tested the effects of adding
TAFI68 to this system. The addition of TAFI68 had no effect on transcription (lane 6). Thus, Rrn3 was at
least one of the activities present in the heat-treated S100 extract. We then examined the possibility that Rrn3 might complement extracts from dexamethasone-treated cells. As shown in Fig. 1C
(lanes 5 and 6), the addition of purified Rrn3 to
extracts of DEX-treated cells (P1798 DEX) did not
reconstitute transcription. This was not a cell line-specific result,
because the purified Rrn3 was active in P1798 cell extracts as
illustrated by the stimulation of transcription when Rrn3 was added to
an extract from untreated P1798 cells (lanes 3 and
4). This observation suggests that Rrn3 may be a limiting
activity in the intact cell. More importantly, these results suggest
that TFIC and TIF-IA are not the same transcription factor.
The simplest model for the effects of CHX and DEX on rDNA
transcription, and one supported by the original complementation experiments using heat-treated extracts, was that the two compounds were inhibiting the same transcription factor. Our observations indicate that cycloheximide and dexamethasone are inhibiting two different components of the rDNA transcription apparatus. If this model
is correct, then an extract from cycloheximide-treated cells (inactive
Rrn3/TIF-IA) should complement an extract from dexamethasone-treated cells (inactive TFIC). The results of an experiment designed to test
this model are presented in Fig. 1D. When otherwise inactive S100 extracts from cycloheximide- and dexamethasone-treated cells (lanes 1 and 2, respectively) were mixed in an
in vitro transcription reaction, they did complement one
another and were capable of carrying out transcription (lanes
4-6).
We next sought to determine how CHX might inhibit the activity of Rrn3.
Studies in yeast and mammalian cells indicate that Rrn3 is the bridge
between pol I and the transcription initiation complex (as discussed in
Refs. 12 and 21). Thus, CHX could act to inhibit the interaction of
Rrn3 with either SL1 or pol I.
Cycloheximide Does Not Inhibit the Interaction between Rrn3 and
TAFI68--
Rrn3 interacts with the 68-kDa subunit
(TAFI68) of SL1 (21). Thus we sought to determine if
cycloheximide affected the interaction between Rrn3 and
TAFI68. We first confirmed that the interaction between
Rrn3 and TAFI68 could be observed in vitro (Fig.
2A). Affinity-purified,
recombinant FLAG-Rrn3-His and FLAG-TAFI68 were mixed and
incubated as described under "Experimental Procedures." FLAG-Rrn3-His was purified with Ni-NTA-agarose beads, and the proteins
that bound to the beads were eluted and analyzed as described under
"Experimental Procedures." Western analysis using anti-FLAG antibodies demonstrated that TAFI68 bound to the beads when
Rrn3 was included in the incubation (lane 3) but was not
bound to the nickel affinity resin in the absence of Rrn3 (lane
4).
We next examined the effects of CHX on the interaction of Rrn3 and
TAFI68 in vitro (Fig. 2B). As
described under "Experimental Procedures," 3T3 cells were
cotransfected with vectors expressing FLAG-Rrn3 and
V5-TAFI68. FLAG-Rrn3 was isolated from the control and
CHX-treated cells using immobilized anti-FLAG antibodies, and the
complexes were analyzed by SDS-PAGE and Western blotting using
anti-FLAG and anti-V5 antibodies. TAFI68 by itself did not bind to immobilized anti-FLAG antibodies (Fig. 2B,
lane 3 and 4), but TAFI68 bound to
FLAG-Rrn3 from control or CHX-treated cells immobilized on the
anti-FLAG-agarose (lanes 5 and 6). These results
indicated that CHX did not affect the ability of Rrn3 to interact with
TAFI68.
Cycloheximide Inhibits the Interaction between Rrn3 and RNA
Polymerase I--
Studies on the role of Rrn3 in both yeast and
mammalian cells (15, 16, 21) indicate that the association of Rrn3 with core RNA polymerase I appears to define a subset of the RNA polymerase I molecules that are capable of initiating transcription. Thus, we
sought to determine if treatment with cycloheximide altered the
association of Rrn3 with pol I.
Because these experiments were use to test the interaction between Rrn3
and RNA polymerase I, we first determined if the tagged-Rrn3 localized
to the nucleolus. Twenty-four hours after CMT3 cells were transfected
with a vector driving the expression of GFP-Rrn3, the cells were
subject to FISH and viewed as described under "Experimental Procedures." As shown in Fig.
3A, GFP-tagged Rrn3 localizes
to the nucleolus of CMT3 cells similar to the result obtained by Bodem
et al. (20). Comparison of the localization pattern and the
signal intensities of the 5'-ETS of the 45 S rRNA in the nucleoli showed no significant difference between the cells expressing hRRN3-GFP
protein and nontransfected cells. This indicates that hRrn3-GFP
expression does not interfere with RNA polymerase I transcription.
We then asked if the tagged Rrn3 associated with RNA polymerase I. NIH3T3 cells were transfected with pcDNA3.1 or pcDNA3.1-Rrn3 driving the expression of polyoma (EYMPME) and FLAG-tagged Rrn3. S100
extracts were prepared from both sets of transfected cells (Fig.
3B, lanes 1 and 2) and applied to
immobilized, anti-FLAG antibodies. Western analysis of the proteins
that bound to the anti-FLAG resin and eluted with FLAG peptide
demonstrated that Rrn3 associated with pol I (Fig. 3B,
lane 6), as indicated by the coelution of A194 (the 194-kDa
subunit of RNA polymerase I) with Rrn3 from the anti-FLAG beads. RNA
polymerase I itself did not bind to the anti-FLAG-agarose beads
(lane 5). Having established that tagged Rrn3 localized to
the nucleolus and associated with RNA polymerase I, we then determined
if treatment with cycloheximide altered that association.
As shown in Fig. 3C, cycloheximide had little or no effect
on the content of Rrn3 in NIH3T3 cells transfected with
pcDNA3.1-FLAG-Rrn3 or on the recovery of Rrn3 with immobilized
anti-FLAG antibodies (Fig. 3C, lanes 1 and
2, and 3 and 4, respectively). As
shown in lanes 1 and 3, pol I (A127, the 127-kDa
subunit of pol I) was coimmunopurified with FLAG-Rrn3 in exponentially
growing cells. However, 3 h after treatment with cycloheximide,
most of the Rrn3·RNA polymerase I complexes were dissociated
(lanes 2 and 4) as demonstrated by the low level
of coimmunoprecipitation of pol I with Rrn3 (<15%, compare the
signals from A127 in lanes 3 and 4).
Peyroche et al. (15) have demonstrated that the
43-kDa subunit of yeast RNA polymerase I (rpa43) is critical for the
interaction of yeast Rrn3 with yeast RNA polymerase I, and we have
found that the mammalian homologue of yeast rpa43, mrpa43, interacts
with hRrn3.2 Thus, we sought to determine if this
interaction were susceptible to CHX in mammalian cells (Fig.
4). Exponentially growing NIH3T3 cells
were cotransfected with vectors driving the expression of FLAG-Rrn3 and
V5-mouse rpa43, and treated with cycloheximide (2 µg/ml). FLAG-Rrn3
was immunopurified from whole cell extracts of control and CHX-treated
cells. The proteins that eluted from the immobilized, monoclonal
antibodies with FLAG peptide were analyzed by Western blotting with
antibodies to the FLAG and V5 tags, to detect Rrn3 and rpa43,
respectively. Cycloheximide did not effect the expression of FLAG-Rrn3
or recovery of FLAG-Rrn3 with immobilized anti-FLAG antibodies (Fig. 4,
lanes 1-4). Similarly, cycloheximide had no effect on the
expression of rpa43 (lanes 5 and 6). Moreover, as
predicted by the results reported by Peyroche et al. (15),
rpa43 coimmunopurified with Rrn3 (lane 7). However, treatment with cycloheximide inhibited the interaction of Rrn3 and
rpa43 as demonstrated by the decreased recovery of rpa43 with the
immunopurified Rrn3 from cells treated with CHX (lane
8).
These two lines of evidence demonstrate that the inhibition of rDNA
transcription that results when cells are treated with cycloheximide is
caused by a dissociation of Rrn3 from the RNA polymerase I complex.
Cycloheximide Inhibits the Phosphorylation of
Rrn3--
Phosphorylation has been implicated in regulating the
association of Rrn3 with pol I in yeast (18). Thus, we sought to
determine whether phosphorylation might contribute to the regulation of the association of Rrn3 with pol I in mammalian cells. When 3T3 cells
were transfected with vectors driving the expression of rpa43 and
metabolically labeled with 32P, immunopurified rpa43 was
not phosphorylated (data not shown), confirming the published
observation that rpa43 is not significantly phosphorylated in mammalian
cells (22).
In contrast, when the same experiment was carried out with Rrn3, we
found that Rrn3 was phosphorylated (Fig.
5A, lane 1). Furthermore, as shown in Fig. 5 (lanes 2 and 3),
the phosphorylation of Rrn3 was rapidly inhibited (~70% within
1 h) when the cells were treated with cycloheximide. It is
unlikely that the inhibition of Rrn3 phosphorylation results from a
change in the phosphate pool for the following two reasons: 1) The
cells had been incubated in medium containing
[32P]orthophosphate for 18 h prior to the addition
of cycloheximide. This period of time would allow for a total
equilibration of the phosphate pools in the cells. 2) Cycloheximide had
no effect on the labeling of various proteins that cross-reacted with
the anti-FLAG antibodies (CRM, Fig. 5A).
The observation that cycloheximide inhibited the phosphorylation of
Rrn3 was consistent with the possibility that the phosphorylation state
of Rrn3 might mediate the interaction between Rrn3 and RNA polymerase
I. Analysis of the phosphopeptides of Rrn3 isolated from control and
CHX-treated cells failed to demonstrate any change in the number of
phosphorylated peptides, although the mobilities of several peptides
were altered in a manner consistent with the loss of one or more
phosphates from a peptide containing multiple phosphorylation sites
(data not shown).
Rrn3 Must Be Phosphorylated to Interact with rpa43--
To
determine if the phosphorylation status of Rrn3 affected its ability to
interact with RNA polymerase I, we focused on the interaction between
Rrn3 and rpa43 (Fig. 6). Control
experiments demonstrated that rpa43 bound to FLAG-Rrn3 immobilized on
anti-FLAG beads but not to anti-FLAG antibody beads alone (Fig.
6A, upper panel, lanes 3 and
4) and that treatment with either calf intestinal alkaline
phosphatase (CIAP) or bacterial alkaline phosphatase (BAP) did not
degrade the immobilized Rrn3 or affect the binding of Rrn3 to the beads
(lanes 5-7 and 8-10, lower panel).
Treatment of the immobilized Rrn3 with increasing amounts of CIAP
(upper panel, lanes 5-7) and BAP (lanes
8-10) decreased the binding of rpa43 to Rrn3 as evidenced by the
decreased recovery of rpa43 in the material eluted with FLAG peptide.
Although CIAP was not as effective as BAP, both phosphatases reduced
the binding of Rrn3 to rpa43. This demonstrates that dephosphorylating
Rrn3 reduces its ability to interact with rpa43. This is consistent
with the hypothesis that Rrn3 must be phosphorylated to function in
transcription.
Rrn3 Produced in Bacteria Can Bind to TAFI68 but Not to
Rrn3--
To test this hypothesis we determined if recombinant Rrn3
produced in bacteria would be able to bind to either TAFI68
or Rrn3 (Fig. 6B). It is unlikely that bacteria would
phosphorylate the same sites on Rrn3 as those that would be
phosphorylated in mammalian cells (as discussed by Fath et
al. (18)). Thus, this form of Rrn3 would serve as a control for
the treatment with phosphatase. FLAG-tagged Rrn3 was purified from
either bacteria or Sf9 cells and immobilized on
anti-FLAG-agarose beads. When the agarose beads containing either of
the two forms of Rrn3 were incubated with TAFI68,
TAFI68 bound to both forms of Rrn3 (Fig. 6B,
lanes 3 and 4). However, when rpa43 was incubated
with the beads, rpa43 did not bind to Rrn3 isolated from bacteria
(lane 7), but it did bind to Rrn3 isolated from Sf9
cells (lane 8). The observation that bacterially expressed
Rrn3 can bind to TAFI68 indicates that the protein has
maintained at least one measurable biochemical property. Moreover, the
ability of Rrn3 expressed in bacteria to associate with
TAFI68 but not rpa43 resembles the effects of cycloheximide on mammalian Rrn3. Thus, this series of experiments provided a second
line of evidence that Rrn3 must be phosphorylated to function in
transcription. To further test this hypothesis we determined if
dephosphorylated Rrn3 would reconstitute transcription when added to an
extract from CHX-treated cells.
Dephosphorylated Rrn3 Cannot Complement Transcription When Added to
Extracts from Cycloheximide-treated Cells--
As described
previously, recombinant Rrn3 purified from Sf9 cells could
reconstitute transcription when added to an S-100 from CHX-treated
cells (Fig. 6C, lane 2). However, when Rrn3 was pretreated with increasing amounts of bacterial alkaline phosphatase and then added to the transcription reaction it was unable to restore
transcription (lanes 3-5). To control for possible affects of BAP on transcription, a mixture of phosphatase inhibitors was added
to the Rrn3 after treatment with phosphatase but before Rrn3 was added
to the S-100. The inclusion of the phosphatase inhibitor mixture when
Rrn3 was incubated with BAP, inhibited the phosphatase and enabled Rrn3
to maintain its activity, i.e. it reconstituted
transcription (lane 6). Thus, Rrn3 must be phosphorylated to
function in transcription. Significantly, Rrn3 expressed in Escherichia coli was inactive in this assay (lane
7). These results provide additional evidence that specific
phosphorylation events are required to enable Rrn3 to participate in
rDNA transcription.
We have established that phosphorylation of mammalian Rrn3
regulates its role in rDNA transcription. We have confirmed that the
ability of TIF-IA/Rrn3 to function in transcription is inhibited when
mammalian cells are treated with CHX. In addition, we demonstrated that
Rrn3/TIF-IA is insufficient to reconstitute rDNA transcription when
added to extracts from dexamethasone-treated P1798 cells. Therefore, we
conclude that Rrn3/TIF-IA and TFIC are not the same factors. We provide
several lines of evidence that the phosphorylation status of mammalian
Rrn3 is critical for its function. We have shown that treatment of
cells with CHX leads to a rapid decrease in the phosphorylation status
of Rrn3 and to the dissociation of Rrn3 from core RNA polymerase I. In
contrast, CHX did not significantly inhibit the ability of Rrn3 to
interact with TAFI68. Our investigation of the interaction
of Rrn3 with pol I demonstrated that mammalian Rrn3 interacts with
mammalian rpa43 and that this interaction is inhibited when cells are
treated with CHX. Moreover, we demonstrated that the interaction of
Rrn3 with rpa43 in vitro is inhibited when Rrn3 is
dephosphorylated and that treatment with phosphatase inhibits the
ability of Rrn3 to reconstitute transcription by extracts of
CHX-treated cells. Furthermore, our observation (data not shown) that
the distribution of Rrn3 becomes dispersed upon cycloheximide treatment
is reminiscent of the morphological alternations of the nucleolus
observed after treatment with the kinase inhibitor (5,6-dichloro- The model presented in Fig. 7 summarizes
the findings of the present report and incorporates several other
findings. First, the model demonstrates a requirement for Rrn3
phosphorylation to interact with RNA polymerase I and to allow pol I to
productively recognize the preinitiation complex formed by SL1 and UBF
on the rDNA promoter. This does not preclude the possibility that RNA polymerase I might interact with the preinitiation complex in the
absence of Rrn3. It is formally possible that TFIC serves a parallel
function to that served by Rrn3 and might be capable of facilitating an
"incomplete" complex between RNA polymerase I and the preinitiation
complex formed by SL1 and UBF. Second, the model incorporates
observations made by Brun et al. (58) as well as some made
by Aprikian et al. (12). These authors have demonstrated
that Factor C* or the Rrn3·pol I complex dissociates during
transcription, and Rrn3 loses its capacity to participate in subsequent
rounds of initiation. Third, the model suggests that dephosphorylated
Rrn3 would remain complexed with SL1. Although this would be consistent
with our fluorescence recovery after photobleaching data,
because it would reduce the rate of Rrn3 diffusion, it would appear to
contradict the model proposed by Aprikian et al. (12).
The evidence accumulated to date indicates that the activity of nearly
every component of the mammalian rDNA transcriptional apparatus is
subject to regulation and that in most cases there are multiple
mechanisms for regulating their activity. UBF activity can be regulated
by phosphorylation (33-38), acetylation (39, 40), and sequestration in
complex with proteins such as Rb and p130 (27, 37, 41-44). The amount
of UBF present in the cell is also regulated by altering the level of
expression of the UBF gene (29). Similarly, SL1 is subject to
regulation (45) via post-translational modifications, including
phosphorylation and acetylation (46). Moreover, there is evidence for
cell cycle-specific patterns of phosphorylation of SL1 (47, 48), UBF
(36, 49), and TTF1 (50, 51) that correlate with mitotic silencing of rDNA transcription. Similarly, studies on RNA polymerase I demonstrate that pol I activity may be subject to regulation through multiple mechanisms affecting both its catalytic activity and ability to initiate transcription (reviewed in Refs. 5, 18, and 22).
Studies on the inactivation of rDNA transcription during
encystment of Acanthamoeba castellanii demonstrated
that inactivation was the result of a modification of RNA polymerase I
(52, 53). Similarly, studies on the regulation of rDNA transcription in mammalian cells undergoing serum starvation or P1798 cells exposed to
DEX demonstrated that a polymerase-associated factor was responsible for the inhibition of rDNA transcription. Interestingly, two
laboratories were able to separate what appeared to be the same
activity from RNA polymerase I (9, 10). Our results demonstrate that
the heat-treated extracts used by Thompson's laboratory (9) contain both Rrn3/TIF-IA and TFIC and that Rrn3/TIF-IA and TFIC are not the
same factors.
The model for rDNA transcription in S. cerevisiae proposes
that the binding of the multisubunit complex (upstream activation factor) to the upstream promoter element is required to recruit the
core factor to the promoter (12, 17, 54). In turn, the core factor
recruits the transcriptionally competent form of pol I to the
transcription initiation site. In this model for transcription initiation, Rrn3 functions as the bridge between pol I and the core
factor. In yeast, Rrn3 is associated with a small fraction of the RNA
polymerase I in the cell, and it is that fraction that is competent for
specific initiation of rDNA transcription. This is similar to the
report of Tower and Sollner-Webb (8) who demonstrated the existence of
two biochemically definable forms of pol I in mammalian cells; one that
was capable of initiating transcription and one that could not. In
stationary yeast cells, in which rDNA transcription has been
down-regulated, there is a decrease in the association of Rrn3 and pol
I, although there is no decrease in the amount of free Rrn3 or pol I
(16). Indeed, Milkereit and Tschochner (16) demonstrated that the
addition of purified pol I·Rrn3 complex from growing cells to an
inactive fraction (PA600s) from stationary cells restored
transcription. Interestingly, neither initiation-inactive pol I nor
recombinant Rrn3 could complement extracts from stationary cells.
Fath et al. (18) examined the model that different
post-translational modifications have to exist in either Rrn3 and/or RNA polymerase I to regulate the formation of transcriptionally competent RNA polymerase I. They demonstrated that phosphorylation is
required to maintain a stable and transcriptionally active pol I·Rrn3
complex. They reported that, although yeast Rrn3 is a
phosphoprotein, nonphosphorylated Rrn3 and Rrn3 produced in bacteria
were able to participate in the formation of the transcriptionally competent RNA polymerase I·Rrn3 complex. Moreover, they demonstrated that treatment of yeast RNA polymerase I with alkaline phosphatase abrogated its ability to interact with Rrn3 and inhibited the formation
of transcriptionally competent pol I. These observations led to the
model that phosphorylation/dephosphorylation at specific pol I sites
mediates the interaction with Rrn3 and the ability of pol I to initiate
rDNA transcription. Although structural and genetic studies indicate
that yeast Rrn3 interacts with the 43-kDa subunit of pol I (rpa43), it
is not yet known if the phosphorylation status of rpa43 regulates the
interaction of Rrn3 with rpa43.
In contrast, our results led to the conclusion that phosphorylation of
Rrn3 is required for the formation of the Rrn3·pol I complex that is
capable of transcription initiation. Although this model for regulating
rDNA transcription would be different from that proposed by Fath
et al. (18), it is consistent with the general model that
the formation of the Rrn3·pol I complex is essential for rDNA
transcription. Moreover, most of the studies on the regulation of
polymerase-associated factors required for rDNA transcription in
mammalian cells are consistent with a model wherein Rrn3, and not pol
I, would be the target of post-translational modification, as in the
experiments presented herein and those of Bodem et al.
(20). We have demonstrated that the addition of recombinant Rrn3
from Sf9 cells to extracts of CHX-treated cells is sufficient to
reconstitute transcription. If the post-translational modification of
pol I was responsible for the inactivation of transcription in that
system, then the addition of Rrn3 would not be sufficient to
reconstitute transcription.
Our observation, that serum starvation significantly reduces the
phosphorylation state of RNA polymerase I in cultured rat hepatoma
cells (22), is consistent with the formal possibility that the
post-translational modification of pol I might also contribute to the
interaction of mammalian Rrn3 with pol I. However, only the A194
subunit of mammalian pol I has been definitively demonstrated to be
phosphorylated (Ref. 22 and discussion therein), and the results from
studies on yeast and mammalian RNA polymerase I indicate that Rrn3
interacts with rpa43 and not A194. It is possible that additional
subunits contribute to the stability of the Rrn3·pol I complex, and
it is interesting to note that several of the yeast RNA polymerase I
subunits are phosphorylated, including A190, A34.5, A23, A19, and A43
(55-57). However, in the absence of evidence to suggest that Rrn3
interacts with additional subunits, our studies have focused on the
interaction between Rrn3 and rpa43. The results obtained in those
experiments are consistent with the model that the phosphorylation
state of Rrn3 determines the formation of the Rrn3·pol I complex in
mammalian cells. The determination of the modified residues in Rrn3 and
the correlation of the state of modification with the ability of Rrn3
to interact with pol I will provide an important tool to understanding
the mechanism of rDNA transcription.
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: Sigfried and Janet
Weis Center for Research, Geisinger Clinic, 100 N. Academy Ave.,
Danville, PA 17821. Tel.: 570-271-6662; Fax: 570-271-6701; E-mail:
lrothblum@geisinger.edu.
Published, JBC Papers in Press, May 15, 2002, DOI 10.1074/jbc.M201232200
2
Q. Hu and L. I. Rothblum, manuscript in
preparation..
The abbreviations used are:
pol I, RNA
polymerase I;
rRNA, ribosomal RNA;
hRrn3, human Rrn3;
DEX, dexamethasone;
CHX, cycloheximide;
SL1, selectivity factor I;
TAFI68, 68-kDa subunit of SL1;
UBF, upstream binding
factor;
TFIC, transcription factor 1C;
TIF-IA, transcription initiation
factor 1A;
mrpa43, mouse homologue of yrpa43;
A194,
Rrn3 Phosphorylation Is a Regulatory Checkpoint for Ribosome
Biogenesis*
,,,,¶
Sigfried and Janet Weis Center for Research,
Geisinger Clinic, Danville, Pennsylvania 17821 and the
§ NCI, National Institutes of Health, Bethesda, Maryland
20892
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
80 °C.
) with
XhoI (24). The antisense hybridization probe (+1370/+1444)
was synthesized by in vitro transcription with biotin-16-UTP
using T7 RNA polymerase. 100 ng of probe and 20 µg of yeast tRNA were
mixed and dried under vacuum. The RNA mixture was dissolved in 10 µl
of deionized formamide, denatured by heating (70 °C, 10 min),
quick-chilled, and diluted with an equal volume of 4× SSC, 2% BSA,
and 20% dextran sulfate. Fixed cells were hybridized at 42 °C in a
humid chamber with 20 µl of probe/coverslip. After 16 h, the
coverslips were rinsed sequentially with 2× SSC/50% formamide at
37 °C, 2× SSC and 1× SSC at room temperature, 30 min each. The
cells were then incubated with avidin-DCS-conjugated with Texas
red (Vector Laboratories, 2 µg/µl) in 4× SSC/0.25% BSA for 1 h, and rinsed in 4× SSC, 4× SSC/0.1% Triton X-100, 4× SSC.
Coverslips were mounted in Mowiol (Calbiochem). Samples were observed
with Zeiss 510 confocal microscope.
45) was prepared by heating S100 extracts for 15 min at 45 °C.
(Note that this treatment does not inactivate Rrn3/TIF-IA or TFIC, but
does inactivate RNA polymerase I (9).) In vitro
transcription reactions (50 µl) were carried out as described previously (6, 26). Complementation assays contained 25-40 µg of
S100 extracts from control, cycloheximide-treated N1S1 or dexamethasone-treated P1798 cells. Where indicated, the reactions were
supplemented with affinity-purified, recombinant Rrn3 (0.1-1.0 µg),
heat-treated extracts (25 µg), or inactive extracts (5-25 µg) and
assayed for the ability to accurately initiate transcription from the
rat rDNA promoter as described (6, 26). The template (0.1 µg/50-µl
reaction) was pU5.1E/X, which contains the rat 45 S rDNA promoter
(286 to +630) truncated with EcoRI to yield a transcript of 632 nt (26). The radiolabeled, in vitro
synthesized RNA was purified and analyzed by urea-polyacrylamide gel
electrophoresis and autoradiography.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
45) can reconstitute transcription when added to an extract from dexamethasone (DEX)-treated P1798 cells
(lane 6). Interestingly, the same heat-treated extract can also rescue transcription when added to extracts from cycloheximide (CHX)-treated cells (lane 5). These results are consistent
with the possibility that TFIC and TIF-IA are the same factor (25).
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Fig. 1.
Rrn3 complements extracts from
cycloheximide-treated cells but not from dexamethasone-treated
cells. A, heating an active extract (N1S1
Control, 5 µl, lane 1) from rat N1S1 cells at
45 °C inactivates its ability to transcribe rDNA (N1S1
45, 5 µl, lane 4). However, the addition of
45, 5 µl, to inactive extracts from CHX-treated N1S1 cells
(N1S1 CHX, 5 µl, lane 2) and from DEX-treated
mouse P1798 cells (P1798 DEX, 5 µl, lane 3)
results in the reconstitution of transcription as shown in as shown in
lanes 5 and 6, respectively. Transcription
reactions, purification of the in vitro synthesized RNA, and
PAGE analysis of the transcription reactions were carried out as
described under "Experimental Procedures." Trans., the
632-nt transcript that results from correct initiation. B,
Rrn3 complements extracts from cycloheximide-treated cells (N1S1
CHX). In lanes 2-4 increasing amounts of Rrn3 (0.01, 0.1, and 1.0 µg) purified from Sf9 cells (inset)
were added to inactive N1S1 CHX extracts (5 µl, lane 1),
and the mixtures were assayed in transcription reactions in
vitro as described under "Experimental Procedures." Purified
Rrn3 (1.0 µg) did not support transcription (lane 5).
Purified TAFI68 (1.0 µg) was added to one reaction
(lane 6). Trans., the 632-nt transcript that
results from correct initiation. The inset is the Coomassie
Blue-stained SDS-PAGE of 5 µg of purified bvRrn3.
C, S100 extracts from cycloheximide-treated N1S1 (5 µl,
lanes 1 and 2), P1798 (5 µl, lanes 3 and 4), or dexamethasone-treated P1798 cells (5 µl,
lanes 5 and 6) were complemented with purified
Rrn3 (1.0 µg), lanes 2, 4, and 6,
and their ability to transcribe rDNA was assayed as described under
"Experimental Procedures." Trans., the 632-nt transcript
that results from correct initiation. D, inactive S100
extracts from dexamethasone-treated P1798 (lane 2, 5 µl)
cells can complement extracts from cycloheximide-treated N1S1 cells
(lane 1, 5 µl). In lane 3, the N1S1 CHX extract
(5 µl) was complemented with 5 µl of N1S145. In lanes
4-6, the N1S1 CHX extract (5 µl) was complemented with 5, 10, and 15 µl of P1798 DEX extract. Transcription reactions, purification
of the in vitro synthesized RNA, and PAGE analysis of the
transcription reactions were carried out as described under
"Experimental Procedures." Trans., the 632-nt transcript
that results from correct initiation.
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Fig. 2.
Cycloheximide does not alter the interaction
between Rrn3 and TAFI68. A, protein
affinity chromatography assays demonstrate that TAFI68
interacts with Rrn3 in vitro. FLAG-Rrn3-His and
FLAG-TAFI68 purified from SF9 cell extracts (lanes
1 and 2) were incubated with Ni-NTA affinity resin, as
indicated, and the proteins bound to the resin were analyzed by
immunoblot with monoclonal anti-FLAG antibody (lanes 3 and
4). B, coimmunoprecipitation assays demonstrate
that Rrn3 isolated from CHX-treated cells interacts with
TAFI68. NIH3T3 cells were cotransfected with vectors
directing the expression of FLAG-Rrn3 and V5-TAFI68.
Forty-two hours after transfection, the cells were treated with vehicle
or cycloheximide (CHX, 2 µg/ml) for 3 h. The cells were then
lysed (lanes 1 and 2), and Rrn3 was purified from
control or CHX-treated cells (lanes 5 and 6,
respectively) using monoclonal anti-FLAG-agarose beads. After
incubation and washing, the immunopurified proteins were fractionated
by SDS-PAGE and analyzed by Western blots with monoclonal, anti-V5
(TAFI68, upper panel) and anti-FLAG (Rrn3,
lower panel) antibodies.
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Fig. 3.
Cycloheximide causes Rrn3 to dissociate from
RNA polymerase I. A, chimeric Rrn3 localizes to
the nucleolus. CMT3 cells transiently expressing hRrn3-GFP (left
panel) were subjected to fluorescence in situ
hybridization to probe for the 5'-external transcribed spacer of
pre-rRNA (FISH). Colocalization of hRrn3-GFP and the 5'-ETS
(overlay) demonstrates that the chimeric protein is
localized to the nucleolus and is predominantly within fibrillar
centers. Bar, 5 µm. B, chimeric Rrn3 associates
with RNA polymerase I. S-100 extracts from 3T3 cells transfected with
pCDNA3.1 (pCDNA; lanes 1, 3,
and 5) or pCDNA3.1-FLAG-polyoma-Rrn3 (Rrn3,
lanes 2, 4, and 6) were
immunoprecipitated with immobilized anti-FLAG antibodies. The agarose
beads were washed, and the immunoprecipitates were eluted with FLAG
peptide. Input (10%), flow-through (FT), and bound
(lanes 5 and 6) samples were fractionated by
SDS-PAGE and blotted to Immobilon P. The blots were probed with
polyclonal antibodies to the 194-kDa subunit of pol I (A194)
(upper panel), stripped, and reprobed with monoclonal
anti-polyoma antibodies (lower panel). C,
cycloheximide (CHX) inhibits the interaction between
chimeric Rrn3 and RNA polymerase I. S-100 extracts from control or
CHX-treated (3 h) 3T3 cells transfected with
pCDNA3.1-FLAG-polyoma-Rrn3 (lanes 1 and 2)
were immunoprecipitated with immobilized anti-FLAG antibodies
(lanes 3 and 4). The starting material and
immunoprecipitates were fractionated by SDS-PAGE and blotted to
Immobilon P. The blots were probed with polyclonal antibodies to the
127-kDa subunit of pol I (A127, upper panel),
stripped, and reprobed with monoclonal anti-polyoma antibodies
(Rrn3, lower panel).
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Fig. 4.
Cycloheximide (CHX) inhibits the interaction
between Rrn3 and rpa43 in vivo. S-100 extracts
from control or CHX-treated 3T3 cells cotransfected with
pCDNA3.1-FLAG-polyoma-Rrn3 and pV5Rpa43 were immunoprecipitated
with immobilized anti-FLAG antibodies. The immunoprecipitated proteins
were eluted with FLAG peptide. The starting extracts (lanes
1, 2, 5, and 6) and the
immunoprecipitates (lanes 3, 4, 7, and
8) were fractionated by SDS-PAGE and blotted to Immobilon P. The blots were probed with monoclonal anti-FLAG antibodies
(FLAG-Rrn3, lanes 1-4) stripped, and reprobed
with monoclonal anti-V5 antibodies (V5-rpa43, lanes
5-8).
View larger version (19K):
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Fig. 5.
Cycloheximide inhibits the phosphorylation of
Rrn3. NIH3T3 cells were transfected with pCDNA3.1FLAG-Rrn3.
Twenty-four hours after transfection, the medium was replaced with
phosphate-free medium containing [32P]orthophosphate (1 mCi/ml). Cells were grown overnight in the radioactive medium, and CHX
(2 µg/ml) was added to the medium at the indicated times prior to
harvesting the cells. Rrn3 was immunoprecipitated with immobilized
anti-FLAG antibodies. The immunoprecipitates were suspended in SDS
sample buffer, fractionated by SDS-PAGE, and blotted to Immobilon P. A, the blots were exposed to x-ray film (lanes
1-3), and then the recovery of Rrn3 was determined by blotting
with monoclonal anti-FLAG antibodies (Rrn3, lanes
4-6). The lower panel in the autoradiogram
demonstrates that the labeling of a nonspecific coimmunoprecipitating
protein (CRM) is unchanged when the cells are treated with
cycloheximide. B, a graph depicting the results of three
similar experiments. The relative intensities of the autoradiographic
signals were normalized to the recovery of Rrn3 in each case. That
ratio was then divided by the ratio for the control sample for each
experiment. The values represent averages of three experiments ± S.D.
View larger version (15K):
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Fig. 6.
Dephosphorylation inhibits the interaction
between Rrn3 and rpa43 and the ability of Rrn3 to complement extracts
from cycloheximide-treated cells. A, FLAG-S-Rrn3
isolated from Sf9 cells was immobilized on anti-FLAG-agarose and
treated with increasing amounts (1, 2, and 5 units) of either calf
intestinal alkaline phosphatase (lanes 5-7,
CIAP) or bacterial alkaline phosphatase (lanes
8-10, BAP) as indicated. After washing, the
immobilized Rrn3 was incubated with Sf9 cell extracts containing
mouse S-rpa43 as described under "Experimental Procedures." The
proteins that bound to the FLAG-agarose were eluted with FLAG peptide
and analyzed by Western blotting as described under "Experimental
Procedures." The recovery of Rpa43 (upper panel) and the
Rrn3 load were detected with S-protein horseradish peroxidase conjugate
(lower panel). B, FLAG-Rrn3 (4 µg) isolated
from either Sf9 cells or bacteria was immobilized on monoclonal
anti-FLAG-agarose beads and incubated with either TAFI68 (4 µg, lanes 1-4) or rpa43 (4 µg, lanes 5-8)
purified from Sf9 cells. After the beads were washed, the
proteins that bound to the FLAG-agarose were eluted with FLAG peptide
and analyzed by Western blotting as described under "Experimental
Procedures." C, Rrn3 purified from Sf9 cells
(bv Rrn3) was added to inactive extracts from CHX-treated
N1S1 cells (CHX S-100), and the mixture was assayed in an
in vitro transcription reaction as described under
"Experimental Procedures." In lanes 3-5, 1 µg of
bvRrn3 was incubated with increasing amounts (0.04, 0.4, and
4.0 units) of bacterial alkaline phosphatase (BAP) for 15 min at
30 °C. 1 µl of phosphatase inhibitor mixture II (Sigma) was added
to the phosphatase-treated Rrn3, prior to addition to the in
vitro transcription reaction. In lane 6, the Rrn3 was
incubated with bacterial alkaline phosphatase (4 units) in the presence
of the phosphatase inhibitor mixture (1 µl/20-µl reaction) prior to
addition to the in vitro transcription reaction. FLAG-Rrn3
(1 µg) purified from E. coli was added to the reaction in
lane 7.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-ribofuranosyl-benzimidazole) DRB
(30-32). This finding is consistent with our model that inhibition of
phosphorylation of Rrn3 inhibits its interaction with pol I via rpa43.
View larger version (25K):
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Fig. 7.
Rrn3 phosphorylation is a checkpoint for rDNA
transcription. Phosphorylated Rrn3 (red circles) binds
to RNA polymerase I (Pol I) and acts as a bridge between RNA
polymerase I and SLI. Following initiation, Rrn3 becomes
dephosphorylated, loses its ability to bind to Pol I (rpa43), and
preferentially binds to SL1. Hence, Rrn3 may remain associated with SL1
at the transcription initiation site. Prior to reinitiation, Rrn3 is
phosphorylated and can again act as the bridge between SL1 and Pol
I.
FOOTNOTES
ABBREVIATIONS
' subunit of RNA
polymerase I;
A127, subunit of RNA polymerase I;
FISH, fluorescence
in situ hybridization;
ETS, external transcribed spacer;
PBS, phosphate-buffered saline;
BSA, bovine serum albumin;
nt, nucleotide(s);
Ni-NTA, nickel-nitrilotriacetic acid;
BAP, bacterial
alkaline phosphatase;
CIAP, calf intestinal alkaline phosphatase;
GFP, green fluorescent protein.
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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