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Vol. 14, Issue 7, 2744-2755, July 2003
¶
*Department of Molecular Microbiology and
Biotechnology, Tel-Aviv University, Ramat Aviv 69978, Israel;
Department of Biochemistry, Dartmouth Medical
School, Hanover, New Hampshire 03755; and
Department of Molecular Microbiology, the Bruce
Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa
31096, Israel
Submitted November 18, 2002;
Revised February 17, 2003;
Accepted February 17, 2003
Monitoring Editor: Pamela A. Silver
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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;
Buratowski, 2000;
Young, 2000). Gene expression,
however, has also been shown to be controlled at several other levels. One
important posttranscriptional event that is used as a regulatory step for gene
expression is the export of mRNA from the nucleus to the cytoplasm (reviewed
in Cole, 2000;
Kuersten et al.,
2001; Reed and Hurt,
2002). Relative to our knowledge on transcription, less is known
about the contribution of the mRNA export machinery to regulation of gene
expression.
The yeast RNA polymerase II (pol II) is composed of 12 subunits, most of
which are essential for viability (Young,
1991). Rpb4p, the fourth largest subunit, has unique features.
Cells lacking RPB4 can transcribe and grow comparably to wild-type
(WT) cells during optimal growth conditions at moderate temperatures
(18–23°C). In contrast, these cells rapidly lose their ability to
transcribe most, if not all, genes when exposed to either nonoptimal
temperatures (<12°C or >32°C), ethanol (10%), or starvation
stresses (Choder and Young,
1993; Rosenheck and Choder,
1998; Maillet et al.,
1999; Sheffer et al.,
1999; Miyao et al.,
2001). Using a promoter-independent transcription assay, we
demonstrated that Rpb4p is required for pol II enzymatic activity at
temperature extremes but not at intermediate temperatures
(Rosenheck and Choder, 1998).
Thus, a main role assigned to Rpb4p is to permit transcription when cells
experience stress or suboptimal growth conditions. Consistent with this, only
a small portion of pol II molecules (
20%) contain Rpb4p during optimal
growth, whereas association of Rpb4p with pol II increases fivefold under some
stress conditions (to near 100%; Choder and
Young, 1993).
Rpb4p is always present in excess over other pol II subunits. Under optimal
conditions, only 2% of Rpb4p molecules are complexed with pol II
complexes, and this increases to 10% under conditions where most pol II
complexes contain Rpb4p (Choder and Young,
1993; Rosenheck and Choder,
1998). Moreover, sucrose sedimentation of a whole cell extract
indicated that Rpb4p does not sediment as a free protein but seems to be
associated with both pol II and, to a much greater extent, smaller complexes
(Rosenheck and Choder, 1998).
This suggests that Rpb4p performs some function(s) independent of its
association with the pol II enzyme. Interestingly, NSP1, a nuclear
pore complex (NPC) protein, was recently identified as a high-copy suppressor
of an rpb4 mutant, suggesting a connection between the NPC and
RPB4 function (Tan et
al., 2000; see DISCUSSION).
The response of eukaryotic cells to heat shock (HS) and to other stress
conditions occurs at both transcriptional and posttranscriptional levels
(Morano et al., 1998;
Cotto and Morimoto, 1999). In
response to a relatively moderate HS (37°C), transcription of most yeast
genes is inhibited for a short period of time and there is a modest induction
of transcription of HS genes (Saavedra
et al., 1996). However, after a short delay, subsequent
gene expression is executed efficiently at all levels and cells continue to
divide. We have previously reported that severe HS (42°C) or 10% ethanol
shock lead to nuclear retention of most poly(A)+ RNA. Under these
conditions, the transcription of HS genes is strongly induced and HS
transcripts are efficiently exported in a process that requires most of the
factors essential for normal poly(A)+ RNA export (Saavedra et
al., 1996,
1997).
A major challenge in current molecular biology is to understand how
sequential steps in gene expression are coupled and how coupling is involved
in regulation of gene expression in response to external signals.
Sophisticated crosstalk between transcription, splicing, polyadenylation, and
mRNA export have been described (recently reviewed by
Komeili and O'Shea, 2000;
Reed and Hurt, 2002). Yra1p
and Sub2p are mRNA export factors that couple the transcription machinery to
splicing and to mRNA export, by virtue of their interaction with THO complex
that functions in transcription elongation
(Jimeno et al., 2002;
Strasser et al.,
2002). Npl3p, a major hnRNP protein in yeast required for mRNA
export, is recruited to the mRNA during transcription
(Lei et al., 2001).
Interestingly, during stress, Npl3p dissociates from the mRNA (or is not
recruited to the mRNA; Krebber et
al., 1999). This is likely to be one of the mechanisms used
to arrest the transport of the bulk mRNA during stress. Nevertheless, the
transport of HS mRNAs, which does not require Npl3p, is executed efficiently
(Saavedra et al.,
1996,
1997). The detailed mechanism
of mRNA export during stress is unknown. Neither is it known how coupling
between various levels of gene expression is affected by the environment.
Here we show that Rpb4p has a dual role during both moderate and severe HS: a role in transcription and a role in the export of mRNAs to the cytoplasm. We suggest that Rpb4p may couple RNA polymerase II function to mRNA export during stress.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Construction of GFP-RPB4, Its Integration at the
RPB4 Locus in the Chromosome, and Its Expression from a Plasmid
A linear fragment encoding GFP-RPB4 under control of RPB4
regulatory elements was constructed from three different DNA fragments, each
obtained by PCR. One is a 398-base pair fragment spanning positions –398
to –1 upstream of RPB4 start codon. The second is a GFP
fragment that contains the entire GFP coding region, except for the stop
codon. The third fragment was placed downstream of the GFP fragment and
encodes 10 alanine residues used as a (structurally flexible) linker, followed
by the entire RPB4 coding region and 145 base pairs of its
3'-noncoding region. A NotI site was introduced in the region
encoding the poly(Ala) linker. The RPB4p-GFP-RPB4 fragment
was introduced into MC11-1 cells by transformation and integrated into the
rpb4 locus by homologous recombination, thus replacing
rpb4
1::HIS3
(Woychik and Young, 1989).
Transformants were selected on histidine-containing plates that were incubated
at 37°C to select for integration of RPB4. Integration of the
chimeric gene in the correct locus was verified by histidine auxotrophy,
PCR-based analyses, and later by a direct sequencing of a DNA fragment that
had been PCR amplified using the transformant's genome as the template.
To express the fusion gene from a plasmid, the gene was inserted in pRS315 as detailed in Table 1.
Immunostaining Assay
Cells were fixed with 4% paraformaldehyde for 15 min at room temperature
(addition of formaldehyde, a more conventional fixer, led to a rapid export of
Rpb4p-Myc2 before fixation had been completed and could not be used).
Spheroplasts were mounted on polylysine-coated slides, permeabilized with cold
methanol, blocked with 1% BSA, 01% Triton X-100 in PBS, and incubated with
primary antibodies in PBS containing 1% BSA for 1 h at room temperature. After
extensive washing, cells were reacted with FITC-labeled secondary antibodies
in PBS containing 1% BSA, followed by extensive washing. Cell nuclei were
strained with DAPI and the slides were examined by fluorescence
microscopy.
Random Mutagenesis of the RPB4 and Identification of
Temperature-sensitive Mutants
The RPB4 coding region was mutagenized using an error-prone PCR
technique (Fromant et al.,
1995), using pMC143 as the template. oMC129
(5'-CTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAAGCGGCCGAGCAGCTGCCGCTGCAGCGGC-3')
was used as the forward primer, consisting the 3' end of the GFP
sequence (italicized) upstream of the NotI site (underlined), which
is present in the poly(Ala) linker (see plasmid construction above).
oMC78(5'-CGCGGATCCCTTAAAAGAGCAAGACGAGATTTAAAATCC-3') was the
reverse primer, located 110 base pairs downstream of RPB4 stop codon.
PCR reaction (22 cycles) was carried out as described
(Fromant et al.,
1995) except that MgCl2 was added to 4.2 mM and the
concentration of the dNTP that was in excess over the others was 1.5 mM. The
resulting mutagenized fragment was digested with BamHI, ligated to
BamHI digest of pMC143, followed by PCR amplification using standard
conditions with the following primers: oMC132
(5'-CTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAG-3') identical
to the 5' end of oMC129 and oMC130
(5'-GCTGGCACGACAGGTTTCCC-3'). This oligo recognizes the vector
sequence located 290 base pairs downstream of the BamHI and 400 base
pairs downstream of the RPB4 stop codon in pMC143. The mutagenized
fragment was integrated into pMC143 by homologous recombination in vivo after
cotransforming the MC11-1 strain with a NotI/SpeI digest of
pMC143 (SpeI is located 20 base pairs downstream of the RPB4
stop codon; hence the digest lacks the entire RPB4 sequence).
Transformants were allowed to grow at 23°C. After tiny colonies appeared,
they were replica plated onto two plates: one plate was incubated at 23°C
and the other at 37°C. Colonies that grew at 23°C but not at 37°C
were selected, and their plasmid DNA was recovered and introduced again into
naïve MC11-1 cells to verify that the ts phenotype was plasmid dependent.
The specific mutations in rpb4 that were responsible for the ts
phenotype were determined by sequencing analyses (see
Fig. 4).
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In Situ Hybridization Assays
In situ hybridization assays to detect poly(A)+ RNA or SSA4 mRNA
were performed essentially as described previously
(Amberg et al., 1992;
Gorsch et al., 1995;
Saavedra et al.,
1996).
In Vivo Labeling of Proteins and Analysis by Gel Electrophoresis
Cells were grown at 22°C in synthetic complete medium lacking leucine
and methionine until midlogarithmic growth phase (
107
cells/ml), collected and resuspended in 4 ml medium, and then divided into
four equal samples in screw-capped 2-ml tubes. One tube was incubated at
22°C and the others at 42°C for the indicated time periods. All tubes
were vortexed occasionally. 35S Trans-label (Amersham, Piscataway,
N.J.) was then added to a final concentration of 100 µCi/ml, 15-min before
cell harvest. Labeling was terminated by the addition of cycloheximide to 50
µg/ml, and cells were immediately collected by centrifugation, resuspended
in ice-cold protein extraction buffer A
(Choder and Young, 1993), and
frozen at –80°C until further use. Proteins were extracted and
30-µg samples were subjected to electrophoresis in 5–15% gradient
polyacrylamide gel (1.5 mm x 16 cm x 20 cm). Gels were
fluorographed and exposed to x-ray films.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Figure 1B shows that, in cells proliferating exponentially at 22°C, GFP-Rpb4p fluorescence was nuclear, because it colocalized with the fluorescence of the DNA stain, DAPI. When cells were exposed to elevated temperatures (ranging from 34 to 42°C), a large portion of GFP-Rpb4p was relocalized to the cytoplasm and the fluorescence was detected all over the cell. (Figure 1C, 37°). Similarly, even at 22°C, starvation or ethanol stress also caused GFP-Rpb4p to relocate and to be detected all over the cell (Figure 1C, Stationary, Ethanol). Thus, relocation of Rpb4p is a characteristic of the stress response and not of high temperature per se. When not fused to Rpb4p, GFP was evenly distributed throughout the cell in both unstressed and stressed cells (our unpublished results), arguing against the possibility that localization of GFP-Rpb4p is governed by the GFP domain.
The kinetics of the relocation of Rpb4p in response to temperature shift
was monitored: after a shift to 37°C (moderate temperature stress),
GFP-Rpb4p relocation was slow (Figure
1D). When cells were shifted from 22 to 42°C
(Figure 1D) or into 15% ethanol
(our unpublished results), GFP-Rpb4p accumulated in the cytoplasm much more
rapidly; within 2–5 min, 50% of the cells exhibited cytoplasmic
fluorescence, and after 30 min all of the cells exhibited cytoplasmic
fluorescence. Significantly, the stress-induced relocation of GFP-Rpb4p was
not due to a change in the level of the protein, because its level was similar
in unstressed and stressed cells (Figure
1A). Addition of cycloheximide concomitantly with HS did not
affect its relocation (Figure
1D), indicating that the GFP-Rpb4p molecules found in the
cytoplasm were not newly synthesized and that no newly synthesized factors are
required for relocation of Rpb4p. Instead, in response to these stress
conditions, most of the nuclear GFP-Rpb4p molecules shifted to the cytoplasm.
The data indicate that GFP-Rpb4p is exported from the nucleus in response to
various stress conditions. Because Rpb4p is required in the nucleus under
these stress conditions, it is quite likely that Rpb4p shuttles during these
conditions.
To examine Rpb4p localization by means other than its fusion to GFP, we
tagged RPB4 at its c-terminus with two copies of the Myc sequence and
determined localization of the tagged molecule by an indirect immunostaining
technique. Figure 2,
RPB4-Myc2 panel, demonstrates once again the HS-induced relocation of
Rpb4p. Unlike Rpb4-Myc2, Rpb2-HA and Rpb3-HA, two other subunits of RNA
polymerase II (Young, 1991),
are localized to the nucleus under both HS and moderate temperatures
(Figure 2, RPB2-HA and
RPB3-HA panels). Thus, the HS-induced export is not a general
characteristics of the RNA polymerase II complex or subunits.
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Cells lacking RPB4 are more sensitive than WT cells to some, but
not all, types of stress conditions. For example, although RPB4 is
required during temperature, starvation, or ethanol stresses, it is not
important for coping with osmotic or oxidative stress under otherwise optimal
conditions (Sheffer et al.,
1999; Maillet et al.,
1999). Interestingly, under conditions where RPB4 is
required, its product is found mainly in the cytoplasm (Figures
1C and
2), whereas under conditions
where RPB4 is dispensable, Rpb4p is nuclear (Figures
1B and
2, and our unpublished
results). Note that when cells are under stress, Rpb4p is required for pol II
activity; under such conditions some of the Rpb4p molecules must remain in the
nucleus. Because there are many more Rpb4p molecules than there are pol II
complexes (Rosenheck and Choder,
1998), the nuclear fluorescence would be masked by the strong
cytoplasmic one, and fluorescence would be detected throughout the cell.
Analyses of rpb4 Mutants Indicate a Posttranscriptional
Role for Rpb4p during Various Stresses
Cells lacking RPB4 are unable to grow at elevated temperatures
(>32°C) or to synthesize HS mRNAs after severe stress because pol
II4 (pol II lacking Rpb4p) has little or no activity under these
conditions (see INTRODUCTION). To determine whether Rpb4p carries a
posttranscriptional function and to study further the functions of Rpb4p,
RPB4 was randomly mutagenized. To this end, a centromeric plasmid was
constructed carrying GFP-RPB4 under control of RPB4 5'
and 3' regulatory regions. The fusion protein expressed from this
plasmid was present at approximately the same level as that expressed in the
YMF1 strain (Figure 3B). Consistently, this plasmid fully restored the proliferation of the
rpb4 strain either at high temperatures or in the presence of
ethanol (Figure 3A). The
plasmid also restored pol II transcriptional capacity during HS (42°C;
Figure 3C and see also
Figure 5A, YTN1 panel). We used
error-prone PCR conditions (Fromant et
al., 1995) and replaced the WT RPB4 coding region
with the mutagenized one, making sure that it was in-frame with
(nonmutagenized) GFP (see MATERIALS AND METHODS). Transformants were first
grown on selective plates at 22°C where Rpb4p function is dispensable.
Colonies were then replica-plated and screened at a nonpermissive temperature
(37°C). Plasmids were isolated from the temperature-sensitive (ts)
transformants and then reintroduced into naive cells of the same
rpb4 strain to ensure that the ts phenotype was due to
mutations in the plasmidencoded RPB4 and not to mutations elsewhere
in the genome. Finally, expression of the fusion proteins was determined by
Western analysis. Mutations in RPB4 that were responsible for the ts
phenotype were determined by DNA sequence analysis. The sequences of these
alleles are shown in Figure 4.
The alleles that were chosen for further studies and the strains that express
these alleles are listed in Table
1.
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Thus far, 13 mutants have been sequenced and further analyzed. Two classes of ts rpb4 mutants were obtained, referred herein as class I and class II: class I mutants (9 of 13 analyzed) are defective in supporting pol II transcription at high temperatures; as shown in Figure 4, all these mutant forms carry at least one mutation in the C-terminal region (position 129–212). Five of them carry mutations exclusively in the c-terminus. Class II mutants, 4 of 13 analyzed, can support transcription at high temperatures like the WT counterpart, yet (like all the mutants identify in this screen) they cannot confer upon the cells the ability to cope with the stress. Mutations in three of these mutant forms fall within the N-terminal region (position 33–80; see Figure 4). GFP-Rpb4-40p carries two cysteines at positions 143 and 149. We suspect that these two closely located cysteines adversely affect the native conformation of Rpb4p. Western analyses of the mutant fusion proteins raised the possibility that part of the reasons why some of these mutants are defective lays in their abnormal turnover (our unpublished results). Nevertheless, abnormal turnover characterizes both classes. We therefore surmise that abnormal turnover is not the only parameter that affects the abnormal function of the various mutant forms.
One example for the different defect of the two classes of mutants is shown
in Figure 5A in which the level
of the global poly(A)+ RNA was assessed by its hybridization to
radiolabeled poly(dT). Although exposure of cells carrying WT
GFP-RPB4 to 37°C had little effect on the global mRNA level, when
cells expressing the GFP-rpb4-5 mutant were shifted from 22 to
37°C, their global mRNA level gradually declined
(Figure 5A; cf. YTN1 with
YTN75). This decline is a characteristics of cells lacking RPB4
(Choder and Young, 1993;
Sheffer et al., 1999;
our unpublished results), indicating that GFP-Rpb4-5p cannot support
transcription. YTN75 was therefore classified as class I mutant and the
protein it carries, GFP-Rpb4-5p, as class I mutant form. The transcriptional
defect of class I mutants during HS is sufficient to explain why cells
carrying these mutants are temperature sensitive and cannot survive stress. In
contrast with YTN75 strain, YTN25 and YTN73 strains could transcribe their
genome at 37°C at a level similar to their WT counterpart
(Figure 5A, compare YTN25 and
YTN73 panels with YTN1 panel). YTN25 and YTN73 were therefore classified as
class II mutants and the proteins they carry, GFP-Rpb4-2p and GFP-Rpb4-3p, as
class II mutant forms.
During abrupt and severe HS (e.g., a shift from 22 to 42°C) yeast cells
express preferentially HS genes. We therefore examined the capacity of our
mutants to function also under severe HS. Results shown in
Figure 5B demonstrate that
although the strain expressing the WT GFP-RPB4 responded to the HS by
a rapid and robust transcription of HS genes SSA3 and SSA4,
transcripts of these genes could not be detected in class I mutants (cf. YTN75
panel with YTN1 panel). Note that within 45 min of the severe HS,
transcription of non-HS genes (e.g., ACT1 and NUP157 shown
in Figure 5B) was compromised
and transcript levels gradually declined, both in the WT and in the mutants,
consistent with previous results (Saavedra
et al., 1997). Unlike class I mutants, transcription of
SSA3 and SSA4 during the HS was as efficient in class II
mutants as it was in WT cells (Figure
5B, cf. YTN25 and YTN73 panels with YTN1 panel). It is worth
noting that Northern blot hybridization analyses indicated that the sizes of
HS mRNAs produced in class II mutant cells were identical to those transcribed
in WT cells under both optimal and suboptimal conditions cells
(Figure 5B and our unpublished
results). Significantly, in spite of their capacity to synthesize full-size
transcripts efficiently, class II mutants were still temperature
sensitive.
The rpb4 mutant were selected based on their inability to form colonies at 37°C. Yet, further study indicated that both class I and class II mutants were also sensitive to the presence of ethanol (see Figure 3D). Moreover, consistent with their inability to proliferate at 37°C, both class I and class II mutants died much more rapidly than WT at 42°C (Figure 3E). In addition, some of the mutants (e.g., YTN25) died much more rapidly than the WT strain during starvation (our unpublished results).
The identification of rpb4 mutants that transcribe normally but are defective in their ability to cope with various stresses (class II) suggests that Rpb4p plays an additional posttranscriptional role under these stress conditions. The nature of this posttranscriptional function is discussed in the following section.
Rpb4p Is Required for the Export of poly(A)+ RNA at
37°C and for Export of Heat Shock mRNAs during Severe Temperature Stress
(42°C)
Most WT lab yeast strains can grow at temperatures as high as 38°C.
Nevertheless, when yeast cells are shifted from moderate temperatures
(18–23°C) to 37°C, a modest stress response occurs: expression
of HS genes is transiently induced and cells transiently decrease
transcription of most other genes. Later, cells adjust to the high
temperature, and the expression of non-HS genes becomes efficient again,
allowing cells to continue to divide, albeit at a slower rate
(Lindquist, 1986;
Nonet et al., 1987;
Choder and Young, 1993). In
light of the observations, discussed in the previous section, that at
37°C Rpb4p performs a posttranscriptional function and is also
exported to the cytoplasm, we tested the possibility that Rpb4p plays a role
in mRNA export when cells are exposed to elevated temperatures. To test this
possibility we used the YTN25 strain expressing GFP-Rpb4p-2, a class
II mutant allele. Although this strain can transcribe efficiently at 37 and at
42°C (see Figure 5), it is
unable to grow at 37°C and rapidly dies at 42°C
(Figure 3E). To determine what
kind of posttranscriptional defect characterizes YTN25, we determined the
location of poly(A)+ RNA. Figure
6, panel MC11-1, shows that 1 h after the shift of
rpb4 cells from 22°C to 37°C, the level of the
poly(A)+ RNA declined, reflecting the transcriptional defect of
these cells (see INTRODUCTION). In contrast, 1 h after the shift of WT to
37°C, cells contained a high level of poly(A)+ RNA detected
mainly in the cytoplasm (Figure
6, YTN1). This indicates that at 37°C, mRNAs were synthesized
and exported efficiently to the cytoplasm in wild-type cells (see also
Lee et al., 1996;
Krebber et al.,
1999). In contrast, after the shift of YTN25–37°C, cells
produced poly(A)+ RNA, but it accumulated in nuclei
(Figure 6, YTN25, 37°C).
Note that not all cells accumulated mRNAs in their nuclei. This suggests that,
at 37°C, the penetrance of the mutation was not complete or that the
function of Rpb4p is merely to enhance export efficiency (see DISCUSSION). In
contrast with nuclear accumulation at 37°C, poly(A)+ RNA was
normally distributed in the cytoplasm of these cells grown at 22°C
(Figure 6, MC11-1 and YTN25,
22°C). These results indicate that, under conditions where RPB4
is dispensable (22°C), both transcription and mRNA export are carried out
efficiently and independently of Rpb4p. However, at 37°C, Rpb4p is
required for the efficient execution of both processes.
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When yeast cells are exposed to 42°C, a strong stress response occurs
and transcription of HS genes is strongly induced. HS transcripts are then
selectively exported, while export of the bulk polyA+ RNA is
blocked (Saavedra et al.,
1996,
1997). A similar pattern was
observed after stressing cells with 10% ethanol
(Saavedra et al.,
1996). The role of Rpb4p in poly(A)+ RNA at 37°C
led us to determine if Rpb4p is required also for HS mRNA export at 42°C.
To this end, we used in situ hybridization to examine the localization of
SSA4 mRNA, encoding one of the yeast Hsp70 proteins. As expected,
very little SSA4 mRNA was visible when these cells were grown at
22°C (Figure 7A, see
columns designated 0, and Figure
7B, 22°C panels). After a shift to 42°C, SSA4
mRNA was not produced in cells lacking RPB4
(Figure 7, A and B, strain
MC11-1). In cells expressing WT Rpb4p, GFP-Rpb4p, or class II mutant Rpb4p,
SSA4 mRNA was produced and readily detected. However, cellular
distribution of the SSA4 transcript was dependent on the nature of
Rpb4p. In cells carrying WT-Rpb4p or WT GFP-Rpb4p, SSA4 mRNA was
exported and detected in the cytoplasm as early as 15 min after temperature
shift (Figure 7A, strains SUB62
and YTN1, and Figure 7B, SUB62
panel). In contrast, strong nuclear accumulation of SSA4 mRNA was
detected within 15 min after shifting isogenic cells expressing GFP-Rpb4-2p or
GFP-Rpb4-3p (class II mutants) to 42°C
(Figure 7A, strains YTN25 and
YTN73, 15 min; Figure 7B YTN25
panel). Thus, class II mutants are defective not only in export of the bulk
poly(A)+ RNA at 37°C (Figure
6), but also in the export of SSA4 mRNA at 42°C. The
two class II mutants differed in their penetrance. Although cells expressing
GFP-rpb4-2 were almost completely defective in SSA4 mRNA
export, those expressing GFP-rpb4-3 supported SSA4 mRNA
export but at a reduced rate. Consequently, cytoplasmic SSA4 mRNA
could be detected in cells expressing GFP-rpb4-3, but only after 60
min at 42°C (Figure 7A, cf.
strains YTN25 and YTN73, 60-min), reflecting a partial export block.
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To examine further the transport defect of cells expressing a class II
mutant (GFP-rpb4-2) and to determine whether the mutations in the
expressed protein affect the export of other classes of HS mRNAs, we
pulse-labeled cells with [35S]methionine+cysteine for 10-min
periods before or at different times after HS. Results are shown in
Figure 8A. In WT cells, the
expected set of HS proteins was synthesized and readily detected within 15 min
of HS (lane 2). As observed previously
(Saavedra et al.,
1997), within 30 min after temperature shift (lane 3) and more
clearly after 45 min (lane 4), the HS proteins were the predominant proteins
synthesized. In contrast, in cells expressing GFP-rpb4-2, the
synthesis of all HS proteins was severely compromised
(Figure 8A, lanes 10–12).
Because SSA4 mRNA accumulates in the nuclei of cells carrying
GFP-rpb4-2, the defect in synthesis of Ssa4p is most likely a
reflection of the block in export of SSA4 mRNA. Most likely, the
defect in the synthesis of the other HS proteins is also due to a defect in
the export of their mRNAs. Figure
8A, YTN75 (lanes 9–12) shows that no HS proteins were
detected in class I mutant cells that do not transcribe HS genes at 42°C
(see Figure 5B, YTN75). Thus,
the inability of class I mutant cells to synthesize proteins during HS is
similar to that of rpb4, as analyzed by two-dimensional gel
electrophoresis (Maillet et al.,
1999). This defect is more severe than that observed in the class
II mutant (which is not defective in transcription), as the latter did exhibit
limited synthesis of HS proteins (Figure
8A, cf. lanes 10–12 with 6–8). This suggests that
leaky export of HS mRNAs did occur in YTN25 cells (unless some translation was
carried out in the nucleus). Nevertheless, the leaky export was insufficient
to protect the cells from the damaging effects of the severe stress, resulting
in their death (Figure 3E).
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Figure 8B shows the localization of class II mutants. GFP-Rpb4-2p was constitutively mislocalized to the cytoplasm and its localization was not affected by the environmental conditions (Figure 8B, YTN25 panels). Note, however, that at least during stress, some GFP-Rpb4-2p molecules should be present in the nucleus to support transcription. Localization of GFP-Rpb4-3 in optimally growing cells was partially defective as it was partly cytoplasmic (Figure 8B, cf. YTN73 panel with that of YTN1); during HS, localization of some GFP-Rpb4-3p molecules was still nuclear. These results demonstrate that the localization of class II mutant proteins is abnormal. Their mislocalization may contribute to their defect in supporting mRNA export or may be the result of their compromised transport capability.
Taken together, our results indicate that Rpb4p plays an important role in
mRNA export both under conditions of moderate temperature stress, under which
cells continue to grow (37°C), and during severe HS (42°C). Export of
both bulk poly(A)+ RNA and HS mRNAs is compromised in these mutants
at temperatures where WT Rpb4p becomes essential for transcription. However,
during optimal growth, Rpb4p is required neither for transcription nor for
mRNA export, and rpb4 cells grow like WT
(Choder and Young, 1993, and
compare MC11-1 with SUB62 in Figure
3A, 22°C).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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What might Rpb4p be doing in transcription and in mRNA export? Does Rpb4p
perform two different functions, one in transcription and one in export, or do
phenotypic defects in both processes reflect a single operational defect in
Rpb4p? The possibility that the interaction of Rpb4p with pol II, which helps
to maintain pol II integrity during stress
(Choder and Young, 1993;
Jensen et al., 1998;
Rosenheck and Choder, 1998),
is responsible for both Rpb4p functions is appealing because of its
simplicity. Indeed, recent studies indicated that pol II plays an important
role in various posttranscriptional processes including capping, 3'
processing, and mRNA maturation (Hammell, Heath, and Cole, unpublished
results; for recent reviews see Cramer
et al., 2001;
Maniatis and Reed, 2002;
Zhao et al., 1999).
Maintaining pol II integrity is therefore important for both transcription and
pre-mRNA processing. RNA processing, in turn, appears to be coupled to export
of the mature mRNA. However, if the sole role of Rpb4p is to maintain pol II
integrity and/or to help recruiting other factors to pol II complex, why would
most Rpb4p molecules be found in the cytoplasm under conditions (stress) when
their function is important in the nucleus? More significantly, class II
mutants are fully functional in transcription, suggesting that pol II
integrity is not impaired in these mutants; yet these mutants are defective in
mRNA export. This suggests that maintaining pol II integrity when Rpb4p is
mutant is not sufficient to ensure mRNA export. We propose that Rpb4p has a
dual role during stress; a role in transcription and a role in mRNA
export.
Previously, Woychik and her coworkers have selected NSP1 as a
high-copy suppressor of the rpb4 ts phenotype at 34°C (a
very mild temperature stress that can readily be tolerated by WT strains but
not by rpb4 strains; Tan
et al., 2000). The identification of NSP1, which
encodes a component of the nuclear pore complex, is intriguing. Nsp1p could
not suppress the in vitro transcriptional defects of rpb4 cell
extracts (Tan et al.,
2000), consistent with the possibility that this suppressor
affects a posttranscriptional function of Rpb4p. Although it was proposed that
suppression by Nsp1p was indirect (Tan
et al., 2000), the results presented here suggest that
overexpression of NSP1 suppresses the export defect that
characterizes rpb4 mutant cells in a direct manner. The
ability of NSP1 to suppress the ts defect of rpb4
under moderate temperature stress suggests that Rpb4p plays a role in
enhancing the efficiency of the export machinery; it is not essential for
export if export is enhanced by other means (e.g., by Nsp1p overproduction).
Indeed, Figure 6 shows that,
even at 37°C, a severe defect in export was observed in some, but not all,
YTN25 cells.
Our attempts to demonstrate a physical interaction of Rpb4p with mRNA
either directly or via the export complex have thus far not been successful.
This suggests that either the interaction of Rpb4p with the export complex is
transient or unstable when extracted from the cell or that the essential role
of Rpb4p in export is indirect. Interestingly, however, Rpb4p complexed with
Rpb7p is capable of binding RNA in vitro
(Orlicky et al.,
2001), which leads us to favor the possibility proposed by Orlicky
et al. (2001) that
the heterodimer interacts (transiently?) with the emerging transcript.
Our study was based, to a substantial degree, on our earlier studies of
mRNA distribution after stress, using in situ hybridization (Saavedra et
al., 1996,
1997). Subsequently, another
study suggested that HS mRNA was not exported selectively after heat shock
(42°C) but instead competed for export with bulk poly(A)+ RNA
(Vainberg et al.,
2000). Unlike our previous results (Saavedra et al.,
1996,
1997), no nuclear accumulation
of poly(A)+ mRNA was detected by Vainberg et al. We tested
the WT and rip1 strains (derivatives of W-303-1a) used by
Vainberg et al. and found that, in our laboratory, they behaved
similarly to our WT strains (FY23 and FY86, derivatives of S288C;
Winston et al.,
1995). We therefore suspect that the different conclusions from
the two studies reflect differences in how the in situ hybridization assays
were performed. Consistent with our previous results (Saavedra et
al., 1996,
1997), selective mRNA export
during stress has also been observed in both Schizosaccharomyces
pombe (Tani et al.,
1996) and mammalian cells
(Imed-Eddine et al.,
2000). In addition, Brodsky and Silver
(2000) used a novel RNA-binding
GFP fusion protein assay to localize individual species of mRNA and reported
efficient export of SSA4 mRNA and nuclear accumulation of
PGK1 and ASH1 mRNAs in stressed cells.
Many studies over the past several years indicate that there is an
intricate coupling between transcription, mRNA processing, and mRNA export.
Recent studies are beginning to define the mechanisms of coordination. The
recruitment of nuclear export factors onto nascent transcripts during
transcription involves a variety of factors, including Npl3p
(Lei et al., 2001),
Yra1p, Sub2p, and the THO complex (containing 4 subunits;
Jimeno et al., 2002;
Strasser et al., 2002
and references therein). Moreover, genetic analyses revealed the possible
involvement of TBP (Lei et al.,
2001) and TAF (ptr6+ of S. pombe;
Shibuya et al., 1999)
whose specific roles in export are not clear. Recently, eight proteins whose
roles in export are not known were reported to be tightly associated with
THO/Yra1p/Sub2p complex (Strasser et
al., 2002). Taken together these data indicate that much
remains to be uncovered about nuclear mRNA metabolism and export. In
particular, how these components are involved in the regulation of both
transcription and export in response to environmental signals is still
unknown. This work assigns a transport role to the pol II subunit Rpb4p.
Because both the transcriptional and export roles of Rpb4p are important only
during stress, we propose that Rpb4p participates in the cellular responses to
stress at the interface of the transcription and export machineries. The
recruitment of Rpb4p to the transcription apparatus in response to starvation
stress has been demonstrated previously
(Choder and Young, 1993). It
remains to be determined whether or not Rpb4p is recruited to the transport
complex, or to auxiliary complexes involved in transport, also only during
stress.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Footnotes |
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Both authors contributed equally to this work. 
¶ Corresponding author. E-mail address: choder{at}tx.technion.ac.il.
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