Supplemental Material can be found at:
http://www.jbc.org/cgi/content/full/M704054200/DC1
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 47, pp. 33902–33907, November 23, 2007
© 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
HSF1-TPR Interaction Facilitates Export of Stress-induced
HSP70 mRNA*□
S⽧
Received for publication, May 16, 2007, and in revised form, September 25, 2007 Published, JBC Papers in Press, September 25, 2007, DOI 10.1074/jbc.M704054200
Hollie S. Skaggs1, Hongyan Xing, Donald C. Wilkerson, Lynea A. Murphy, Yiling Hong2, Christopher N. Mayhew3,
and Kevin D. Sarge4
From the Department of Molecular and Cellular Biochemistry, Chandler Medical Center, University of Kentucky,
Lexington, Kentucky 40536-0084
The up-regulation of heat shock proteins such as HSP70 that
occurs in response to exposure to elevated temperature and
many other stress conditions is vital for the ability of cells to
survive these stresses. Because of their crucial cytoprotective
function, it is very important that up-regulation of HSP expression after stress occur as rapidly and as efficiently as possible.
An intriguing finding of past studies is that stress conditions
inhibit the export of many mRNAs from the nucleus, but
mRNAs encoding heat shock proteins continue to be efficiently
exported during stress (1–7). However, the mechanism by
which HSP mRNAs bypass this stress-associated export inhibition was not known.
* This work was supported by National Institutes of Health Grants GM61053
and GM64606 (to K. D. S.), Training Grant T32ES007266 (Department of
Toxicology; to H. S. S.), and Postdoctoral Grant HD50043 (to D. C. W.). The
costs of publication of this article were defrayed in part by the payment of
page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
⽧
This article was selected as a Paper of the Week.
□
S
The on-line version of this article (available at http://www.jbc.org) contains
supplemental Fig. S1.
1
Present address: Div. of Hematology and Oncology, Markey Center, University of Kentucky, Lexington, KY 40536-0084.
2
Present address: Dept. of Biology, University of Dayton, Dayton, OH 45469.
3
Present address: Dept. of Cell and Cancer Biology, University of Cincinnati,
Cincinnati, OH 45267.
4
To whom correspondence should be addressed: Dept. of Molecular and
Cellular Biochemistry, Chandler Medical Center, University of Kentucky,
741 S. Limestone St., Lexington, KY 40536-0084. Tel.: 859-323-5777; Fax:
859-323-1037; E-mail: [email protected].
33902 JOURNAL OF BIOLOGICAL CHEMISTRY
HSF1 is the transcription factor responsible for up-regulating transcription of HSP70 and other genes in response to elevated temperature and other stress conditions. HSF1 performs
this function by undergoing stress-induced trimerization to the
DNA-binding form and then interacting with heat shock elements in the promoters of these genes to increase their transcription (8, 9). TPR is a 270-kDa polypeptide that is associated
with the nuclear basket on the nucleoplasmic face of the nuclear
pore complex (10 –17). Previous data suggest that TPR is
involved in the export of both mRNAs and proteins from the
nucleus (13, 16, 18 –20). During the course of yeast two-hybrid
experiments in our laboratory, we identified the existence of an
interaction between HSF1 and the TPR protein. The results
presented here suggest that the HSF1-TPR interaction could be
important for the export of HSP mRNAs during stress
conditions.
EXPERIMENTAL PROCEDURES
Yeast Two-hybrid Assay—The interactions between pGBDHSF1 and pVP16-TPR-(14 –117) and pVP16-TPR-(1218 –
1320) were characterized by streaking yeast (strain pJ694A)
containing these constructs or pGBD-HSF1 bait and empty
pVP16 plasmid (as a negative control) on plates lacking tryptophan and leucine; tryptophan, leucine, and histidine; or
tryptophan, leucine, and alanine.
In Vitro Binding Assay—In vitro translated 35S-labeled TPR(14 –117) or TPR-(1218 –1320) was incubated with GST5HSF1 and GST bound to glutathione-agarose. After washing,
bound proteins were analyzed by SDS-PAGE and autoradiography to detect the 35S-labeled TPR proteins. The amounts of
GST-HSF1 and GST proteins bound to the beads were determined by SDS-PAGE followed by Western blotting using goat
anti-GST polyclonal antibody (Amersham Biosciences).
Immunoprecipitation Analysis—HeLa cells (American
Type Culture Collection) were heat-shocked at 42 °C for 1 h,
and extracts of these cells were then subjected to immunoprecipitation using rabbit anti-HSF1 polyclonal antibody (21) or
rabbit nonspecific IgG followed by Western blotting using
mouse anti-TPR monoclonal antibody (Oncogene Research
Products).
Chromatin Immunoprecipitation Assay—ChIP assays were
performed with Jurkat cells as described previously (22) using
5
The abbreviations used are: GST, glutathione S-transferase; ChIP, chromatin
immunoprecipitation; RSV, Rous sarcoma virus; GFP, green fluorescent
protein; RIPA, radioimmune precipitation assay.
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Stress conditions inhibit mRNA export, but mRNAs encoding
heat shock proteins continue to be efficiently exported from the
nucleus during stress. How HSP mRNAs bypass this stress-associated export inhibition was not known. Here, we show that
HSF1, the transcription factor that binds HSP promoters after
stress to induce their transcription, interacts with the nuclear poreassociating TPR protein in a stress-responsive manner. TPR is
brought into proximity of the HSP70 promoter after stress and
preferentially associates with mRNAs transcribed from this promoter. Disruption of the HSF1-TPR interaction inhibits the
export of mRNAs expressed from the HSP70 promoter, both
endogenous HSP70 mRNA and a luciferase reporter mRNA.
These results suggest that HSP mRNA export escapes stress
inhibition via HSF1-mediated recruitment of the nuclear poreassociating protein TPR to HSP genes, thereby functionally connecting the first and last nuclear steps of the gene expression
pathway, transcription and mRNA export.
HSF1-TPR Interaction Facilitates Export
NOVEMBER 23, 2007 • VOLUME 282 • NUMBER 47
genomic contamination. cDNA was prepared from samples
using ImProm-II reverse transcriptase (Promega Corp.) and
oligo(dT)16 primers. cDNA samples were assayed by quantitative real-time PCR as described below.
RNA Immunoprecipitation Analysis—HeLa cells (1.5 ⫻ 106)
were transfected with 2 ␮g of either the HSP70i-luciferase or
RSV-luciferase plasmid using Effectene following the manufacturer’s instructions. Cells were then heat-shocked for 1 h at
42 °C, washed once with ice-cold phosphate-buffered saline,
and cross-linked with 2% paraformaldehyde for 12 min while
rotating. Cross-linking was quenched with 125 mM glycine for 5
min; and cells were washed twice with ice-cold phosphate-buffered saline, harvested by scraping, and snap-frozen in liquid
nitrogen. Cells were resuspended in 2 ml of low stringency
radioimmune precipitation assay (RIPA) buffer (50 mM TrisHCl (pH 7.5), 1% Nonidet P-40, 0.5% sodium deoxycholate,
0.05% SDS, 1 mM EDTA, 150 mM NaCl, 1⫻ Complete protease
inhibitor mixture (Roche Applied Science), and 80 units of
RNaseOUT (Invitrogen)), pipetted 20 times, and incubated on
ice for 10 min. Cells were then sonicated three times (80 –90%
output) for 20 s and centrifuged at 16,000 ⫻ g for 10 min at 4 °C.
The supernatant was precleared with 20 ␮l of protein
G-Sepharose (GE Healthcare) and washed with low stringency RIPA buffer and 100 ␮g/ml yeast tRNA (Ambion, Inc.)
for 2 h at 4 °C. During the preclearing, the low stringency
RIPA buffer-washed protein G-Sepharose beads were coated
with 5 ␮g of either anti-TPR antibody or mouse IgG (Sigma)
in low stringency RIPA buffer. The precleared supernatant
was then incubated with the antibody-coated beads for 90
min at 4 °C with rotation. Complexes were washed five times
for 10 min each at room temperature with 1 ml of high stringency RIPA buffer (50 mM Tris-HCl (pH 7.5), 1% Nonidet
P-40, 1% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 1 M
NaCl, 1 M urea, and 0.2 M phenylmethylsulfonyl fluoride).
Beads were then resuspended in 100 ␮l of 50 mM Tris-HCl
(pH 7.5), 5 mM EDTA, 10 mM dithiothreitol, and 1% SDS, and
cross-links were reversed for 1 h at 70 °C. mRNA was isolated as described above, and samples were analyzed by
quantitative PCR as described below.
Quantitative Real-time PCR—This was performed with the
Mx4000 system (Stratagene) using Brilliant SYBR Green QPCR
Master Mix (Stratagene). Samples were checked for specific
amplification using dissociation curve analysis included with
the software. PCR products were also assayed on polyacrylamide gels with ethidium bromide staining to ensure that they
were of the expected size. For quantitative PCR analysis of the
TPR-HSP70 promoter ChIP assay, the primers used were as
follows: HSP70, 5⬘-caacacccttcccaccgccactc-3⬘ and 5⬘-ctgattggtccaaggaaggctgg-3⬘; and histone H4, 5⬘-gagagggcggggacaattga-3⬘ and 5⬘-ggtcatgtccggctgtggaaag-3⬘. The Ct values were
normalized to input DNA (DNA before the immunoprecipitation step) and IgG controls. Data are represented as -fold
differences above mouse control IgG relative to input DNA
using the formula 2((Ct(IgG)⫺Ct(Input))⫺(Ct(TPR)⫺Ct(Input))). For the
experiments analyzing luciferase cDNA, primers used for
quantitative PCR analysis were 5⬘-gtctgaattccagtcgatgtacacgttcg-3⬘ and 5⬘-cacgaagcttgcatgcgagaactccacgc-3⬘. The
Ct values were normalized to input cDNA (cDNA made from
JOURNAL OF BIOLOGICAL CHEMISTRY
33903
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mouse anti-TPR monoclonal antibody and mouse nonspecific
IgG (as a negative control). The primers used to amplify the
promoter regions of the stress-inducible HSP70i gene and histone H4 gene were as follows: HSP70i, 5⬘-ctcagggtccctgtccc-3⬘
and 5⬘-tgagccaatcaccgagc-3⬘; and histone H4, 5⬘-gagagggcggggacaattga-3⬘ and 5⬘-ttggcgtgctcggtgtaggt-3⬘. PCR products
were then separated on polyacrylamide gels and stained with
ethidium bromide. ChIP samples were also analyzed by quantitative real-time PCR as described below.
HSP mRNA Export Analysis—pEGFP-TPR-(14 –117) and
pEGFP-TPR-(1218 –1320) were generated using a PCR-based
strategy to amplify the relevant regions while adding restriction
sites to each end to allow ligation into the pEGFP-C2 vector.
Cloning junctions were checked by sequencing. One ␮g of each
plasmid or empty vector was cotransfected with either the
HSP70i-luciferase (luciferase expressed from the stress-inducible human HSP70i promoter) (21) or RSV-luciferase plasmid
using Effectene (Qiagen Inc.) following the manufacturer’s
instructions. The RSV-luciferase plasmid was a kind gift of Dr.
Dan Noonan and was generated by replacing the chloramphenicol acetyltransferase coding sequence from the RSV-chloramphenicol acetyltransferase plasmid (23) with the luciferase coding sequence. Cells were heat-shocked at 42 °C for 1 h, and
cytoplasmic and nuclear fractions were then prepared by hypotonic lysis as follows. Cells were swollen in 5 packed cell volumes of 10 mM HEPES (pH 7.9), 1.5 mM MgCl2, 10 mM KCl, 1
mM dithiothreitol, and 1 mM phenylmethylsulfonyl fluoride
(Buffer A) for 10 min on ice. Cells were then centrifuged at 2000
rpm for 10 min, resuspended in 2 packed cell volumes of Buffer
A, and lysed by 20 strokes of a Dounce homogenizer (type B
pestle). The nuclei and cytoplasm were separated by centrifugation at 2000 rpm for 10 min. Separation was verified by viewing fractions with a microscope. mRNA was extracted from
each fraction using TRIzol reagent (Invitrogen) following the
manufacturer’s instructions. To analyze mRNA concentrations, each pool was subjected to an RNase protection assay
using SuperSignal RPA III (Ambion, Inc.) following the manufacturer’s instructions with a probe for either luciferase or L32
ribosomal protein mRNA. The probe for luciferase mRNA was
constructed via in vitro transcription using MAXIscript
(Ambion, Inc.) and biotinylated UTP (Roche Applied Science).
The template for in vitro transcription was created by PCR with
the HSP70-luciferase plasmid and primers 5⬘-cacggaaagacgatgacg-3⬘ and 5⬘-taatacgactcactataggttgggtaacgccaggg-3⬘. This
PCR product contained the 3⬘-end of the luciferase mRNA,
ending with the polyadenylation signal (yielding a protected
fragment of 325 bp) and untranscribed vector sequence (resulting in an unprotected fragment of 438 bb). The construction of
the L32 probe was described previously (24).
To determine whether expression of the two HSF1-binding
regions of TPR affects endogenous HSP70 mRNA levels, GFPTPR-(14 –117) and GFP-TPR-(1218 –1320) or empty vector
were transfected into HeLa cells according to the manufacturer’s protocol. Cells were heat-shocked and fractionated as
described above. Total RNA was extracted with TRIzol according to the manufacturer’s instruction. RNA was resuspended in
10 mM Tris-HCl (pH 7.5), 2.5 mM MgCl2, and 0.5 mM CaCl2 and
incubated with RNase-free DNase I to remove possible
HSF1-TPR Interaction Facilitates Export
total RNA before the immunoprecipitation step) and IgG
controls, which were set as 1 unit. Data are represented as
-fold differences relative to these two values using the formula 2((Ct(IgG)⫺Ct(Input))⫺(Ct(TPR)⫺Ct(Input))).
For experiments analyzing endogenous HSP70 mRNA levels,
the following primers were used: human HSP70, 5⬘-caccttgccgtgttgga-3⬘ and 5⬘-ttctcgcggatccagtg-3⬘; and human L32,
5⬘-catctccttctcggcatca-3⬘ and 5⬘-aaccctgttgtcaatgcctc-3⬘. The
data presented represent two independent experiments in
which values obtained utilizing the formula 2⫺⌬⌬Ct were
averaged. The results are graphed as relative differences
between cytoplasmic and nuclear HSP70 mRNA levels compared with the control samples (GFP alone), which were set
to a value of 1.
A
HSF1
+ Tpr
(14-117)
HSF1
+ pVP16
-TL
HSF1
+ Tpr
(1218-1320)
HSF1
+ pVP16
-TLH
B
-TLA
14-117
1218-1320
HSF1-interacting
regions from 2-hybrid
Tpr
33904 JOURNAL OF BIOLOGICAL CHEMISTRY
GS
T-H
SF
1
GS
T
GS
T-H
SF
1
GS
T
C
- GSTHSF1
35S-Tpr
- (14-117)
35
S-Tpr
- (1218-1320)
- GST
Autoradiogram
α-GST Western
FIGURE 1. HSF1 interacts with the TPR protein. A, yeast strain pJ694A transformed with pGBD-HSF1 and pVP16-TPR-(14 –117), pVP16-TPR-(1218 –1320),
or pVP16 alone was streaked on plates lacking tryptophan and leucine (⫺TL);
tryptophan, leucine, and histidine (⫺TLH); or tryptophan, leucine, and alanine
(⫺TLA). B, the schematic depicts the location of the N-terminally and centrally
located TPR segments identified as HSF1-interacting regions. C, in vitro translated 35S-labeled TPR-(14 –117) or TPR-(1218 –1320) was incubated with GSTHSF1 or GST that was bound to glutathione-agarose beads; and then after
washing, the amount of bound 35S-labeled TPR-(14 –117) or TPR-(1218 –1320)
was determined by SDS-PAGE and autoradiography. The amounts of GSTHSF1 and GST bound to beads were determined by GST Western blotting.
Input
37
42
α-HSF1
37
42
IgG
37
42
-Tpr
FIGURE 2. HSF1 interacts with the TPR protein in a stress-regulated manner. Extracts of non-stressed (37 °C) or stressed (42 °C, 60 min) HeLa cells were
immunoprecipitated using anti-HSF1 antibody or nonspecific IgG, and the
immunoprecipitates were subjected to anti-TPR Western blotting.
association between TPR and the HSP70 promoter in response
to exposure to stress conditions.
On the basis of previous results indicating a role for TPR in
mRNA export (13, 18, 19), including the finding that the yeast
TPR ortholog Mlp1p interacts with the mRNA export heterogeneous nuclear ribonucleoprotein Nab2p (19), we hypothesized that the recruitment of TPR to the HSP70 promoter might
function as a way to specifically promote association between
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To further the understanding of the regulation and function
of HSF1, we performed a yeast two-hybrid screen using the
HSF1 protein as a bait. Two of the interacting clones that were
obtained from this screen represented two different regions of
the TPR protein. One of the HSF1-interacting regions of TPR
identified by the yeast two-hybrid screen comprises a sequence
near the N terminus (amino acids 14 –117), whereas the other is
located close to the middle of the protein (amino acids 1218 –
1320) (Fig. 1, A and B). As an independent test of the interaction
between HSF1 and these two regions of TPR and to determine
whether the interaction is direct, in vitro binding experiments
were performed in which in vitro translated 35S-labeled TPR(14 –117) and TPR-(1218 –1320) were incubated with GSTHSF1 or GST bound to glutathione-agarose beads. The results
confirmed the ability of both regions of TPR to interact with
HSF1 (Fig. 1C).
To determine whether endogenous HSF1 and TPR proteins
interact and, if so, whether the interaction between these proteins is regulated in a stress-dependent manner, immunoprecipitation analysis was performed using extracts of cells kept at
37 °C or cells subjected to heat stress at 42 °C for 1 h. Fig. 2
shows that endogenous HSF1 and TPR did associate and that
more HSF1-TPR complex was observed in extracts of stressed
cells compared with non-stressed cells.
In multicellular eukaryotes, HSF1 binds to heat shock gene
promoters in response to stress conditions. Therefore, the data
presented in Fig. 2 indicating that HSF1 interacts with TPR in a
stress-induced manner prompted the question of whether TPR
might also associate with the promoter of the stress-inducible
HSP70 gene when cells are exposed to stress. We tested this
hypothesis using the ChIP assay. The results demonstrate that a
low level of TPR association was detected within cross-linking
distance of the HSP70 promoter in cells kept at 37 °C and that a
higher level of TPR was associated with the HSP70 promoter in
cells that were subjected to stress treatment at 42 °C for 1 h (Fig.
3A, upper panels). TPR was not found to associate with the
promoter region of the histone H4 gene, indicating the specificity of its HSP70 promoter association (Fig. 3A, lower panels).
As a complementary approach, we repeated this experiment
and used quantitative real-time PCR for the analysis. The
results (Fig. 3B) are consistent with the finding of increased
2363
1
Inp
ut
RESULTS
HSF1-TPR Interaction Facilitates Export
A
37
42
α-Tpr
Control
IgG
hsp70i
primers
Input
α-Tpr
Input
Relative Tpr association
B
FIGURE 4. TPR interacts with mRNAs transcribed from the HSP70 promoter. RNA immunoprecipitation analysis was performed to measure the
amounts of luciferase (Luc) mRNA transcribed from the HSP70 promoter versus the RSV promoter that are associated with the TPR protein. The results
were normalized to the levels of each mRNA and to control IgG samples
(which were set to a value of 1). Data are from triplicate experiments. Data are
shown as means ⫾ S.E.
3.0
2.0
1.0
0.0
37
42
37
hsp70i
42
H4
FIGURE 3. TPR associates with the HSP70 promoter in response to stress.
A, ChIP assay was performed on non-stressed (37 °C) or stressed (42 °C, 60
min) Jurkat cells using anti-TPR antibody or control IgG and PCR primers specific to the promoter region of the stress-inducible HSP70i gene (upper panels)
or the histone H4 gene (lower panel). PCR products were separated on a polyacrylamide gel and stained with ethidium bromide. B, ChIP assay was performed as described for A, but analysis was done by quantitative real-time
PCR . Data are from triplicate experiments. Data are shown as means ⫾ S.E.
NOVEMBER 23, 2007 • VOLUME 282 • NUMBER 47
TPR and the stress-induced transcripts that arise from this
gene. We tested this hypothesis using an RNA immunoprecipitation approach. In this experiment, HeLa cells were transfected with expression constructs in which the luciferase gene is
transcribed from either the stress-inducible human HSP70
gene promoter or the RSV promoter. The transfected cells were
subjected to 1 h of heat shock treatment (42 °C), after which
they were incubated with the chemical cross-linking agent
paraformaldehyde, and extracts of these cells were then immunoprecipitated using anti-TPR antibody. RNA isolated from the
TPR-containing complexes was reverse-transcribed into
cDNAs, which were then analyzed by quantitative real-time
PCR using a luciferase primer pair. The results of this experiment (Fig. 4) indicate that significantly more luciferase mRNA
transcripts generated from the HSP70 promoter were associated with the TPR protein compared with luciferase mRNAs
transcribed from the RSV promoter.
The results described above indicate that stress conditions
result in increased interaction between HSF1 and TPR,
increased association of TPR with the HSP70 promoter, and the
preferential association of TPR with mRNAs arising from transcription from the HSP70 promoter. In light of the data suggesting a role for TPR in mRNA export (13, 18, 19), we hypothesized
that these events could be part of a mechanism for specifically
enhancing the export of mRNAs transcribed from heat shock
gene promoters. To test this hypothesis, we sought to deterJOURNAL OF BIOLOGICAL CHEMISTRY
33905
Downloaded from www.jbc.org by on January 14, 2008
H4
primers
Control
IgG
GF
P
(14 -Tp
-11 r
GF 7)
(12 P-Tp
18 r
-13
20
)
GF
P
HSF1-TPR Interaction Facilitates Export
A
-GFP-Tpr segment
-GFP
-β-actin
Inp
ut
α-H
SF
1
IgG
Inp
ut
α-H
SF
1
IgG
GFP-Tpr
(1218-1320)
GFP-Tpr
(14-117)
GFP
Inp
ut
α-H
SF
1
IgG
B
-Tpr
C
GFP
N
GFP-Tpr
(1218-1320)
N Probe
C
Luc
hsp70-Luc
L32
Luc
RSV-Luc
D
Relative hsp70 mRNA
(Relative to control (=1))
L32
C
N
GFP-Tpr
(14-117)
C
N
GFP-Tpr
(1218-1320)
FIGURE 5. Expression of HSF1-interacting regions of TPR inhibits
export of endogenous HSP70 mRNA and a reporter transcript
expressed from the HSP70 promoter. A, HeLa cells were transfected
with GFP-TPR-(14 –117), GFP-TPR-(1218 –1320), or GFP alone and then
subjected to Western blot analysis using anti-GFP antibody. B, HeLa cells
transfected with GFP-TPR-(14 –117), GFP-TPR-(1218 –1320), or GFP alone
were subjected to heat treatment at 42 °C for 60 min, and HSF1 immunoprecipitates from extracts of these cells were then subjected to anti-TPR
Western blotting. C, HeLa cells were cotransfected with the GFP-TPR-(14 –
117), GFP-TPR-(1218 –1320), or GFP-alone construct along with either a
HSP70 promoter-driven (upper panels) or RSV promoter-driven (lower panels) reporter plasmid and subjected to heat shock treatment at 42 °C for 60
min, and mRNA from the cytoplasmic (C) and nuclear (N) fractions of these
transfected cells was then analyzed by RNase protection assay using a
probe that detects luciferase (Luc) mRNA or L32 ribosomal protein mRNA
(control). D, HeLa cells were transfected with the GFP-TPR-(14 –117), GFPTPR-(1218 –1320), or GFP-alone construct and subjected to heat shock
treatment at 42 °C for 60 min, and mRNA from the cytoplasmic and nuclear
fractions of the transfected cells was then reverse-transcribed into cDNA
and analyzed by quantitative real-time PCR using primers that amplify a
segment of the HSP70i nucleotide sequence or the L32 ribosomal protein
33906 JOURNAL OF BIOLOGICAL CHEMISTRY
nucleotide sequence (normalizing control). The results of this experiment
are presented as the relative levels of endogenous HSP70 mRNA in the
nucleus or cytoplasm, normalized for L32 mRNA levels, compared with the
results for the control cells (transfected with GFP alone), which were set to
a value of 1. Data are from duplicate experiments. Data are shown as
means ⫾ S.E.
VOLUME 282 • NUMBER 47 • NOVEMBER 23, 2007
Downloaded from www.jbc.org by on January 14, 2008
C
GFP-Tpr
(14-117)
N
C
mine whether export of the HSP70 promoter-driven luciferase
mRNAs described above is affected by cotransfecting the cells
with expression constructs encoding the two regions of the
TPR protein (amino acids 14 –117 and 1218 –1320) that were
shown by our data to interact with HSF1 (Fig. 1). We reasoned
that if HSF1-TPR interaction is important for export of mRNAs
expressed from HSP gene promoters, then expressing either of
these two HSF1-binding regions of TPR could inhibit export of
these mRNAs expressed from HSP gene promoters by decreasing the ability of HSF1 and TPR to associate. Fig. 5A shows that
both GFP fusion constructs were expressed at levels similar to
that of GFP alone. Fluorescence microscopy analysis of cells
transfected with these constructs revealed that a significant
proportion of each GFP-TPR fragment fusion construct was
found in the nuclei of these cells, where they would need to be to
exert their effects in this experiment (supplemental Fig. S1).
Co-immunoprecipitation analysis confirmed that transfection
of the GFP-TPR-(14 –117) and GFP-TPR-(1218 –1320) constructs, but not the GFP expression construct, resulted in
decreased levels of the HSF1-TPR complex in stressed cells
(Fig. 5B).
Next, cells were cotransfected with the GFP-TPR-(14 –117),
GFP-TPR-(1218 –1320), or GFP-alone construct along with the
construct containing luciferase expressed from either the
HSP70 promoter or the RSV promoter (non-heat shock element-containing) and subjected to heat shock treatment at
42 °C for 60 min, and mRNA from the cytoplasmic and nuclear
fractions of these cells was then analyzed by RNase protection
assay using a probe that detects luciferase mRNA or mRNA
encoding the L32 ribosomal protein (as a control). The results
indicate that cells transfected with GFP-TPR-(14 –117) or GFPTPR-(1218 –1320) exhibited a decrease in the cytoplasmic levels of luciferase mRNA expressed from the heat shock elementcontaining HSP70 promoter compared with cells transfected
with GFP (Fig. 5C, upper panels). Transfection of GFP-TPR(14 –117) or GFP-TPR-(1218 –1320) did not change the
nuclear versus cytoplasmic levels of luciferase mRNA expressed
from the non-heat shock element-containing RSV promoter
compared with transfection of GFP alone, indicating the HSP70
promoter selectivity of the effect (Fig. 5C, lower panels).
Finally, to test whether expression of these two regions of the
TPR protein is able to inhibit export of endogenous HSP70i
mRNAs, we performed an experiment similar to the one shown
in Fig. 5D, except that the cells were transfected with only the
GFP-TPR-(14 –117), GFP-TPR-(1218 –1320), or GFP expression construct (no luciferase reporter constructs). After subjecting the transfected cells to heat shock treatment at 42 °C for
60 min, mRNA from the cytoplasmic and nuclear fractions of
these cells was reverse-transcribed and then analyzed by quantitative real-time PCR using primers that amplify the HSP70i
sequence or the L32 sequence (normalizing control). The
results of this experiment (Fig. 5D) are presented graphically as
HSF1-TPR Interaction Facilitates Export
the relative levels of endogenous HSP70 mRNA in the nucleus
or cytoplasm, normalized for L32 mRNA levels, compared with
the results for the control cells (transfected with GFP alone),
which were set to a value of 1. The results indicate that the
export of endogenous HSP70i mRNA, like that of the heat
shock element-driven luciferase mRNAs above, was
decreased in cells transfected with GFP-TPR-(14 –117) or
GFP-TPR-(1218 –1320) compared with cells transfected
with GFP (Fig. 5D).
DISCUSSION
Acknowledgments—We are grateful to Dan Noonan and Brian Finlin
for providing plasmid constructs and to other members of the laboratory for insightful discussions.
NOVEMBER 23, 2007 • VOLUME 282 • NUMBER 47
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The results presented in this work indicate that, in response
to stress, the TPR protein interacts with the stress gene transcriptional regulator HSF1, is recruited to the HSP70 promoter
region, and preferentially associates with mRNAs transcribed
from this promoter compared with those expressed from a
non-stress-induced promoter and that the HSF1-TPR interaction is required for efficient export of HSP mRNAs from the
nucleus during stress. The association of TPR with these
mRNAs may be assisted by its interaction with mRNA-binding
heterogeneous nuclear ribonucleoproteins such as Nab2p (19).
The TPR protein is known to be able to form homodimers (25).
Thus, once it is complexed with HSP mRNAs, TPR could facilitate their export by docking with TPR found at the nucleoplasmic face of nuclear pore complexes (10 –17).
These results reveal the existence of a direct functional connection between the first and last nuclear steps in the gene
expression pathway, transcription and export of mRNAs from
the nucleus. The HSF1-TPR interaction and its downstream
events could serve as a mechanism for bypassing the inhibition
of mRNA export that occurs in response to stress and/or to
increase the kinetics of export of HSP mRNAs so that cells can
express these crucial cytoprotective proteins as soon as possible. One intriguing question for future studies is whether the
TPR protein is recruited to other genes to aid in the export of
their mRNAs from the nucleus.
REFERENCES