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INTRODUCTION |
Mammalian cells respond to a variety of stress conditions with
elevated synthesis of a remarkably similar set of proteins, called the
stress or heat shock proteins
(Hsp)1 (1, 2). Among these,
members of the ATP-binding 70-kDa Hsp family are one of the most highly
conserved proteins known in biology with greater than 50% overall
amino acid sequence homology between Escherichia coli and
man (3, 4). A large body of evidence has emerged during the past decade
which indicates that members of the 70-kDa Hsps may mediate crucial
cellular functions under non-stress conditions, such as the clathrin
uncoating in mammalian cells (5), post-translational translocation of
proteins to the endoplasmic reticulum lumen (6), selective import of proteins into lysosomes (7), or folding assistance to nascent polypeptides during translation (8-10). As well, the E. coli 70-kDa Hsp homologue, DnaK, was demonstrated to mediate a DNA
helicase function in
phage replication in association with
additional protein components (11).
A unique subfamily of mammalian Hsp70 proteins comprises large stress
response proteins, members of the Hsp110/SSE subfamily. The mammalian
Hsp110 protein has been cloned from Chinese hamster ovary cells and
characterized in detail in our laboratory, and a number of unique
features that distinguish this diverged member from the DnaK-Hsp70
family have been identified (12-13). One of the most prominent
structural elements present in Hsp110 and related proteins is a
100-amino acid-long
-helical loop found between the peptide-binding
domain and the C-terminal
-helical region (12). Hsp110 has been
shown to protect heat-denatured proteins from aggregation as well as to
confer cellular thermoresistance in vivo (13).
It is widely believed that the primary function of the four major
groups of heat shock proteins (Hsp27, Hsp60, Hsp70, and Hsp90) is to
assist protein folding in vivo. Functions include the
initial folding of newly synthesized proteins to refolding of proteins
damaged by environmental stress. A common theme in this molecular
chaperoning function is the transient binding or association of Hsp
proteins with partially folded or misfolded peptide stretches of
proteins (14). In case of Hsp70, binding of ATP by the N-terminal
domain facilitates association of the peptide substrate, whereas ATPase
activity of the chaperone is required for substrate release (15).
Little is known, however, if molecular targets other than proteins
exist that interact with members of the Hsp70 protein family and
additional molecular chaperones. Several lines of evidence suggest
that, in the cell, these proteins also recognize and bind macromolecules other than proteins. For example, it has recently been
demonstrated that GroEL, the E. coli Hsp60 homologue, can associate with lipid membranes while remaining functional as a protein
folding chaperone (16). This mechanism has been proposed to be a
membrane-protective component during heat stress (16). Equally
important, heat shock proteins have been recently associated with
various aspects of RNA metabolism. GroEL has been suggested to be part
of a protein complex that protects bacterial transcripts from RNase
E-mediated degradation (17, 18). Intriguingly, Miczak and co-workers
(19) demonstrated that DnaK and in some instances GroEL co-purify with
the bacterial degradosome, although no functional implications have
been proposed in mRNA metabolism. The 60-kDa chaperonin of the
thermophilic archaeon, Sulfolobus solfataricus has been
identified as an RNA-binding protein with specific interaction with the
16 S rRNA (20). Additionally, an in vitro reconstituted
endonuclease activity is associated with this chaperonin, which cleaves
pre-rRNA at a specific 5' site (20). Earlier studies on heat response
regulation in Drosophila demonstrated that expression of the
major heat-induced protein, Hsp70, is self-regulated at both the
transcriptional and post-transcriptional levels (21). Binding of Hsp70
to its own mRNA has been proposed to explain the rapid alterations
in mRNA stability observed in the cytoplasm (21). Finally, a
previously described 102-kDa RNA-binding protein, conserved throughout
higher plants, has recently been identified as being identical with
Hsp101 (22). Through direct binding and complex formation with the 5'
leader sequence of tobacco mosaic viral RNA, Hsp101 acts as a
translational enhancer, a function that can be recapitulated in yeast
and that is independent of its role in conferring thermoresistance
(22). Each of these studies independently implicates the involvement of
various heat shock proteins in regulatory mechanisms of RNA metabolism
through their direct or indirect association with RNA.
Our previous label transfer studies using cytoplasmic lysates of human
lymphocytes demonstrated that a 70-kDa complex was invariably detected
with AU-rich 3'-UTR RNA sequences of various lymphokine and
proto-oncogene mRNAs (see e.g. in Ref. 23). In addition,
during attempts of expression and purification of AU-rich RNA-binding
fusion proteins in E. coli, an intense 70-kDa activity was
co-purified with these proteins. Based on these results, we tested
AU-rich RNA binding capacity of mammalian Hsp/Hsc70 proteins. In this
study we report direct RNA binding of mammalian Hsp70 and Hsp110
molecules by their N-terminal ATP-binding region. We demonstrate that
these proteins preferentially bind AU-rich 3'-UTR sequences and that
this interaction can be regulated by ATP at physiological
concentrations. We also show that engagement of the peptide-binding
domain by a peptide substrate abrogates RNA binding by the N terminus
of the protein. Additionally, RNA binding activity and sequence
specificity can be modified by deletion or rearrangement of C-terminal
domain structures. Finally, Hsp70 proteins can be immunoprecipitated
from protein-RNA complexes formed between cytoplasmic proteins of human
lymphocytes and AU-rich RNA probes. These results suggest that binding
of Hsp70 family members to 3'-UTR ARE sequences may be mechanistically
important in the cytoplasmic metabolism of lymphokine and other
short-lived mRNAs.
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EXPERIMENTAL PROCEDURES |
Reagents--
RPMI and Dulbecco's modified Eagle's tissue
culture media, protein A-Sepharose, bovine serum albumin, reduced
carboxymethylated lactalbumin (RCMLA), and homoribopolymers (poly(A),
poly(C), poly(G), and poly(U)) were purchased from Sigma. Fetal calf
serum was from Protein GMK., Hungary. Recombinant Hsp proteins and
monoclonal antibody against human Hsp70 were from StressGen
Biotechnologies. [32P]UTP (3000 Ci/mmol) was purchased
from Izotóp Kft., Hungary, and from NEN Life Science Products.
Unlabeled nucleotides were from Roche Molecular Biochemicals, Germany.
Generation of Recombinant Hsp110 Proteins--
Hsp110, a
diverged member of the Hsp70 stress protein family, was cloned and
identified earlier in our laboratory (12). Both the wild type and
targeted deletion mutants of Hsp110 were expressed in E. coli as His-tagged proteins as described elsewhere (24).
Cell Culture and Cytoplasmic Lysate Preparation--
Peripheral
blood mononuclear cells were separated from whole blood of healthy
volunteers on a discontinuous Ficoll-Paque gradient (Amersham Pharmacia
Biotech, Austria) and cultured immediately in RPMI 1640 medium
supplemented with 10% fetal calf serum. In case of activation, PHA was
added to cultures at a final concentration of 1 µg/ml for 12 h.
HeLa cells were maintained in monolayer cultures in Dulbecco's
modified Eagle's medium supplemented with 8% fetal calf serum.
Cytoplasmic fractions were prepared as described elsewhere (25) with
minor modifications. Briefly, 2-3 × 107 cells were
washed in ice-cold serum-free medium, and pellets were resuspended in
buffer A containing 10 mM PIPES, pH 6.8, 100 mM
KCl, 2.5 mM MgCl2, 300 mM sucrose,
1 mM phenylmethylsulfonyl fluoride and lysed on ice for 3 min with the addition of 1% Triton X-100. Following centrifugation for
3 min at 900 × g, supernatants were collected and
frozen immediately until use.
In Vitro RNA Transcription and Label Transfer Studies--
The
350-nucleotide-long IFN-
3'-UTR and 270-nucleotide-long IL-2 3'-UTR
RNAs were transcribed from linearized plasmids containing the 3'-UTR
AU-rich instability determinant sequences (ARE) of IFN-
and IL-2
lymphokine mRNAs as described elsewhere (26, 27). The plasmid
pRK5-myc containing the 400-nucleotide-long c-myc 3'-UTR was
a gift of Dr. John Hesketh, and the probe RNA was generated as
described elsewhere (28). The pT7/T3
-19-IL-10hu plasmid containing
the full-length IL-10 cDNA (ATCC, 68191) was constructed by
inserting into the BamHI site of pT7/T3
-19 vector the
IL-10 cDNA. Full-length IL-10 RNA (IL-10) or IL-10 RNA lacking the
3'-UTR (IL-10 (
3'-UTR)) was transcribed from the pT7/T3
-19-IL-10hu plasmid linearized with SacI or SspI, yielding a
respective 1580- and 875-nucleotide-long transcript. The
90-nucleotide-long
2R1 and
2H3 probes were generated from the
same DNA template except that
2H3 was transcribed in the antisense
orientation yielding a 5× UAAAU repeat sequence, a corresponding sense
sequence of which (AUUUA motif in a 5× tandem repeat) is found within
the 3'-UTR of granulocyte-macrophage-colony-stimulating factor mRNA (29). All transcription reactions were performed in the presence of
[32P]UTP. Sequences of the "AUUUA"-containing ARE
found in these probes are given in Fig. 1. 105 cpm RNA
probes with specific activity of ~2-5 × 108
cpm/µg (~10 fmol) were incubated with 0.5 µg of recombinant
proteins or 5 µg of total lysate proteins in 12 mM HEPES,
pH 7.9, 15 mM KCl, 0.2 mM dithiothreitol, 0.2 µg/ml yeast tRNA, and 10% glycerol at 30 °C for 10 min. In
experiments where the effects of ATP, homoribopolymers, or peptide
substrates were tested, these agents were added to the reaction
simultaneously with the RNA probe. In some experiments, the amount of
trace magnesium was increased to 1 mM which did not have a
noticeable effect on the extent of RNA binding. Protein-RNA complexes
were covalently fixed with ultraviolet light (3000 microwatts/cm2, 5 min, UV Stratalinker model 1800, Stratagene) on ice and were subsequently RNase-treated (7.5 units of
RNase T1 and 15 µg of RNase A/sample) for 15 min at 37 °C. Samples
were then separated on 12.5% SDS-PAGE under reducing conditions, and
gels were analyzed by autoradiography or PhosphorImaging (Molecular Dynamics).
Immunoprecipitation--
Protein A-Sepharose was conjugated with
either a monoclonal antibody against human Hsp70 or control antibody
(P3, parent hybridoma supernatant). Beads were washed with buffer IP
(10 mM Tris-HCl, pH 7.4, 100 mM NaCl, 2.5 mM MgCl2, 0.5% Triton X-100, 1 mM
PMSF, and 1 µg/ml pepstatin A). 10 µg of total cytoplasmic lysate
proteins were incubated with 3 × 105 cpm of
32P-labeled RNA probes, UV cross-linked, and digested with
RNases as described above. Immunoprecipitation of RNA-protein complexes was performed in buffer IP at 4 °C by incubating the reaction mixture with the prepared beads for 45 min under gentle agitation. Beads were then pelleted, and the supernatants were collected (depleted
fractions). After washing extensively with buffer IP, beads and
depleted fractions were analyzed by 12.5% SDS-PAGE and subsequent autoradiography.
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RESULTS |
Hsp70 and Hsp110 Binds AU-rich RNA in Vitro--
Two independent
lines of experimental observations turned our attention to examine
in vitro RNA binding by Hsp70 proteins. First, during
examination of RNA binding properties of various deletion mutants of
AU-rich RNA-binding fusion proteins, an intense 70-kDa RNA binding
activity was co-purifying with these proteins from bacterial extracts
(data not shown). Second, our earlier label transfer studies showed
that, among others, a 70-kDa complex was frequently detected using
cytoplasmic lysates of human lymphocytes and other cell types with
AU-rich 3'-UTR RNA sequences of various lymphokine and proto-oncogene
mRNAs (see e.g. in Ref. 23).
Therefore, to test the possibility that mammalian Hsp70 proteins are
capable of binding to AU-rich RNA in vitro, recombinant Hsp70 was analyzed in a label transfer assay using
[32P]UTP-labeled 3'-UTR RNA probes derived from various
lymphokine as well as c-myc mRNAs. Fig.
1 illustrates the ARE sequences of the
RNA probes used in this analysis. Fig.
2A suggests that Hsp70 binds
to these various AU-rich RNA sequences with a comparable intensity
(1st 3 lanes). RNA binding is restricted to the
3'-UTR and is not likely to involve open reading frame sequences as
Hsp70 binds to the full-length IL-10 probe but not to IL-10 RNA without the 3'-UTR (4th and 5th lanes). To
ensure that Hsp binding required the presence of the AUUUA motifs and
not the AU context per se,
2R1, which encodes the
90-nucleotide-long ARE of granulocyte-macrophage-colony-stimulating factor with five reiterated AUUUA pentamers and
2H3, its antisense sequence with five "UAAAU" motifs were compared. Whereas
2R1 displayed comparable Hsp70 binding relative to the other ARE probes,
2H3 was similar to tRNA in that they are not recognized by Hsp70 (lanes 6-8). These data strongly suggest that, in
vitro, Hsp70 preferentially binds AU-rich 3'-UTR sequences common
to many unstable mRNAs.

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Fig. 1.
Sequences of the 3'-UTR AU-rich instability
determinant elements of the various RNA probes used in this study.
Characteristic AUUUA pentamers are in boldface. Linearized
plasmid templates harboring these ARE sequences were used to generate
[32P]UTP-labeled RNA probes in in vitro
transcription reactions. Length of each probe RNA is indicated under
"Experimental Procedures."
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Fig. 2.
AU-rich RNA binding by Hsp70 and Hsp110
proteins. A, recombinant human Hsp70 protein was
analyzed in a label transfer assay using various AU-rich RNA probes
(see Fig. 1). 0.5 µg of protein was incubated with
[32P]UTP-labeled RNA, exposed to monochromatic UV light,
and digested with RNase A and T1. RNA-protein complexes were analyzed
by 12.5% SDS-PAGE and autoradiography. 3'-UTR-less IL-10 RNA, 2H3,
and tRNA were used as negative controls. B, equal amounts
(0.5 µg) of recombinant human Hsp70 and hamster Hsp110 proteins were
analyzed in a label transfer assay, using IL-2 3'-UTR RNA probe
(indicated at the top) as described above.
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Since Hsp110 is a unique and recently characterized member of the Hsp70
superfamily, its ability to bind RNA was also examined. By sequence
analysis and molecular graphics, Hsp110 has been shown to contain the
primary elements common to other members of the Hsp70 family of
proteins (12, 13). Fig. 2B demonstrates that, like Hsp70,
recombinant Hsp110 also binds the AU-rich 3'-UTR RNA sequences of the
IL-2 mRNA but not tRNA (data not shown). This suggests that RNA
binding might be associated with a region that is common to and
homologous in the two proteins. However, it is also seen that despite
comparable molar ratios of protein and RNA in the reaction, the binding
activity of Hsp110 is notably weaker than that of Hsp70. This
represents a qualitative difference between members of these two
related, but distinct, stress protein families.
ATP Modulates in Vitro RNA Binding by Hsp/Hsc70 and
Hsp110--
Previous studies have shown that the di-nucleotide-binding
domain (also called the Rossmann fold) of glyceraldehyde-3-phosphate dehydrogenase represents an RNA-binding motif and that NAD+
or NADH as well as ATP were effective in modulating in vitro RNA binding by this metabolic enzyme (26).2foot;f2;10>
Since ATP is also an important modulator of Hsp70 function through its
association with the N-terminal ATP-binding motif in this protein, we
examined the possibility whether ATP had a modulatory effect on AU-rich
RNA binding by Hsps. Fig. 3 illustrates
the result of this study. It is seen that RNA binding by Hsp70, Hsc70,
and Hsp110 proteins was substantially reduced by ATP in the 0.5-5
mM range. The autoradiogram shown for Hsp110 required an
approximately 4× exposure to provide a signal intensity comparable to
those of Hsp70 and Hsc70. It is also seen that the half-maximal RNA
binding by all three proteins was observed within the physiological ATP
concentration range (~1 mM, Fig. 3B). The presence of Mg2+ in the RNA-binding reaction did not have
any noticeable effect on the ATP modulation of Hsp RNA binding (data
not shown). These results indicate that ATP is a common and potent
modulator of RNA binding by both Hsp/Hsc70 and Hsp110 proteins and that
the ATP-binding domain common to these proteins may also be involved in
RNA binding.

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Fig. 3.
Inhibition of RNA binding activity of Hsp70,
Hsc70, and Hsp110 proteins by ATP. A, 0.5 µg of total
proteins was analyzed for IL-2 3'-UTR RNA binding in the presence of
various concentrations of ATP as indicated at the top of the
panel. Following UV-cross-linking and RNase digestion, complexes
were analyzed by SDS-PAGE and PhosphorImaging. B,
PhosphorImage values for each band were expressed and plotted as
percent of controls, where no ATP was present in the RNA-binding
reaction.
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Binding of RNA to Targeted Deletion Mutants of Hsp110--
To show
a direct involvement of the ATP-binding domain in RNA binding, deletion
mutants of Hsp110 that contain different predicted functional domains
were assessed for RNA binding in label transfer assays. The regions
indicated have been determined by comparative sequence alignment and
molecular graphics using DnaK as a model (12, 31). Briefly, Hsp110
contains basic ATP-binding sequences (residues 0-375), a predicted
peptide-binding domain (residues 375-508), a loop domain (residues
508-608), and a predicted series of
-helices making up its C
terminus (residues 608-858). The domain composition of wild type
Hsp110 (WT) and mutants 1-5 as well as of Hsp70 are
presented schematically in Fig.
4A. These mutants and their
ability to exhibit chaperoning functions have been described elsewhere
(24). The RNA binding experiments revealed that the N-terminal
375-amino acid-long ATP-binding domain is required for RNA binding
based on the observation that deletion mutants 1 and 2, which lack the
ATP-binding domain, showed no RNA binding activity (Fig.
4C). Interestingly, these experiments also demonstrated that
removal of the 100-amino acid-long
-loop structure (mutant 4) or the
C-terminal
-helical domain (mutant 5) of Hsp110 further enhanced its
ability to bind RNA, by 5.8- and 3.8-fold, respectively (Fig.
4C), i.e. making it more comparable in RNA
binding to that seen for Hsp70 in Fig. 2C. RNA binding activity by deletion mutant 3, in which both of these distal elements were deleted, was only marginally increased (by 1.3-fold) relative to
that of wild type Hsp110 (Fig. 4C). These data,
nevertheless, further illustrate direct RNA binding by these heat shock
proteins.

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Fig. 4.
A, schematic illustration of domain
structure of wild type (wt) Hsp110 and its various deletion
mutants. Amino acid positions of each structural domain are given at
the top of the panel. A unique structural feature
of Hsp110 is a 100-amino acid-long (amino acids 508-608) -loop
structure, not present in any other members of the Hsp70 family. Domain
structure of Hsp70 and proportional length of each domain relative to
Hsp110 is given at the bottom of the panel.
R indicates the most C-terminal regulatory domain of Hsp70.
B, Coomassie Brillant Blue-stained gel of the label transfer
assay shown in C illustrates size differences of wild type
(lane 1) and deletion mutants 1-5 (2nd to 6th
lanes, respectively). This gel served as a loading control
for the evaluation of relative RNA binding intensities of each protein.
C, wild type (1st lane) and all Hsp110
deletion mutants (del 1-5 in 2nd to 6th
lanes, respectively) were analyzed for RNA binding in a
label transfer assay using the IL-2 3'-UTR RNA probe. Relative
intensities of RNA binding measured by densitometric analysis of each
bands are shown below the corresponding lanes and expressed
relative to that of the wild type which was chosen 1. Asterisks indicate the positions of deletion mutants
#1 and #2 as verified from the alignment of the
autoradiogram with the Coomassie-stained gel.
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To characterize further binding of Hsp110 deletion mutant proteins to
AU-rich RNA, we also examined the effect of ATP on this parameter.
Similar to that of wild type Hsp110, ATP was capable of diminishing
in vitro RNA binding by all three mutants (del 3-5, Fig. 5). Similar to the
studies on wild type Hsp110 and Hsp70, an ATP concentration of 1 mM largely abrogated RNA binding of all proteins which
demonstrates that regardless of the presence of downstream C-terminal
portions of the molecule, RNA binding by the N-terminal domain is still
sensitive to and regulated by ATP.

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Fig. 5.
Effect of ATP on in vitro
RNA binding by wild type and mutant Hsp110 proteins. 0.5 µg total of each protein was analyzed in a label transfer experiment
using IL-2 3'-UTR RNA probe in the presence of ATP as indicated at the
top of the panel. RNA-protein complexes were
analyzed using SDS-PAGE and autoradiography.
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We also examined the competition of homoribopolymers on RNA binding by
Hsp110 and its deletion mutants and found that AU-rich RNA binding by
all three mutant proteins, but not that by the wild type Hsp110, was
markedly diminished by poly(U) (data not shown). Whereas no effect of
poly(A) or poly(C) was evident on RNA binding by either proteins,
interestingly, poly(G) was a strong competitor of AU-rich RNA binding
by both the wild type and truncated proteins (data not shown) (also see
"Discussion").
Peptide Substrate Interferes with RNA Binding by Hsp70 and Hsp110
Proteins--
The most noticeable function of Hsps under physiological
conditions is the recognition and binding of misfolded or partially unfolded regions of polypeptide chains. Since the N-terminal
ATP-binding domain and the C-terminal peptide-binding region have been
suggested to interact through the transmission of conformational
changes (15, 31-35), we assessed whether peptide binding is capable of modulating or interfering with RNA binding by Hsps in vitro.
For this purpose, we introduced reduced carboxymethylated lactalbumin (RCMLA) into the binding reaction. This fully extended, non-native conformational homologue of
-lactalbumin has been shown to be an
effective peptide substrate of Hsp/Hsc70 (36). Fig.
6 illustrates that even at a 1:1 molar
ratio for Hsp70 or a 1:6 molar ratio for Hsp110 (0.1 µg of RCMLA),
peptide substrate reduced RNA binding by ~20-25%. RCMLA, when
examined in a similar assay, did not bind RNA (data not shown). Peptide
substrate binding by these Hsps is not affected by the buffer used in
the RNA binding assay as prevention of thermal aggregation of
luciferase by both Hsp70 and Hsp110 proteins is comparable in this
buffer to that obtained in the usual aggregation buffer (data not
shown). These experiments indicated that peptide binding by either
Hsp70 or Hsp110 chaperones interferes with their RNA binding.

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Fig. 6.
Peptide substrate binding modifies in
vitro RNA binding activity of Hsp70 and Hsp110
proteins. RCMLA was added to the label transfer reaction at the
indicated concentrations, in which 0.5 µg of Hsp70 or Hsp110 proteins
were analyzed for binding to IL-2 3'-UTR RNA probe. RNA-protein
complexes were analyzed by SDS-PAGE and autoradiography.
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Immunoprecipitation of AU-rich RNA-Protein Complexes by Anti-Hsp70
Antibody--
Many regulatory steps in the metabolism of unstable
mRNAs have been implicated to occur via interaction of ARE with
various protein factors (AUBP) (see e.g. Refs. 37 and 38).
Formation and/or dissociation of specific ARE·AUBP complexes might be
mediated by additional components as shown with certain cytoskeletal
elements (39). Because of the demonstrated affinity of members of the Hsp70 family of proteins to ARE and to answer the question as to
whether Hsp70 proteins might potentially be part of an ARE·AUBP complex, we used a direct immunoprecipitation approach. As previously demonstrated, RNA binding activity by many AUBPs is markedly
up-regulated upon activation of lymphocytes by a variety of stimuli
(see e.g. Ref. 40). Therefore, cytoplasmic extracts of both
resting and PHA-activated human lymphocytes as well as HeLa cells were
prepared as described under "Experimental Procedures," and equal
amounts of total proteins were incubated with IFN-
mRNA 3'-UTR
probe. RNA-protein complexes were UV cross-linked and exposed to RNase treatment. Prior to SDS-PAGE, reactions were immunoprecipitated using
monoclonal antibodies against human Hsp70. Fig.
7 illustrates a result of a typical
experiment and shows that a 70-kDa complex immunoprecipitates from all
three label transfer reactions. Fig. 7 also shows that, in accordance
with our previous findings, it is the activity of a 36-kDa complex that
is most notably increased upon lymphocyte activation. No radioactive
signal could be detected in the precipitated fraction in any of
multiple immunoprecipitation assays using P3 supernatant as a negative
control. Occasionally we observed additional, less intense bands in the
precipitated fractions. Although we think these represent degradation
products of the complex which still retain some of the labeled RNA
probe, the possibility that they arise from other AUBPs entrapped in Hsp·RNA complexes and released during denaturation cannot be ruled out. Nevertheless, these experiments demonstrate that AU-rich 3'-UTR
RNA probes are recognized and bound in vitro by endogenous Hsp70, i.e. proteins derived from different mammalian cell
types. Thus, these results indicate that the formation of Hsp70·ARE
complexes may also occur in the cell.

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Fig. 7.
Immunoprecipitation of RNA-protein complexes
by anti-Hsp70 antibody. Equal amounts of total cytoplasmic
proteins of HeLa and resting (rest) or PHA-activated
(act) human lymphocytes were incubated with radiolabeled
IFN- 3'-UTR RNA probe as described. Reactions were subsequently
immunoprecipitated as described under "Experimental Procedures,"
and proportional aliquots of immunodepleted and precipitated fractions
were analyzed by 12.5% SDS-PAGE and autoradiography.
Arrowhead indicates the position of a 70-kDa complex that
was specifically immunoprecipitated from each cell fraction.
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DISCUSSION |
The 70-kDa heat shock protein family has received considerable
attention in the past decade. Recognition of members of the Hsp70
family as molecular constituents in a wide range of important cellular
mechanisms (reviewed in Refs. 2 and 41) has long emphasized the
universal importance of these proteins in cell physiology. In this
report we extend these findings by providing evidence for direct
in vitro RNA binding by members of the mammalian Hsp70
protein family, a feature that has not previously been described. We
demonstrate that both the stress-inducible (Hsp70) and cognate (Hsc70)
forms as well as Hsp110, the distantly related member of the mammalian
Hsp70 family, bind AU-rich 3'-UTR RNA sequences that function as
instability determinants of various lymphokine and proto-oncogene
mRNAs. By using a series of deletion mutants of Hsp110, we were
able to localize RNA binding to the N-terminal ATP-binding domain. This
finding was strengthened by the capability of ATP to reduce RNA binding
in vitro at physiological ATP concentrations (1-2
mM). The ATP concentrations required for noticeable
competition with RNA binding is considerably higher than the
Km value reported for Hsp70 and related proteins
with chaperone ATPase activity (Km
~10
6 M, Ref. 31). This suggests that RNA
binding by the N terminus of the chaperone molecule involves other,
presumably larger regions within the ATP-binding domain than those
responsible for the binding of ATP. This view is also consistent with
our previous finding that actin, whose structure and folding topology
are nearly identical to that of the ATPase fragment of Hsc70 (32), does
not bind RNA (39).
We also concluded that the C-terminal regions of Hsp110 can influence
the interaction with RNA based on the following observations. First,
removal (mutants 3 and 5) or rearrangement (mutant 4) of the C-terminal
portion of Hsp110 considerably increased the RNA binding activity of
the protein. ATP sensitivity of RNA binding by these mutant proteins
was retained, suggesting that the ATP-binding domain is involved in a
way similar to that of the wild type Hsp110. Second, engagement of the
C-terminal peptide-binding domain by a substrate paralleled with
considerable decrease in RNA binding activity (Fig. 6). Although in
light of the present results one can only speculate how RNA binding is
influenced by C-terminal structures, these data appear to be consistent
with the view that intramolecular interactions between the ATP-binding
and peptide-binding domains trigger conformational rearrangements
within the chaperone molecule. Such conformational changes have been
suggested to elicit the transduction of important information in
chaperoning function (31-35). The functional role of the 100-amino
acid-long
-loop structure unique to Hsp110 is not known. It is
mechanistically possible that this unit may serve as a `connector'
between the large N-terminal region harboring both the ATP- and
peptide-binding domains and the C-terminal lid structure. This putative
function would allow flexible communication of the C terminus with the domains that mediate ATP and peptide binding. In deletion mutants, this
putative interaction may be impaired due to the lack of the loop
(mutant 4), the C terminus (mutant 5), or both (mutant 3). Therefore,
the ATP-binding domain, responsible for RNA binding, may become simply
more exposed or it may take up a different conformation favored by RNA
recognition and binding.
We observed that in all cases, poly(G) appeared to be the strongest
competitor of RNA binding. It may well be that under the in
vitro conditions used, oligo(G) stretches are the structural preference for these proteins to bind to, but since such sequence is
not likely to exist in vivo, this interaction per
se may not be a major element of possible in vivo
function. The observed interaction of Hsp70 with poly(G) is in good
agreement with the earlier observations obtained with
Drosophila Hsp70 using a homoribopolymer affinity
chromatographic approach.3
The U preference, however, may be of considerable importance as long U
stretches as well as U-rich RNA sequences are featured in many
determinants of mRNA stability, including ARE (38). The question
remains, however, what is the physiological relevance of AU-rich RNA
binding by Hsps? The finding that Hsp70 can be immunoprecipitated from
a label transfer reaction of RNA-protein complexes that had been formed
between AU-rich RNA probes and cytoplasmic lysates of lymphocytes or
HeLa cells (Fig. 7) suggests that Hsp70 may potentially associate with
such (and perhaps many other) RNA entities in vivo.
One clearly relevant observation that links Hsp70 proteins to
activation-related lymphokine mRNA metabolism is work by Di et al. (43) who have established that Hsp70 translocates
into a cytoplasmic spectrin-based aggregate upon lymphocyte activation and that this association is abolished by ATP both in vitro
and in vivo (42). It is possible that the activation-induced
formation of the large cytoplasmic spectrin aggregate is in favor of
providing a preferential cytoplasmic environment where regulatory
mechanisms of mRNA stability and translation take place. Hsp70 and
likely other chaperones may be recruited into this compartment to
facilitate, through direct RNA binding, the unwinding of complex
secondary structures. This would aid in the exposure of critical
cis-acting sequences for other protein factors to bind and,
hence, allow proper and efficient assembly of polysomes or complexes
involved in mRNA degradation. Such "nucleic acid chaperone"
function had been designated to other non-chaperone proteins, such as
nucleocapsid proteins of RNA viruses (44), heteronuclear
ribonucleoprotein A1 (40, 45), and glyceraldehyde-3-phosphate
dehydrogenase (26).2
Alternatively, Hsp70 and Hsp110 may also function to modulate the
interaction of a given mRNA with regulatory proteins that would
influence mRNA stability and/or translation (49). In support of
this, recently Scandurro and co-workers (46) have suggested that Hsp70
is a potent regulator of complex formation between the 3'-UTR of
erythropoietin mRNA and its specific binding protein, erythropoietin mRNA-binding protein. Assembly of this complex is
necessary for hypoxia-induced stabilization of erythropoietin mRNA.
Under normoxic conditions, when stress-protective chaperoning activity
of Hsp70 is not operant, this protein may sequester erythropoieton mRNA-binding protein to prevent its binding to erythropoietin mRNA
and the mRNA is being degraded (46). Moreover, it has recently been
shown that evolutionarily highly conserved regions identified through
extensive data base screening overlap with AU-rich sequences with known
function in post-transcriptional regulatory mechanisms (30). Using a
modular retroviral vector, highly conserved regions of various origins
were analyzed for post-transcriptional regulatory activity under
different conditions in vivo. Results from these studies
allowed the conclusion that some highly conserved regions may function
as stress sensor elements to promote rapid adaptation of cells to
various stress conditions (30). It is, therefore, feasible to speculate
that certain Hsps might also be integral parts of such
adaptation/sensor systems at the post-transcriptional level of gene expression.
In conclusion, based on these results, we propose that members of the
Hsp70 family, including Hsp110, may directly and specifically participate in molecular events underlying lymphokine mRNA
stability and/or translation following lymphocyte activation. Hsps
may bind lymphokine mRNAs, mediating an "RNA chaperone"
function that might be required for proper folding of the RNA to expose
critical motifs involved in regulatory events during translation and/or
decay. Alternatively, Hsp70 chaperones may be required in the fine
regulation of messenger ribonucleoprotein complex formation with other
RNA-binding proteins, through the modulation of their folding state or
facilitation of their microcompartmentation. It is also possible that
direct protection of RNA and/or stabilization of RNA-protein complexes by chaperones are relevant mechanisms during various stress conditions.