(Received for publication, August 3, 1994; and in revised form, October 10, 1994)
From the
A 36-kDa protein that binds AU-rich RNA was purified from human
spleen and identified as glyceraldehyde-3-phosphate dehydrogenase
(GAPDH). GAPDH has been previously demonstrated to bind tRNA with high
affinity. Competition studies suggested that cytoplasmic GAPDH binds
the AU-rich elements (AREs) of lymphokine mRNA 3`-untranslated regions
with higher affinity than tRNA. The AUUUA-specific RNA binding activity
of GAPDH was inhibited by NAD, NADH, and ATP in a
concentration-dependent manner, suggesting that RNA binding of GAPDH
might involve the NAD
-binding region, or
dinucleotide-binding (Rossmann) fold. This hypothesis was supported by
experiments that localized RNA binding to the predicted N-terminal
6.8-kDa peptide, known to be involved in the formation of the
NAD
-binding domain. The direct demonstration of
AREspecific binding protein activity localized to the
NAD
-binding region of GAPDH supports the general
concept that enzymes containing this domain may exhibit specific RNA
binding activity and play additional roles in nucleic acid metabolism.
Finally, cytoplasmic GAPDH was found in the polysomal fraction of T
lymphocytes. Thus, the RNA binding specificity of GAPDH as well as its
localization within the cell merit its strong consideration as a
protein important in the regulation of ARE-dependent mRNA turnover and
translation in addition to its well described role in glycolysis.
Post-transcriptional regulation of gene expression has been
demonstrated to play a major role in the regulation of cell growth and
differentiation in eukaryotic cells (reviewed in (1) ). In T
lymphocytes, mRNA turnover represents an important mechanism by which
lymphokine production is modulated following
activation(2, 3, 4) . The
post-transcriptional regulation of lymphokine and proto-oncogene gene
expression has resulted in the discovery of highly conserved AU-rich
sequences in the 3`-untranslated region that function in cis as destabilizing determinants ((5, 6, 7) ; reviewed in (8) ). These
AU-rich elements (AREs) ()consist of reiterations of the
pentanucleotide AUUUA alone or oligo(U)
sequences
in an AU-rich context. AREs have been shown to serve as binding sites
for cytoplasmic and nuclear proteins that may function as trans-acting factors in regulating ARE-dependent mRNA turnover
and translation. A variety of ARE-specific binding proteins (AUBPs)
have been
described(9, 10, 11, 12, 13, 14, 15, 16, 17, 18) ,
together with the recent identification of hnRNP A1 as a cytoplasmic
AUBP in activated T lymphocytes(19) .
Despite these
findings, the molecular and cellular mechanisms by which these AUBPs
modulate the turnover of labile mRNAs remain unclear. Recent work
correlated a marked increase in the stability of provirally modified
IL-2 mRNA with the enhanced binding of hnRNP A1 to AREs in
vitro(20) . These findings suggested that hnRNP A1 may
serve multiple roles in RNA metabolism in addition to its role in RNA
processing, perhaps as a function of its location (nuclear versus cytoplasmic) in the cell(19, 21) . Indeed, many
of the proteins important in the regulation of the turnover and
translation of mRNA may serve additional and quite dissimilar roles as
demonstrated with cellular iron metabolism and the reciprocal
regulation of the iron response element-binding protein and aconitase
activity(22, 23) . In this regard, the number of
enzymes with specific RNA binding activity is steadily growing,
including glutamate dehydrogenase(24) ,
NAD-dependent isocitrate dehydrogenase (25) ,
thymidylate synthetase(26) , dihydrofolate
reductase(27) , catalase(28) , and
thiolase(15) . Thus, abundant metabolic enzymes may be involved
in or regulate many processes involved in cellular homeostasis
independent of the activity that characterized their initial
description (reviewed in (29) ).
In an attempt to identify
novel AU-rich RNA-binding proteins, a 36-kDa AUBP purified from human
spleen was identified by N-terminal microsequencing as
glyceraldehyde-3-phosphate dehydrogenase (GAPDH). GAPDH is a key
glycolytic enzyme, utilizing NAD as a coenzyme for the
oxidative phosphorylation of glyceraldehyde 3-phosphate to
1,3-diphosphoglycerate when assembled as a tetramer of identical 36-kDa
subunits. Of interest, numerous non-glycolytic activities have been
attributed to GAPDH, including nucleic acid
binding(30, 31, 32, 33, 34) ,
helicase activity(31) , DNA repair(35) , and
interaction(s) with actin cytoskeleton (36) and
microtubules(37) . Of particular pertinence is the report that
the 36-kDa subunit of GAPDH was purified from the nuclei of HeLa cells
as a tRNA-binding protein implicated in nuclear export of
tRNA(38) . In this study, we characterize the AUBP activity of
GAPDH and demonstrate that RNA binding occurs in the Rossmann fold or
the NAD
-binding region, a structural feature highly
conserved among NAD
-dependent
dehydrogenases(39) . This observation thus permits a rationale
for the RNA binding activity recently attributed to several
dehydrogenases and other enzymes.
Sequencing of the N-terminal
23 amino acids of the 36-kDa AUBP revealed identity with the N terminus
of human GAPDH. Immunoblotting with the 40.10.09 monoclonal antibody
raised against human GAPDH reacted with the purified 36-kDa protein,
but not with rabbit GAPDH (Fig. 1A). To demonstrate
that the RNA binding was mediated by GAPDH, we determined that the
anti-human GAPDH monoclonal antibody immunoprecipitated the
radiolabeled RNA-protein complex (Fig. 1B). A 25-kDa
protein with AUBP activity that was copurified with the 36-kDa
protein/GAPDH was not recognized by the anti-GAPDH antibody. Its
identity is unknown. Finally, purified 36-kDa AUBP and rabbit muscle
GAPDH bound the ARE of the IFN- 3`-UTR with similar activity (Fig. 1C). In other studies, we were able to
specifically immunoprecipitate GAPDH complexed to radiolabeled RNA by
UV cross-linking from cytoplasmic lysates of
phytohemagglutinin-activated human lymphocytes (data not shown),
indicating the activity of GAPDH as an RNA-binding protein in these
cells.
Figure 1:
Identification of the
36-kDa AU-rich RNA-binding protein as glyceraldehyde-3-phosphate
dehydrogenase. A, Western blotting. 1.5 µg of protein of
the 0.25 M NaCl/poly(U) fraction and 1.5 µg of rabbit
muscle GAPDH (Sigma) were analyzed by Western blotting using the
anti-uracil-DNA glycosylase/GAPDH antibody (40.10.09). B,
immunoprecipitation. 1.5 µg of protein of the 0.25 M NaCl/poly(U) fraction was UV-cross-linked to the
[P]UTP-labeled IFN-
3`-UTR, and the
RNA-protein complex was immunoprecipitated with control (P3) or
specific antibody (40.10.09) as described under ``Materials and
Methods.'' The depleted supernatants and the immunoprecipitated
complexes were analyzed by SDS-PAGE and autoradiographed. C,
binding of rabbit muscle GAPDH to the IFN-
3`-UTR. 0.5 µg of
commercial GAPDH or 0.5 µg of the 0.25 M NaCl/poly(U)
fraction was assayed for binding to the
[
P]UTP-labeled IFN-
3`-UTR.
Figure 2:
Specificity
of binding of GAPDH to AU-rich RNA. A, schematic
representation of the sequences of the RNA probes used in RNA binding
assays. B, binding of GAPDH to different AU-rich lymphokine
and cytokine RNAs. [P]UTP-labeled IFN-
3`-UTR, GM-CSF, and c-myc 3`-UTR RNA probes were incubated
with 0.75 µg of splenic GAPDH (0.25 M NaCl/poly(U)
fraction), UV-cross-linked, and analyzed by SDS-PAGE. C,
binding of GAPDH to separated AUUUA motifs. 0.2 µg of splenic GAPDH
was incubated with
P-labeled RNA probes containing one,
two, three, or four AUUUA pentamers as described under ``Materials
and Methods.'' (Equal molars amount of radioactive probes were
added, corrected for the number of U nucleotides.) D,
competition of lymphokine-cytokine mRNA 3`-UTRs with binding of GAPDH
to the IFN-
3`-UTR. 0.2 µg of splenic GAPDH was assayed for
binding to the [
P]UTP-labeled IFN-
3`-UTR
in the presence of the indicated unlabeled RNA sequences. 2.5 µg/ml
poly(I) was used to block nonspecific RNA
binding.
The specificity of the GAPDH-ARE interaction was further studied in
competition studies (Fig. 2D). The AUUUA sequence
specificity was confirmed by demonstrating that the unlabeled in
vitro transcribed IFN- and IL-2 3`-UTRs as well as a
ribo-oligonucleotide containing the sequence 5`-AUUUAUUUAUUUAUUUA-3`
(
2R1 probe) effectively competed for binding, while the addition
of the nonspecific inhibitor
2H3 (antisense
2R1) had little
or no effect on complex formation. In other studies, GAPDH binding to
the IFN-
and c-myc 3`-UTRs was found to greatly exceed
that found in a ribo-oligonucleotide with five continuous reiterated
AUCUA sequences, further indicating specificity for oligouridine or
AUUUA sequences (data not shown). Further information on the
specificity of GAPDH binding to RNA was obtained with the testing of
the ability of unlabeled ribohomopolymers to compete for binding to the
P-labeled IFN-
3`-UTR. Poly(U) was superior to either
poly(C) or poly(A) at reducing binding, with poly(A) demonstrating an
intermediate level of inhibition (Fig. 3).
Figure 3:
Competition of binding with
ribohomopolymers. A, 0.2 µg of rabbit muscle GAPDH was
assayed for binding to the [P]UTP-labeled
IFN-
3`-UTR in the absence or presence of the indicated amounts of
poly(U), poly(C), or poly(A) RNA. B, the RNA-protein complexes
were analyzed as described under ``Materials and Methods,''
and the results of densitometric reading of the autoradiogram are
expressed as percent binding to GAPDH in the presence of the indicated
competitors relative to control.
GAPDH was recently
identified as a nuclear tRNA-binding protein(38) . The binding
of the two types of RNA ligands (tRNA and the ARE-containing 3`-UTR) by
GAPDH was examined with selected tRNAs, ptRNA (human
precursor serine tRNA) and ptRNA
(yeast
mitochondrial precursor methionine tRNA). GAPDH bound the IFN-
3`-UTR more strongly than the tRNA probes, exhibiting a rank order of
binding of IFN-
3`-UTR > ptRNA
>
ptRNA
(Fig. 4A). Consistent with this
observation, both unlabeled in vitro transcribed
ptRNA
and the yeast tRNA mixture were less
efficient than the unlabeled IFN-
3`-UTR at competing with GAPDH
binding to the radiolabeled IFN-
3`-UTR (Fig. 4B).
Figure 4:
Comparison of binding of GAPDH to the
ARE-containing 3`-UTR and tRNA. A, binding of GAPDH to the
radiolabeled IFN- 3`-UTR, ptRNA
, and
ptRNA
. 0.2 µg of rabbit muscle GAPDH was incubated
with the indicated [
P]UTP-labeled RNA probes in
the presence of 0.5 µg/ml poly(I). (Equal molar amounts of
radioactive probes were added, corrected for the number of U
nucleotides of the different probes.) B, competition of
binding of GAPDH to the IFN-
3`-UTR with tRNAs. 0.2 µg of
splenic GAPDH was assayed for binding to the
[
P]UTP-labeled IFN-
3`-UTR in the presence
or absence of the indicated molar excess of the unlabeled in vitro transcribed IFN-
3`-UTR and ptRNA
and yeast tRNA (Sigma). 0.5 µg/ml poly(I) was used to block
nonspecific RNA binding.
Figure 5:
RNA binding activity of GAPDH is regulated
by redox changes. A, effect of 2-mercaptoethanol (2-me) on GAPDH binding to both the ARE and tRNA. 0.25 µg
of splenic GAPDH was preincubated for 5 min at room temperature in the
absence or presence of 1% 2-mercaptoethanol prior to incubation with
the radiolabeled IFN- 3`-UTR, ptRNA
, and
ptRNA
(as described in the legend to Fig. 4A). RNA-protein complexes were analyzed by
nonreducing SDS-PAGE. B, effect of diamide on the AUBP
activity of GAPDH. 0.2 µg of splenic GAPDH was preincubated with 10
mM diamide for 10 min at room temperature, and then the
indicated amount of 2-mercaptoethanol was added for 5 min at room
temperature. Radiolabeled GAPDH
IFN-
3`-UTR RNA complexes
were analyzed as described under ``Materials and
Methods.''
NAD and NADH are
necessary for the glycolytic function of GAPDH. It has been reported
that incubation of GAPDH with NAD
reduces its RNA
binding activity(30, 38) . Experiments were therefore
undertaken to determine if the same was true of AUBP binding activity.
Increasing concentrations of NAD
and NADH decreased
GAPDH binding of both [
P]UTP-labeled IFN-
3`-UTR (Fig. 6A, upper panel) and
ptRNA
(lower panel) RNA probes.
These data indicate that NAD
and NADH may regulate RNA
binding by GAPDH to both types of RNAs. The specificity of this
inhibitory effect on GAPDH-RNA interactions was supported by the lack
of effect of these coenzymes on AUBP activity on the serendipitously
copurified 25-kDa protein.
Figure 6:
Inhibition of RNA binding of GAPDH by
coenzymes NAD and NADH and by ATP. A, 0.4
µg of splenic GAPDH was preincubated for 5 min at room temperature
with the indicated concentrations of NAD
and NADH
prior to binding to the [
P]UTP-labeled IFN-
3`-UTR (upper panel) or ptRNA
(lower panel). B, 0.25 µg of splenic GAPDH
was assayed for binding to the [
P]UTP-labeled
IFN-
3`-UTR in the presence of the indicated concentrations of ATP
or UTP. RNA-protein complexes were analyzed as described under
``Materials and Methods.''
It has been reported that GAPDH is
capable of autophosphorylation in vitro(50) . This
prompted us to study whether the AUBP activity of GAPDH can be
modulated by autophosphorylation. GAPDH was preincubated with ATP
and/or Mg under conditions to promote
autophosphorylation (50) and then analyzed for binding to the
[
P]UTP-labeled IFN-
3`-UTR probe.
Preincubation with 1 mM ATP alone or ATP + Mg
reduced the binding of GAPDH to AU-rich RNA dramatically (data
not shown). This effect was equally apparent whether the incubation was
carried out at 0 or 30 °C, further suggesting that ATP binding to
GAPDH, and not phosphorylation, altered AUBP activity. To examine the
specificity of this effect, ATP or UTP was added to the binding
solution at increasing concentrations without preincubation or
Mg
. ATP progressively decreased GAPDH binding to RNA,
with half-maximal inhibition at
50 µM, while a
20-fold higher concentration of UTP was needed to mediate comparable
inhibition (Fig. 6B).
Figure 7:
Fingerprinting of the RNA-binding domain
of GAPDH. Shown is schematic representation of the predicted V8
protease peptide map of GAPDH (A). The subunit of GAPDH
consists of two domains. The first half of the subunit is the
NAD-binding domain, and the second half is for
substrate binding, specificity, and catalysis. Cysteine 147 (in rabbit
muscle GAPDH) occurs at the junction between the two domains in the
center of the subunit. Digestion of 2 µg of rabbit muscle GAPDH
with S. aureus V8 protease at the indicated protein/protease
ratios was performed before (B) or after (C) binding
to the radiolabeled IFN-
3`-UTR for the indicated lengths of time
as described under ``Material and Methods.'' Following UV
cross-linking and RNase digestion, the proteins and peptides were
analyzed by 17% Tris/Tricine-PAGE.
It is worth noting that the generation
of peptide fragments by V8 protease digestion was influenced by
GAPDHRNA complex formation (Fig. 7C). V8 protease
digestion after RNA binding (but prior to UV cross-linking) inhibited
the generation of the 6-kDa peptide relative to that observed when V8
protease digestion was performed prior to RNA binding (Fig. 7, B and C, digestion, 1:5 ratio). This effect was
accompanied by a corresponding change in the rate of loss of the 10-kDa
peptide with AUBP activity, suggesting that RNA binding has made it
more resistant to proteolytic attack by V8 protease. One interpretation
of this observation is that access of the protease is blocked by RNA
binding, thereby localizing the RNA-binding domain of GAPDH to the
C-terminal end of the predicted N-terminal 6.8-kDa peptide (Glu-61).
The selectivity of the RNA binding activity of the five observed
fragments of GAPDH is underscored by the complex digestion pattern
demonstrated by silver staining, which reveals at least 15-20
peptides ranging from 3 to 33 kDa (data not shown).
Identification
of the observed 6-kDa RNA-binding peptide as the N terminus of GAPDH
cannot be made solely on the basis of the predicted size of a
proteolytic fragment. Therefore, we determined the amino acid sequence
of the observed 6-kDa RNA-binding peptide. GAPDH was digested with V8
protease, and the digestion mixture was separated by electrophoresis
and transferred to nitrocellulose membrane. RNA binding performed in situ on the membrane-attached peptides (Northwestern
blotting) revealed an 6-kDa fragment (Fig. 8A).
Microsequence analysis of the excised 6-kDa peptide identified by RNA
binding and Coomassie Brilliant Blue staining (Fig. 8) yielded
an amino acid sequence that is 100% identical to that of the N terminus
of rabbit muscle GAPDH. These experiments unambiguously localize the
RNA-binding domain of GAPDH to its NAD
-binding region
as well as demonstrate RNA binding by GAPDH in the absence of UV
cross-linking.
Figure 8:
Identification of the IFN-
3`-UTR-binding peptide fragment of GAPDH as the
NAD
-binding region (Rossmann fold). Rabbit muscle
GAPDH was digested with V8 protease and then transferred to
nitrocellulose membrane. RNA-binding proteins were detected by
Northwestern blotting (as described under ``Materials and
Methods'') (A); total proteins were visualized by
Coomassie Brilliant Blue staining (B). The 6-kDa peptide
(indicated by the arrows) was excised from the membrane for
microsequencing.
Figure 9:
Polysomal location of GAPDH and its
modulation by transcriptional inhibition. Proteins of the polysomal
fractions of 7 10
peripheral blood mononuclear
cells (
1 µg) were separated by 12% SDS-PAGE and analyzed by
immunoblotting with the anti-GAPDH antibody (40.10.09) as described
under ``Materials and Methods.'' Lane1,
resting human lymphocytes; lane2, human lymphocytes
activated with phytohemagglutinin for 16 h; lane3,
phytohemagglutinin-activated human lymphocytes treated with
5,6-dichlorobenzimidazole riboside (100 µM) for 1 h.
The presence of the ARE in the 3`-UTR of labile cytokine,
lymphokine, and proto-oncogene mRNAs has been shown to be important in
the regulation of both mRNA stability (5, 6, 7, 8) and
translation(52, 53, 54, 55) . The
ability of specific AUBPs to interact with these sequences has been
correlated with changes in both nuclear and cytoplasmic mRNA stability (11, 20) . In this paper, we report the identification
of a 36-kDa AUBP purified from human spleen as
glyceraldehyde-3-phosphate dehydrogenase based on its N-terminal amino
acid sequence, immunoprecipitation of GAPDHIFN-
3`-UTR
complexes with the anti-GAPDH monoclonal antibody, and the
demonstration that rabbit muscle GAPDH shares similar RNA binding
activity.
Here we demonstrate that GAPDH binds to AU-rich RNA
sequences present in the 3`-untranslated region of IFN-,
c-myc, GM-CSF, and IL-2 mRNAs. The observation that GAPDH
formed complexes with the 3`-UTR of c-myc very efficiently is
intriguing because of the reported dysregulation of c-myc expression in Bloom's syndrome(56) , a condition in
which structural alterations in GAPDH have been described(57) .
GAPDH was able to bind RNA probes with discontinuous AUUUA pentamers,
although three reiterations were necessary for optimalbinding. This is
in contrast to observations with hnRNP A1, where little or no binding
was observed. (
)
Of relevance to this finding is the
previous report that GAPDH is a tRNA-binding protein implicated in
nuclear export of tRNA(38) . Competition studies indicate that
both human splenic and rabbit muscle GAPDHs bind the 3`-UTR of
IFN- with somewhat higher affinity than two selected tRNA probes.
Although these assays do not permit the affinity of GAPDH for the
3`-UTR of IFN-
to be measured, previous work demonstrated that
GAPDH bound tRNA with a high affinity (K
1.8
10
M for
tRNA
)(38) . Based on these data, it would seem
reasonable to infer that the GAPDH-ARE binding was also a high affinity
interaction. In addition, ptRNA
and the
ptRNA
used in this study contain oligo(U)
sequences (CUUUUUUUA and AUUUUA, respectively). In this regard, it is
of interest that combinatorial selection studies have demonstrated that
the HELN-1 protein, a member of the AUBP family, recognizes oligo(U)
sequences (3, 4, or even 5 U nucleotides) flanked by other nucleotides
than A(14) . The stronger binding to lymphokine 3`-UTRs
relative to tRNAs may therefore have been the result of higher avidity
given the presence of reiterated AU-rich sequences that could serve as
multiple binding sites for GAPDH, which exists as monomers, dimers, and
tetramers in solution. Unfortunately, measurement of RNA binding
affinity under native conditions in the absence of UV cross-linking was
not successful because of the aggregation of GAPDH in the presence of
RNA. However, these studies do permit the identification of AUUUA
pentamers and U-rich sequences as sites at which GAPDH can specifically
interact with RNA with high affinity. This is in contrast to studies of
tRNA binding by GAPDH, where the nature of the sequence recognized by
GAPDH was not established(38) .
It is worth noting that multiple AUBPs between 32 and 40 kDa have been described in cytoplasmic and nuclear extracts(9, 10, 11, 13, 17, 18) . Based on similar binding characteristics, one of these RNA-binding proteins may represent GAPDH. We have previously described a cytoplasmic 36-kDa AUBP activity in activated T lymphocytes that consisted of trypsin-sensitive (hnRNP A1) and trypsin-resistant components(19) . Given their relatively comparable, but not identical AUBP activity (see above), and their similar size, it may be that the trypsin-resistant component of the 36-kDa AUBP represented GAPDH, instead of a closely related molecule or isoform of hnRNP A1(58) .
The demonstration that NAD, NADH,
and ATP were able to diminish the specific AUBP activity of GAPDH
suggested that the dinucleotide-binding (Rossmann) fold of GAPDH might
serve as an RNA-binding domain. This was further supported by the
finding that inhibition by UTP occurred at a 20-fold higher
concentration, consistent with the lower binding affinity of this
compound for the NAD
-binding region of
GAPDH(59) . To date, we are unaware of direct evidence that
identifies the Rossmann fold as an RNA-binding domain. Through V8
protease-mediated proteolysis, Northwestern blotting, and
microsequencing, we have demonstrated that the AUBP activity of GAPDH
was exhibited by the predicted N-terminal 6.8-kDa peptide. As RNA
binding of GAPDH retarded generation of this predicted N-terminal
6.8-kDa peptide, it is likely that glutamate 61 was masked from
proteolytic attack by the GAPDH
RNA complex. Thus, it can be
inferred that a component of the RNA-binding domain extends beyond the
predicted N-terminal 6.8-kDa peptide. This observation would be
consistent with the structure of the NAD
-binding
domain, which is made up of two roughly identical
mononucleotide-binding sites in the NAD
-dependent
dehydrogenases(39) . In GAPDH, one of the
mononucleotide-binding areas is in the predicted 6.8-kDa V8 peptide
fragment, with the other residing in the C-terminal half of the
dinucleotide-binding fold. Hydrophobic interactions have been shown to
be important in coenzyme binding in the NAD
-binding
domain (39) . Consistent with these findings is our observation
that the AUBP activity of GAPDH is unaffected over a wide pH range
(from 3.5 to 9.7) (data not shown), suggesting that specific ARE
binding by GAPDH is not a consequence of its overall cationic nature
(pI 8.1-8.7) or ionic interactions. This insensitivity to pH
changes suggests the importance of hydrophobic interactions in RNA
binding by GAPDH.
Concurrent with these studies, other members of
the family of dehydrogenases (lactate dehydrogenase, yeast
mitochondrial NAD-dependent isocitrate dehydrogenase,
and glutamate dehydrogenase) and
NAD(P)
/NAD(P)H-binding enzymes (catalase and
dihydrofolate reductase) have been demonstrated to have
sequence-specific RNA binding capacity (reviewed in (29) ).
Yeast isocitrate dehydrogenase binds the AU-rich 5`-untranslated region
of all major yeast mitochondrial mRNAs(25) , while glutamate
dehydrogenase has a specific RNA binding activity for cytochrome c oxidase mRNA(24) . Given the absence of RNA-binding
domains with characteristic ribonucleoprotein consensus sequences
(reviewed in (60) and (61) ) and the similarity in
three-dimensional structure of the dinucleotide-binding regions of
different dehydrogenases(39, 62) , our data suggest
that the Rossmann fold is capable of serving as an RNA-binding domain.
The strong evolutionary conservation of the Rossmann fold and its
presence in kinases, tRNA synthetases, and NAD(P)
- and
FAD-dependent dehydrogenases (39, 63) may therefore be
relevant to the ability of these proteins to regulate RNA metabolism.
Characterization of the NAD
-binding region as an
RNA-binding domain may therefore have broad implications for both mRNA
metabolism and translation, as evidenced by the reciprocal
identification of GAPDH as a tRNA-binding protein (38) and some
tRNA synthetases as mRNA-binding proteins(64) .
In human T
lymphocytes, GAPDH can be localized to the polysomes, supporting the
notion that GAPDH binds RNA in vivo. Furthermore, polysomal
levels of GAPDH are increased by transcriptional inhibition, similar to
data reported for other AUBPs(18, 19) . Thus, the
potential role of cytoplasmic GAPDH as a functionally relevant AUBP is
strengthened by demonstrating both its polysomal location as well as a
common pattern of modulation by RNA polymerase II inhibition. Based on
this finding, the local concentration of NAD, NADH,
and ATP in different subcellular compartments (polysomes, cytosol, and
nucleus) may regulate GAPDH activity. Our data suggest that the
Rossmann fold of GAPDH is reciprocally regulated between its RNA
binding (inactive in glycolysis) and NAD
binding
(active in glycolysis) states in vivo. This mechanism would
favor glycolytic activity of GAPDH in the cytosol (where the
concentration of NAD
and ATP is high) by inhibiting
RNA binding. Conversely, GAPDH may bind RNA in the polysomal and
nuclear microenvironments due to lower concentrations of
NAD
, NADH, and free ATP. This interpretation is
supported by the finding that NAD
and NADH blocked
IFN-
binding to GAPDH at concentrations (
1-10
µM) at which they have been reported to associate with its
Rossmann fold(65) .
Alternatively, post-translational
modifications (redox or otherwise) might influence the intracellular
location of GAPDH as well as differentially regulate its RNA or
NAD binding activity. In this regard, it is worth
noting that purified splenic GAPDH consisted of a single isoform on
two-dimensionalPAGE, in contrast to what has been demonstrated for the
multiple isoforms of total cellular GAPDH(30, 31) .
Thus, isoforms of GAPDH may differ considerably in their affinity for
specific RNA ligands. Indeed, Karpel and Burchard (31) reported
that only the most basic isoform of yeast GAPDH possesses poly(U)
binding capacity and helix destabilizing activity in vitro.
GAPDH has been shown to possess a wide range of biological activities in addition to its well characterized role as a specific dehydrogenase essential in glycolysis. Our studies potentially extend these activities into mRNA metabolism. In this regard, it is intriguing that hnRNP A-type proteins have been shown to shuttle between the nucleus and cytoplasm and are implicated in mRNA export(66, 67) . Given the relatively reciprocal distribution of GAPDH and hnRNP A1 between the nucleus and cytoplasm, their demonstrated AUBP activity, and the elevation of polysomal GAPDH levels with RNA polymerase II inhibition, GAPDH may play a similar role not only in nuclear tRNA export(38) , but also in the export of AU-rich mRNAs, as reported for other AUBPs(19, 68) . In addition to this potential role in nucleocytoplasmic export of tRNA and mRNA, the polysomal location of GAPDH and its RNA binding specificity suggest a role in ARE-dependent mRNA turnover and translation. Given the ability of GAPDH to decrease the melting point of RNA(31) , GAPDH may be important for RNA unwinding during translation or accessibility of the 3`-UTR to endoribonuclease attack and may therefore influence the translatability or stability of mRNA through its binding to the ARE. In contrast to GAPDH, hnRNP A1 has been shown to have annealing activity(69) . As the ability of hnRNP A1 to bind the ARE in vitro correlates with increased mRNA stability in vivo(20) , these data suggest the intriguing possibility that GAPDH and hnRNP A1 may compete for similar, but not identical, RNA ligands in vivo, with profoundly different consequences in terms of mRNA stability and translation.
In conclusion, identification of the Rossmann fold of GAPDH as an RNA-binding site provides new insights into the regulation of this multifunctional protein. Moreover, this finding may be relevant to the RNA binding activity that has been recently described in other dehydrogenases, given the conservation of the Rossmann fold among these enzymes. The RNA binding specificity of GAPDH as well as its polysomal localization not only prompt consideration of this protein as a regulator of ARE-dependent mRNA turnover and translation, but also extend the conceptual framework of cytoplasmic mRNA metabolism to include proteins with previously defined roles in glycolysis.