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J. Biol. Chem., Vol. 277, Issue 38, 35738-35745, September 20, 2002
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From the
Received for publication, April 24, 2002, and in revised form, June 27, 2002
Post-transcriptional pathways provide a major
means of regulating eukaryotic gene expression. Reiterations of the
AU-rich element (ARE) within the 3'-untranslated region of many
cytokine and proto-oncogene mRNAs serve as signals for rapid
degradation and translational repression. The identification of this
cis-acting stability determinant has fueled the search for
ARE-binding proteins (AUBP) that function as trans-acting
factors that transduce this function. Previous work identified
heterogeneous nuclear ribonucleoprotein (hnRNP) A1 as a major AUBP
capable of binding the ARE of granulocyte-macrophage colony stimulating
factor (GM-CSF) RNA in the context of a full-length mRNA. We report
here that functional studies failed to indicate a role for hnRNP A1 in
ARE-dependent mRNA turnover. In an effort to identify
other functionally relevant AUBP, the major GM-CSF ARE-specific binding
protein in cells lacking hnRNP A1 was purified from CB3 mouse
erythroleukemia cells. Microsequencing identified this protein as the
glycolytic enzyme lactate dehydrogenase (LDH) M. RNA binding by LDH was
shown to occur in the NAD+-binding region (Rossmann fold).
Polysome gradient analysis demonstrates that LDH is found in the
translationally active fraction. Polysomal localization of LDH was
dependent on RNA binding. Moreover, polysomal LDH exists in a complex
with AUF1 and hsp-70, which has been implicated previously in the
regulation of mRNA turnover. The interaction between LDH and AUF1
is direct as it can be demonstrated in vitro with purified
proteins. Collectively these data implicate a role for LDH in the
post-transcriptional regulation of gene expression.
The regulation of mRNA turnover is a key mechanism of
modulating eukaryotic gene expression. Labile cytokine, lymphokine, and proto-oncogene messages contain AU-rich sequences in their 3'-untranslated regions that are highly conserved across mammalian species. As demonstrated conclusively by Shaw and Kamen (1), these
AU-rich elements (ARE)1
compose a major class of cis-acting stability determinants.
These ARE consist of reiterations of the pentamer AUUUA or oligo(U) sequences in an AU-rich context (1-3). Significantly, cytoplasmic and
nuclear proteins have been identified that bind specifically to the
ARE; these AU-binding proteins (AUBP) are thought to regulate mRNA
stability in trans (reviewed in Ref. 4). Although a variety of these AUBP has been described, functional roles in
ARE-dependent turnover have been attributed to a
comparatively small number of proteins, including HuR and AUF1
(5-12).
HuR, a ubiquitously expressed member of the Elav family of
RNA-binding proteins, interacts in vitro with the ARE of
c-fos and IL-3 mRNA (5), as well as with synthetic
mRNA-destabilizing ARE (6). Several studies (7, 8) have demonstrated
that overexpression of HuR results in stabilization of reporter
transcripts containing the ARE of GM-CSF, c-fos, and
vascular endothelial growth factor. These studies did not demonstrate
altered protein production as a consequence of increased mRNA
accumulation (5-8).
Similarly, in vitro ARE binding activity has been
demonstrated for AUF; this protein was initially purified because of
its ability to accelerate the degradation of c-myc mRNA in an
in vitro decay system (9, 10). However, recent work (11, 12)
has suggested AUF1 may play multiple roles in the regulation of
mRNA turnover. For example, Kiledjian et al. (11) have
demonstrated that AUF1 is a component of the Our laboratory has been examining potential trans-acting
factors involved in mediating ARE-dependent mRNA
turnover. Initial studies centered on investigating the role of hnRNP
A1, prompted by its discovery as the major cytoplasmic protein capable
of binding the GM-CSF ARE in activated T lymphocytes (13, 14). In
addition, IL-2 overproduction by a retrovirally infected T cell line,
MLA-144, was shown to be due to increased mRNA stability (15).
Stabilization of IL-2 mRNA correlated with a proviral insertion in
its 3'-UTR which enhances the binding of hnRNP A1 to its ARE relative
to that of native IL-2 (15).
To examine the role of hnRNP A1 in ARE-dependent mRNA
turnover, we utilized the DP28-9, CB7, and CB3 murine erythroleukemia (MEL) cell lines, which vary in their expression of hnRNP A1 (16). DP28-9 MEL cells derive from DBA/2 adult mice infected with a polycythemia-inducing strain of the Friend leukemia virus (FV-P) and
contain two functional hnRNP A1 genes (16). In contrast, the CB7 MEL
cell line has only one active hnRNP A1 gene, due to silencing of one
allele by a downstream insertion of F-murine leukemia virus helper
virus at the Fli-2 locus. In CB3 MEL cells, proviral
integration occurred at both Fli-2 loci, resulting in the
absence of hnRNP A1 mRNA and protein (16). Transient as well as
stable transfection experiments of CB3 MEL cells with hnRNP A1 failed
to indicate an effect of hnRNP A1 on ARE-dependent mRNA
turnover or expression of reporter gene constructs. Similarly, overexpression of hnRNP A1 in human Jurkat T cells did not result in
alteration of ARE-dependent gene expression.
As we were unable to detect an effect of hnRNP A1 on
ARE-dependent gene regulation, we examined the AUBP
profiles of the three cell lines. The major cytosolic protein capable
of binding the ARE of GM-CSF in the context of its full-length RNA in
CB3 erythroleukemia cells was identified as L-lactate
dehydrogenase (LDH) isozyme M. Lactate dehydrogenase, which catalyzes
the reversible conversion of pyruvate to lactate, is a tetrameric
enzyme that may exist in several enzyme forms (17). Each LDH subunit is
one of two types, designated H (for heart) and M (for skeletal muscle).
The five tetrameric isozymes of LDH are five different combinations of
these subunits (17). The isozyme of LDH identified in these studies is
LDH-M, which consists of five M polypeptide subunits. (For purposes of
ease of reference, the LDH-M will be referred to as simply LDH
throughout the text.). LDH was shown to bind specifically to the ARE of
GM-CSF RNA using both UV cross-linking and filter binding assays. LDH
is polysomally associated and coimmunoprecipitates with AUF1 and
hsp-70. These finding suggest that LDH may serve multiple roles in RNA
metabolism beyond its role in glycolysis. Indeed, non-overlapping roles
of LDH are suggested by the fact that the binding of the ARE by LDH is
competed by NAD+, indicating that each utilizes the
Rossmann fold. Similar roles have been well defined for the
iron-response element-binding protein and its dual role as an aconitase
(18, 19). Collectively, these data implicate LDH as a functionally
relevant AUBP and prompt consideration of an expanded role of this
enzyme in the post-transcriptional regulation of gene expression.
Cell Culture and Transfection--
Murine erythroleukemia (MEL)
cell lines (DP28-9, CB3, and CB7) were generously provided by Benoit
Chabot and Yaacov Ben-David. MEL, Jurkat, and THP-1 monocytic cell
lines were maintained in RPMI 1640 medium (Cellgro) supplemented with
10% heat-inactivated newborn calf serum (HyClone) and 50 µg/ml
gentamycin sulfate (Sigma) at a density of 5 × 105
cells/ml.
CB3C7-11 and CB3C7-20 MEL cells (stably transfected with antisense and
sense hnRNP A1, respectively, generously provided by Benoit Chabot)
were transiently transfected with 0.1 µg of pGL3 luciferase reporter
DNA (Promega) containing six reiterations of either AUUUA or AUGUA in
their 3'-UTR by LipofectAMINE (Invitrogen). Jurkat cells were
cotransfected with either of the AUUUA or AUGUA pGL3 constructs as well
as pCMV-A1 vector (gift of Benoit Chabot). CB3 MEL cells were stably
transfected with human GM-CSF cDNA (generously provided by James
Malter). Transient transfections were incubated in serum-free RPMI 1640 for 21/2 h; at the conclusion of this incubation, RMPI-1640
medium supplemented with 15% fetal calf serum (HyClone) was added to
the transfections. After 20 h, cells were analyzed for luciferase
expression using luciferase assay kit (Promega).
Preparation of in Vitro Transcripts--
RNA transcripts were
generated from linearized DNA templates in the presence of 50 µCi of
[ RNA Binding Analysis by UV Cross-linking--
RNA probes (8 × 105 cpm) were incubated with either 25 µg of purified
cytoplasmic lysate or 1 µg of commercially prepared rabbit muscle
lactate dehydrogenase (Roche Molecular Biochemicals) in 12 mM Hepes, pH 7.9, 15 mM KCl, 0.2 µM dithiothreitol, 0.2 µg yeast tRNA, and 10% glycerol
(all purchased form Sigma) for 10 min at 30 °C and then UV
cross-linked on ice for 5 min with a Stratalinker 1800 (20). Reaction
mixtures were digested with 15 µg of RNase A and 7.5 units of RNase
T1 (Roche Molecular Biochemicals) for 30 min at 37 °C, solubilized
in Laemmli SDS sample buffer (21), and analyzed by 15% SDS-PAGE and
autoradiography. Two-dimensional NEPHGE was performed with separation
in the first dimension accomplished utilizing a pH 3-10 ampholyte
gradient (Bio-Rad) for 2 h and 15 min at 400 V (22). Separation in
the second dimension was achieved by 15% SDS-PAGE.
Protein Purification--
For p36 AUBP purification, a 20-liter
culture of CB3 MEL cells was harvested. Cells were washed in ice-cold
1× PBS, and cell 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 Pefabloc (Invitrogen), and then lysed by the addition of
Triton X-100 (1% final) (Sigma) on ice for 3 min (20). MEL cells were
centrifuged for 5 min at 1500 rpm; supernatants were collected,
aliquoted, and stored at Immunoprecipitation--
UV cross-linking reactions contained
CB3 polysomes (10 A260) to GM-CSF RNA. Reactions
were digested with RNase and then immunoprecipitated with a rabbit
polyclonal antibody directed against LDH (AB1222) (Chemicon).
Immunocomplexes were captured with protein G-agarose beads (Bio-Rad)
overnight at 4 °C, and beads were washed six times in 100 mM NaCl. Proteins were resolved by 15% SDS-PAGE and
electrotransfered to nitrocellulose membrane in CAPS (Sigma) buffer, pH
11.0, with 15% methanol. Immunoblots were washed with Tris-buffered
saline, 0.1% Tween 20 (Sigma) and blocked in 5% milk overnight at
4 °C. Membranes were then probed with AB1222 and goat Nitrocellulose Filter Binding Assay--
Purified LDH and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Roche Molecular
Biochemicals) were incubated with 80,000 cpm radiolabeled GM-CSF RNA in
binding buffer (10 mM Hepes/KOH, pH 7.4, 100 mM
KCl, 2.5 mM MgCl2, and 50 mg/ml competitor
yeast tRNA) in a final volume of 20 µl for 30 min at 30 °C as
described previously (23). For binding competition experiments,
NAD+ (Sigma) was preincubated with reactions for 5 min at
room temperature. Reactions were then applied to nitrocellulose under
negative pressure and briefly washed with binding buffer. Filter-bound
RNA complexes were then counted by liquid scintillation. Binding curves
from five different experiments (each performed in triplicate) were used to determine the dissociation constant (Kd)
(24).
Polysome Profile Analysis by Sucrose Density
Gradient--
4 × 108 CB3 MEL or THP-1 monocytic
cells were resuspended in buffer C (10 mM Tris-HCl, pH 7.5, 1 mM potassium acetate, 1.5 mM magnesium
acetate, 2 mM dithiothreitol; 1.5 ml per 108
cells) (25). MEL and THP-1 cells were lysed with 20 strokes of type B
Dounce homogenizer, and mitochondria and nuclei were removed by
centrifugation at 10,000 × g for 10 min. Samples were then treated with either 30 mM EDTA (Sigma) or 2 µg of
RNase A (Roche Molecular Biochemicals) as indicated. Post-mitochondrial supernatant was loaded onto the top of sucrose gradient (10-40% (w/v)
sucrose) in buffer A (25). The samples were centrifuged at 4 °C for
5 h at 36,000 rpm. Polysome gradient fractions were collected in
500-µl volumes and assessed for LDH and AUF1 by immunoblotting.
Recombinant AUF1/LDH Binding Assay--
pTCR-HisB-AUF1 p40 was
expressed in BL 21 Escherichia coli, and p40 AUF1 was
purified as follows: 20 ml of bacterial sonicate was bound to 2 ml of
Talon metal affinity beads (CLONTECH) in PBS
containing 1 mM Pefabloc, 3 µg per ml leupeptin, and
0.5% aprotinin overnight at 4 °C. Beads were then washed five times with PBS. His-tagged AUF1 (coupled to metal affinity beads) was incubated with either 1 µg of commercially prepared rabbit muscle lactate dehydrogenase protein (Roche Molecular Biochemicals) or 1 µg
of bovine serum albumin (Sigma) in 0.3 M NaCl for 1 h
at 4 °C with rotation, washed six times in PBS containing 0.3 M NaCl, and resolved by 15% SDS-PAGE. In addition, beads
alone were incubated with His-tagged AUF1 as negative control. Proteins
were then visualized by staining with 0.1% Coomassie Brilliant Blue
R-250 (Pierce).
Characterization of ARE-dependent mRNA Turnover in
an hnRNP A1-deficient Cell Line--
Previous studies (13, 14)
identified hnRNP A1 as the dominant cytoplasmic protein capable of
specifically interacting with the ARE of full-length GM-CSF RNA in
activated T lymphocytes. To examine the role of hnRNP A1 in mRNA
turnover, we utilized MEL cell lines in which hnRNP A1 expression had
been silenced by retroviral insertion (16). Insertion of the
Friend-murine leukemia virus helper virus at the Fli-2 locus
silenced expression from either one (CB7) or both (CB3) hnRNP A1
alleles. Immunoblotting established the reported absence of hnRNP A1 in
the CB3 cell line was not due to altered subcellular distribution (Fig.
1A). In contrast, the CB3 cell
line overexpressed hnRNP A2, particularly in the cytoplasmic and
polysomal fractions (Fig. 1A), perhaps to compensate for the
absence of hnRNP A1.
The role of hnRNP A1 in ARE-dependent mRNA turnover was
analyzed. CB3 cells stably transfected with hnRNP A1 or a control cDNA were analyzed by transient transfection for different
expression of luciferase reporter genes into which 6 consecutive
reiterations of AUUUA or AUGUA had been engineered into their 3'-UTR.
The presence of reiterated AUUUA pentamers reduced luciferase
expression by 70-90% relative to controls or vectors with AUGUA
sequences, indicating the post-transcriptional activity of this ARE.
There was no difference in ARE-dependent inhibition of
luciferase expression in CB3 cells that lacked or contained hnRNP A1
(data not shown). Similarly, ARE-mediated inhibition did not differ
between the DP28-9, CB7, and CB3 cell lines (data not shown). Moreover,
no consistent effect on GM-CSF mRNA turnover was observed with CB3
cells (that differed only in hnRNP A1 expression) stably transfected
with the human GM-CSF cDNA (data not shown). Finally,
overexpression of hnRNP A1 in the human Jurkat T cell line had no
effect on ARE-dependent luciferase expression (data not
shown). Based on these studies, we concluded that in these cell lines
and under these conditions, hnRNP A1 plays either a redundant or minor
role in the post-transcriptional regulation of GM-CSF and
ARE-dependent mRNA turnover.
Characterization of AUBP Proteins in an hnRNP A1-deficient Cell
Line--
Given the lack of effect of hnRNP A1 on
ARE-dependent gene expression, the presence of other AUBP
which mediated ARE-dependent mRNA turnover was examined
by concurrent UV cross-linking and immunoblotting. In these
experiments, in vitro transcribed, 32P-labeled
full-length GM-CSF RNA was incubated with cytoplasmic lysate from
DP28-9, CB7, and CB3 cells, UV cross-linked, RNase-digested, resolved
by SDS-PAGE, and blotted onto nitrocellulose. As described previously
(13) in T lymphocyte cytosols, a single 36-kDa protein capable of
binding the ARE of GM-CSF RNA in the context of full-length RNA was
observed, which colocalized with hnRNP A1. In DP28-9 and CB7 cytosols,
(Fig. 1, B and C), a 36-kDa GM-CSF RNA binding
activity paralleled hnRNP A1 levels, whereas the identity of the
36-kDa-binding protein (hereafter referred to as p36) in the CB3 cells
was unknown, as no hnRNP A1 was present. The p36 binding activity was
ARE-specific, as no RNA binding activity was seen with mutant GM-CSF
RNA probe (in which the central uridine of the canonical ARE had been
changed to guanosine) (Fig. 1B). Immunoprecipitation
approaches and two-dimensional NEPHGE immunoblots of the UV
cross-linked binding activity indicated that the p36 binding activity
was not hnRNP A2, a member of the Elav family (HuR and Hel-N1)
of RNA-binding proteins, GAPDH or AUF1 (see Ref. 26 and data not
shown). We therefore concluded that this protein represented a novel
AUBP.
Identification of the p36 AU-specific RNA-binding Protein as
Lactate Dehydrogenase--
The p36 AUBP was initially purified by
ammonium sulfate precipitation of CB3 cytoplasmic lysate (Fig.
2A). The majority of 36-kDa
AUBP activity eluted in the 50% ammonium sulfate fraction (Fig.
2B). Carboxymethylcellulose chromatography resulted in the elution of this RNA binding activity at 0.25 M KCl (Fig.
2B). This 0.25 M fraction was further
purified by poly(U)-Sepharose chromatography, with the 1 M
KCl elution containing the RNA binding activity (Fig. 2B).
Immunoblotting the 1 M poly(U)-Sepharose elution demonstrated no HuR or AUF1 (data not shown). Two-dimensional NEPHGE
analysis of AUBP activity in the 1 M poly(U) fraction
revealed the presence of three isoforms of a 36-kDa protein, with pI
values between 6.6 and 8.0 (Fig. 2C). This
pattern was identical to that found using CB3 polysomes,
indicating purification of the targeted binding activity. The 36-kDa
protein in the 1 M poly(U) fraction resolved as a single
band on 15% SDS-PAGE that was excised, digested in situ
with trypsin, and analyzed by matrix-assisted laser
desorption/ionization-mass spectrometry. This analysis identified the
36-kDa AUBP as murine L-lactate dehydrogenase M with a
probability score of 9.9 × 10 LDH Binding Specificity for GM-CSF mRNA in a Non-UV
Cross-linking Assay--
Nitrocellulose filter binding assays examined
the ability of LDH to bind specifically to the ARE of GM-CSF RNA
independent of cross-linking. LDH/GM-CSF RNA complex formation
plateaued at 750 nM with ~75% of input RNA bound (Fig.
3). Binding of the mutant AUGUA GM-CSF
RNA probe was markedly diminished, approximating that seen with a
radiolabeled globin pre-mRNA transcript, which lacks an ARE. Based
on these studies, the Kd of purified LDH binding to
the wild type GM-CSF RNA was 501.9 nM. In contrast, the
Kd of the LDH binding to the mutated GM-CSF RNA was determined as 1879 nM, confirming its ARE specificity.
Studies have demonstrated the RNA binding capabilities of several
glycolytic enzymes (reviewed in Ref. 27). Each of these enzymes utilize
NAD+, which is bound in a region known as the Rossmann
fold, to accept electrons from their substrates (reviewed in Ref. 28).
Intriguingly, proteolysis studies have implicated the Rossmann fold as
the RNA binding domain of GAPDH (20). The identification of this
putative RNA binding domain was supported by the ability of
NAD+ to interfere with RNA binding (20). Incubation of
purified LDH with NAD+ prior to UV cross-linking inhibited
AUBP activity in a concentration-dependent manner and was
eliminated at 10 µM NAD+ (Fig.
4A). This effect was
independent of UV cross-linking, as similar results were seen in filter
binding assays (Fig. 4B). Collectively, these data show that
occupancy of the Rossmann fold by NAD+ inhibits RNA binding
by LDH, suggesting that LDH utilizes the Rossmann fold for binding
either NAD+ or the ARE of GM-CSF RNA.
Polysomal Localization of LDH Is Dependent on RNA
Binding--
With the identification of LDH as a cytoplasmic GM-CSF
RNA-binding protein, its subcellular localization was examined.
Immunoblotting demonstrated that a discrete percentage of LDH is
polysomal (Fig. 5A).
Comparison of various cell equivalents of S130 to a fixed amount of CB3
polysomes indicated that polysomal levels of LDH are ~10% those of
cytoplasmic LDH in CB3 cells (data not shown). Discontinuous (10-40%)
sucrose density gradient analysis demonstrated that similar ratios of
polysomal LDH were detected in cells containing hnRNP A1, including
THP-1 monocytic cells and activated human T lymphocytes (Fig.
5B and data not shown). LDH was predominantly found in the
40% sucrose density fraction, suggesting association with mRNA
that are being actively translated. Disruption of mRNP complexes from
ribosomes by EDTA treatment (30 mM) prior to polysome sucrose density gradient fractionation resulted in LDH exhibiting a
reduced density (Fig. 5B). The altered sedimentation of LDH as a consequence of EDTA treatment is typical of those proteins whose
polysome association is dependent on RNA binding (29). RNase A
treatment of CB3 cytosol resulted in a similar shift of LDH (data not
shown). Moreover, preincubation of polysomes with 100 µM
exogenous NAD+ disrupted the association of LDH with this
fraction (Fig. 5C). These findings are corroborated by
studies performed with GAPDH (30). Collectively, these data demonstrate
LDH is a polysome-associated protein localized to this compartment by
RNA-protein interactions.
Polysomal LDH Interacts with AUF1--
The dependence of the
polysomal localization of LDH on RNA binding suggests that LDH
interacts directly with RNA in vivo. In the absence of RNA
binding, some LDH remained in the 20% sucrose density fraction (refer
to Fig. 5B), suggesting it is part of a larger complex of
proteins. In addition, the polysomal distribution of AUF1 to the most
dense polysomal fraction (fraction number 18, as indicated by sucrose
density continuous gradient analysis) was similar to that seen with LDH
in MEL cells as well as THP-1 monocytic cells, suggesting their
possible interaction (Fig. 6). Interestingly, there appears to be a greater distribution of LDH to the
dense polysomal fractions of CB3 versus THP-1 cells. This may reflect differences in the cellular distribution of LDH between erythroid (CB3) and myeloid (THP-1) cells and/or species (mouse versus human). Alternatively, the absence of hnRNP A1 in the
CB3 cell line may play a factor and would suggest that LDH serves some
other compensatory function or competes with hnRNP A1 for mRNA-binding sites in the cytoplasm. This association was confirmed by finding that polysomal LDH and AUF1 could be coimmunoprecipitated (Fig. 7A). Based on
densitometry, 50% or greater of polysomal LDH is bound to AUF1 in the
CB3 and THP-1 cell lines. Immunoprecipitation of LDH from THP-1
polysomes demonstrated the presence of AUF1 and hsp-70, which were
shown previously to interact in HeLa cytosols (31).
In order to distinguish if the interaction between LDH and AUF1 was
direct, and not due to coassociation of an mRNA, in
vitro binding studies were undertaken in the absence of RNA.
His-tagged recombinant p40 AUF1 was bound to metal affinity beads and
incubated with purified LDH. Following washing, bead-bound complexes
were eluted, resolved by SDS-PAGE, and visualized. As demonstrated in
Fig. 7B, ~73% of the input LDH (determined
densitometrically) bound the p40 isoform of AUF1. Thus LDH and AUF1
interact directly in the absence of RNA and other proteins. Given the
immunoprecipitation results, the data demonstrate the interaction of
LDH, AUF1, and hsp-70 occur in the polysomal compartment and suggest it
is mediated through direct protein-protein interaction.
Although identified as a major AUBP in human and gibbon T
lymphocytes as well as mouse erythroleukemia cells (13, 14), a variety
of experiments failed to demonstrate a role for hnRNP A1 on
ARE-dependent turnover or translation of reporter gene
constructs or GM-CSF mRNA. We concluded therefore that another AUBP
besides hnRNP A1 mediates ARE-dependent gene regulation.
Experiments to identify the trans-acting factor responsible
for mediating the rapid turnover of GM-CSF message were undertaken.
Despite the absence of hnRNP A1, CB3 cytosols contain a 36-kDa protein
that binds specifically to the ARE of GM-CSF RNA. All previously
characterized AUBP of comparable size (GAPDH, HuR, and AUF1) were
excluded using immunoprecipitation and two-dimensional NEPHGE
immunoblotting approaches.
Matrix-assisted laser desorption/ionization-mass spectrometry
identified the p36 AUBP as LDH. This finding was confirmed by immunoprecipitation of LDH-GM-CSF RNA complexes from CB3 cytosols as
well as by in vitro binding (UV cross-linking and filter
binding) assays with purified LDH. In addition, incubation of either
LDH or CB3 cytosol (data not shown) with increasing concentrations of
NAD+ inhibited p36 AUBP activity. This latter finding is
consistent with the interpretation that the Rossmann fold serves as the
RNA binding domain of LDH, as has been reported previously (20) for
other glycolytic enzymes. LDH localized to the polysomes in multiple
cell types besides CB3 cells. Thus, the polysomal location of LDH did
not occur due to the absence of hnRNP A1. Moreover, in all cell types
examined, LDH localized to the bottom of the sucrose gradients,
consistent with its association with mRNA being actively
translated. In addition, direct association of LDH with AUF1 was demonstrated.
The role of LDH in intermediary metabolism has been well documented.
LDH is a tetrameric enzyme with five isoforms, each consisting of
combinations of two subunits, LDH-M and LDH-H (17). The M subunit
catalyzes the conversion of pyruvate to lactate under anaerobic
conditions, whereas the H subunit kinetically favors the conversion of
lactate to pyruvate and predominates in aerobic tissue, such as heart
muscle (17). The biosynthesis of each subunit and thus the amount of
each LDH isozyme in a given tissue type is subject to genetic
regulation. Although each isozyme catalyzes the same reaction, they
have markedly different Km values for pyruvate (17).
The isozyme identified in these studies consists of four identical M
subunits and is therefore designated M4. This isozyme
predominates in skeletal muscle and has a low Km for
pyruvate; hence it readily transfers electrons from lactate to
NAD+, yielding pyruvate and NADH (17). This reaction is
dependent on the binding of NAD+ to LDH in the Rossmann
fold (17).
Other enzymatic proteins have been identified that bind RNA; these RNA
binding activities appear distinct from their functions in metabolism
(reviewed in Ref. 27). In previous work from this laboratory (20),
GAPDH was purified as a polysomal p36 AUBP from human spleen. A number
of features of LDH led us to conclude that LDH and GAPDH play differing
roles in post-transcriptional gene regulation. First, GAPDH appeared to
be less specific in its interaction with ARE than LDH, as it was shown
to bind a variety of ARE, including those lacking reiterated AUUUA
pentamers (IFN- The localization of LDH to the polysomes of human lymphoid and
monocytic cells, which express GM-CSF, supports a functional role for
LDH in the regulation of gene expression in vivo. The functional relevance of LDH AUBP activity is supported by its specific
interaction with mRNA undergoing active translation. As levels of
LDH on polysomes were approximately one-tenth those in the S130
fraction across cell types and species (data not shown), levels of
polysomal and cytosolic LDH appear to be tightly regulated. Interestingly, the NAD+ concentration (1 µM)
required to significantly inhibit RNA binding by purified LDH is lower
than cytosolic NAD+ levels (30-70 µM) (32,
33). Displacement of LDH from the polysome compartment required much
higher NAD+ concentrations (100 µM) than
those necessary to inhibit RNA binding by purified enzyme. These data
suggest that polysomal LDH may have an enhanced affinity for RNA or a
decreased affinity for NAD+, perhaps mediated through
post-translational modification or protein-protein interactions.
In this regard, we have shown that polysomal LDH exists in a large
protein complex in the absence of RNA binding. By immunoprecipitation, we demonstrated AUF1 and LDH exist as a complex in vivo.
This finding is consistent with the sedimentation of LDH in the most dense gradient of the polysomes, indicating active translation, because
AUF1 exists in a complex including heat shock proteins hsp-70,
translation initiation factor eIF4G, and poly(A)-binding protein (31).
In support of this model is our finding that hsp-70 coimmunoprecipitates with AUF1 and LDH in THP-1 monocytic cells (Fig.
7A).
These data are especially intriguing in light of the diverse cellular
functions which have been described for AUF1, which range from telomere
maintenance to transcription, as well as mRNA turnover (9-12, 34).
In the K562 erythroleukemia cell line, distinct effects on
mRNA stability have been noted. Kiledjian et
al. (11) demonstrated that AUF1 is a component of the These studies are central to consideration of the functional role of
the ARE, as protein-protein interactions may influence the role(s) AUF1
serves in DNA and RNA metabolism. In this regard, our data indicate
that LDH is associated with AUF1 on mRNA undergoing active
translation. Nevertheless, LDH was initially characterized because of
its potential role in mediating rapid ARE-dependent turnover in erythroleukemia cells that lack hnRNP A1. One potential model for LDH function is that it provides further specificity for the
translation and turnover of certain ARE-containing mRNA through its
direct interaction with both the ARE and AUF1 protein. The importance
of such a role by LDH is suggested by the finding that recombinant AUF1
binds an array of RNA ligands, including those lacking ARE (11, 12, 34,
37). Alternatively, LDH may influence, through its direct interaction
with AUF1, the makeup of this protein complex or the functional nature
of its interaction with ARE. For example, a model has been proposed in
which AUF1 mediates ARE-dependent turnover through
proteasomal targeting and degradation of the RNP complex (31). In this
regard, it is important to note that LDH did not copurify with
proteasomes following EDTA treatment of polysomes gradients (data not
shown). The possibility that LDH may serve a distinct role in mRNA
translation and turnover independent of AUF1 is not excluded by this model.
Additionally consistent with its association with both AUF1 and hsp-70
in vivo, LDH may mediate effects on mRNA turnover as a
component of eukaryotic degradation machinery, in a manner analogous to
enolase in E. coli. The degradosome of E. coli
consists of a high molecular weight complex of proteins including RNase
E, an endoribonuclease; polyribonucleotide
nucleotidyltransferase (PNPase); an ATP-dependent helicase
and 3'-5'-exoribonuclease; RhlB, a member of the DEAD box family;
polyphosphate kinase; and enolase, a glycolytic enzyme (38). Additional
proteins are often associated with the degradosome, notably the heat
shock chaperones GroEL and DnaK (38). The metabolic function of enolase
has been defined as the catalysis of 2-phosphoglycerate to
phosphoenolpyruvate (17), although its role in mRNA degradation is
not yet clear (39).
In conclusion, the discovery of LDH as an RNA-binding protein points to
an expanded role for this protein in the regulation of gene expression.
Its binding specificity, polysomal localization, and association with
AUF1 collectively suggest a role in ARE-dependent mRNA
turnover beyond its function in metabolism. The ability of LDH to serve
as an AUBP may represent a global mechanism for regulating ARE-mediated
decay, perhaps by modulating the effects of AUF1. Of particular note,
c-Myc overexpression results in the up-regulation of LDH at the level
of gene transcription (40). Elevated levels of LDH are frequently
detected in human cancers (41-44). The overexpression of LDH may thus
confer neoplastic growth advantage, either through its enzymatic or
gene regulatory function. Understanding the dual functions of this
protein may lead to greater understanding of carcinogenesis.
We thank Drs. Benoit Chabot and Yaacov
Ben-David for provision of DP28-9, CB7, and CB3 murine erythroleukemia
cell lines, as well as CB3C7-11 and CB3C7-20 transfectants.
*
This work was supported by National Institutes of
Health Grants R01 A134928 (to W. R.) and R01 CA52443 (to G. B.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: Dept. of Medicine,
Dartmouth-Hitchcock Medical Center, Lebanon, NH 037556. Tel.: 603-650-7912; Fax: 603-650-6223; E-mail:
william.rigby@dartmouth.edu.
Published, JBC Papers in Press, July 9, 2002, DOI 10.1074/jbc.M204002200
The abbreviations used are:
ARE, A + U-rich
element;
AUBP, AU-rich sequence-binding proteins;
GAPDH, glyceraldehyde-3-phosphate dehydrogenase;
GM-CSF, granulocyte-macrophage colony-stimulating factor;
LDH, lactate
dehydrogenase;
UTR, untranslated region;
PBS, phosphate-buffered
saline;
NEPHGE, non-equilibrium pH-gradient electrophoresis;
hnRNP, heterogeneous nuclear ribonucleoprotein;
Pipes, 1,4-piperazinediethanesulfonic acid;
MEL, murine erythroleukemia;
IL, interleukin;
CAPS, 3-(cyclohexylamino)propanesulfonic acid.
Lactate Dehydrogenase Is an AU-rich Element-binding Protein That
Directly Interacts with AUF1*
,,,¶
Departments of Medicine and Microbiology,
Dartmouth Medical School, Lebanon, New Hampshire 03756 and
§ Department of Molecular Genetics and Microbiology,
University of Medicine and Dentistry, Robert Wood Johnson Medical
School, Piscataway, New Jersey 08854
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-globin mRNA
stabilization complex, whereas other studies (12) have implicated AUF1
in B-cell transcriptional activation. The diversity of functions
attributed to AUF1 suggests its activity may be regulated by
protein-protein interactions.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]UTP (PerkinElmer Life Sciences), 20 µM UTP, and 4 mM each ATP, GTP, and CTP
(Amersham Biosciences) in 20 µl as described previously (20).
Following transcription, reaction mixtures were digested with
RNase-free DNase I (Roche Molecular Biochemicals), and
phenol/chloroform was extracted, ethanol-precipitated, and applied to
P30 BioSpin Columns (Bio-Rad). The XhoI fragment of the pXM
vector containing human GM-CSF DNA (provided by Genetics Institute) was
subcloned into the multiple cloning site of the pT7/T319 plasmid
at its BamHI site. The wild type GM-CSF RNA probe was
synthesized by T7 polymerase transcription of this plasmid linearized
with EcoRI. The mutated GM-CSF RNA probe (containing AUGUA
reiterations in lieu of AUUUA pentamers) was generated by PCR and
confirmed by sequencing.
80 °C. Nuclei were pelleted, and
cytoplasmic lysate was sequentially precipitated with the addition of
25, 35, 45, 50, and 75% ammonium sulfate and then dialyzed against
0.5× PBS, 1 mM Pefabloc, 5% glycerol. The 50% ammonium
sulfate fraction (which contained the 36-kDa AUBP activity) was
resuspended in buffer B (50 mM Hepes, pH 7.9, 25 mM KCl, 5 mM MgCl2, 0.5 mM dithiothreitol, 1 mM Pefabloc), and
subjected to carboxymethylcellulose column chromatography with stepwise
salt elution. The 0.25 M KCl eluent was desalted with a P6
BioSpin Column (Bio-Rad), resuspended in 0.1 M KCl, and
applied to a poly(U)-Sepharose (Amersham Biosciences) column. The
36-kDa AUBP activity was eluted from poly(U)-Sepharose in the 1 M KCl fraction. This chromatographic fraction was aliquoted and stored at 80 °C. Protein concentrations were determined by BCA
protein assay (Pierce). The RNA binding assay delineated above was used
to assess the RNA binding activity of each fraction.
rabbit
horseradish peroxidase-conjugated secondary antibody (Bio-Rad).
Coimmunoprecipitation of LDH with AUF1 was detected by
immunoprecipitating from either THP-1 polysomes or CB3 polysomes (10 A260) with AUF1 polyclonal antibody,
followed by immunoblotting with AB1222. The reciprocal experiments were
conducted utilizing the same antibodies. The interaction between LDH
and hsp-70 was detected by immunoprecipitating with AB1222 and then
immunoblotting with -hsp-70 monoclonal antibody (W27) (Santa Cruz
Biotechnology). Depleted lysates represent supernatants of
immunoprecipitations. Reactive antigens were visualized with Supersignal chemiluminescence substrate (Pierce).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (26K):
[in a new window]
Fig. 1.
CB3 erythroleukemia cells retain 36-kDa AUBP
activity despite absence of hnRNP A1. A, nuclear
(Nuc, 10 µg), cytoplasmic (Cyto, 25 µg),
polysomal (Poly, 0.1 A260), and S130 fractions
of DP28-9, CB7, and CB3 murine erythroleukemia cells were separated by
12% SDS-PAGE and analyzed by immunoblotting with the polyclonal
antibodies specific for hnRNP A1 (Act-1) and hnRNP A2 (Act-2) as
described under "Experimental Procedures." B, binding of
p36 to wild type and mutated ARE. [32P]UTP-labeled wild
type and mutated (AUGUA) GM-CSF RNA probes were incubated with 25 µg
of cytoplasmic lysate from DP28-9, CB7, and CB3 cells, UV cross-linked,
RNase-digested, and separated on 15% SDS-PAGE, transferred to
nitrocellulose, and analyzed by autoradiography. C,
immunoblotting for hnRNP A1 of nitrocellulose used in autoradiograph in
B.
1. To verify that
the p36 RNA binding activity in the CB3 cytosol was LDH, a rabbit
polyclonal anti-LDH antibody was used to immunoprecipitate the
radiolabeled RNA-protein complex from UV cross-linked CB3 cytoplasmic lysates (Fig. 2D).
View larger version (18K):
[in a new window]
Fig. 2.
Isolation of p36 AUBP and identification as
LDH-M. A, purification scheme. B, AUBP analysis
of each purification stage, as described in Fig. 1B.
C, CB3 polysomes (0.5 A260) and 1 M poly(U)-purified eluate (50 µg) were UV cross-linked to
[32P]UTP-labeled wild type GM-CSF RNA, RNase-digested,
and then analyzed by two-dimensional NEPHGE and autoradiography.
D, CB3 cytoplasmic lysate (200 µg) was UV cross-linked to
[32P]UTP-labeled wild type GM-CSF RNA, RNase-digested,
and immunoprecipitated with polyclonal anti-LDH antibody (AB1222).
Beads were washed and then analyzed by 15% SDS-PAGE and
autoradiography. Lanes labeled 2, 4, and
6 represent second, fourth and sixth washes, respectively,
indicating elimination of nonspecific binding activity.
View larger version (12K):
[in a new window]
Fig. 3.
LDH specifically binds GM-CSF RNA AURE in
nitrocellulose filter binding assay. Indicated amounts of purified
LDH protein were incubated with [32P]UTP-labeled (80, 000 cpm) wild type or mutant GM-CSF RNA, or globin pre-mRNA and
analyzed for binding to nitrocellulose by scintillation counting. Data
are shown as the % bound of input RNA.
View larger version (21K):
[in a new window]
Fig. 4.
NAD inhibits RNA binding by LDH.
A, inhibition of AUBP activity by UV cross-linking assay.
Purified LDH protein (0.5 µg) was preincubated for 5 min at room
temperature with the indicated concentrations of NAD prior to binding
and UV cross-linking to [32P]UTP-labeled wild type GM-CSF
RNA. B, purified LDH protein (0.5 µg) was preincubated
with indicated concentrations of NAD for 5 min at room temperature and
then analyzed for binding to radiolabeled wild type GM-CSF RNA by
nitrocellulose filter binding and scintillation counting.
View larger version (14K):
[in a new window]
Fig. 5.
Polysomal localization of LDH is dependent on
RNA binding. A, CB3 polysomes (0.2 A260) and a cell equivalent S130 were analyzed
by immunoblotting for LDH with AB1222. B, discontinuous
sucrose density analysis of CB3 cytosol with and without prior EDTA
treatment and analysis by LDH immunoblotting with AB1222. C,
preincubation of polysomes (0.2 A260) with
indicated concentrations of NAD+, analyzed by
immunoblotting with AB1222.
View larger version (15K):
[in a new window]
Fig. 6.
Continuous sucrose density gradient polysome
analysis of THP-1 and CB3 cytosol. Post-mitochondrial and nuclear
supernatant from CB3 MEL and THP-1 monocytic cells was loaded onto
continuous sucrose gradients (10-40% (w/v) sucrose). Following
centrifugation, 18 fractions were collected and analyzed by
immunoblotting with AB1222 and -AUF1 antibodies.
View larger version (28K):
[in a new window]
Fig. 7.
LDH and AUF1 associate in vivo
and in vitro. A,
coimmunoprecipitation of LDH and AUF1. CB3 erythroleukemia and THP-1
monocyte polysomes (0.5 A260) were
immunoprecipitated (IP) with -AUF1 antibody and then
analyzed by immunoblotting using AB1222. Depleted refers to
supernatant unbound to beads; 20% of this fraction was run on
SDS-PAGE, as indicated. B, coimmunoprecipitation of LDH,
AUF1, and hsp-70. THP-1 monocyte polysomes (0.5 A260) were immunoprecipitated with AB1222 and
then analyzed by immunoblotting with -AUF1 antibody and -hsp-70
(W27). Depleted refers to supernatant unbound to beads; 20%
of this fraction was run on SDS-PAGE, as indicated. C, AUF1
interacts directly with LDH in vitro. His-tagged p40
recombinant AUF1 (1.0 µg) coupled to metal affinity beads was
incubated with either 1.0 µg of purified LDH or 1.0 µg of bovine
serum albumin (BSA) as indicated, washed extensively, and
then resolved on a 15% SDS-PAGE gel. Proteins were visualized by
staining with 0.1% Coomassie Brilliant Blue R-250.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and c-Myc) in their 3'-UTR (20). Second,
nitrocellulose filter binding experiments performed with purified
protein revealed the Kd of GAPDH for GM-CSF RNA is
approximately twice that of LDH for GM-CSF RNA (data not shown). These
data indicate that although GAPDH and LDH are capable of binding ARE,
LDH binds the 3'-UTR of GM-CSF RNA with greater affinity than GAPDH
when analyzed as a purified protein. The interaction of LDH in
vivo with other proteins may further enhance this difference in
affinity, as we were unable to detect any GM-CSF RNA binding by GAPDH
in CB3 cytosols, despite operating under conditions of probe excess. In
this regard, immunoprecipitation studies demonstrate that LDH, but not
GAPDH, interacts with AUF1 in vivo (data not shown). These
data reflect the specificity of the LDH/AUF1 association, as well as
suggest that LDH and GAPDH may play different roles in
ARE-dependent gene regulation, with distinct substrate
specificity and function.
-globin mRNA stabilization complex. Similar to our findings with hnRNP A1,
overexpression of AUF1 in the K562 erythroleukemia cell line had no
effect on ARE-mediated mRNA turnover (35). However, when induced by
hemin to undergo erythroid differentiation, ARE-mediated turnover in
K562 erythroleukemia cells was inhibited. Overexpression of AUF1 led to
a restoration of ARE-mediated turnover (35). It is unclear if the
changes in ARE-dependent turnover induced by hemin
treatment or AUF1 were due to effects on translation and loading onto
polysomes. This observation is potentially important as ARE-mediated
turnover has been reported to be dependent on either translation or
ribosomal transit (36). Thus, it is possible that overexpression of
AUF1 restored ARE-dependent mRNA turnover by permitting
polysomal loading of these mRNA.
ACKNOWLEDGEMENTS
FOOTNOTES
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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