From the Program in Molecular Pharmacology and Therapeutics,
Memorial Sloan-Kettering Cancer Center,
New York, New York 10021
The Elav-like proteins are specific
mRNA-binding proteins that regulate mRNA stability. The
neuronal members of this family (HuD, HuC, and Hel-N1) are required for
neuronal differentiation. In this report, using purified HuD protein we
have localized a high affinity HuD binding site to a 42-nucleotide
region within a U-rich tract in the 3'-untranslated region
p21waf1 mRNA. The binding of HuD to this site is
readily displaced by an RNA oligonucleotide encoding the HuD binding
site of c-fos. The sequence of this binding site is well
conserved in human, mouse, and rat p21waf1 mRNA.
p21waf1 is an inhibitor of cyclin-dependent kinases and
proliferating cell nuclear antigen and induces cell cycle arrest at
G1/S, a requisite early step in cell differentiation. The
identification of an Elav-like protein binding site in the
3'-untranslated region of p21waf1 provides a novel link
between the induction of differentiation, mRNA stability, and the
termination of the cell cycle.
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INTRODUCTION |
The Elav-like proteins are tumor antigens that are the human
homologues of the Drosophila protein Elav (1).There are four members of the human Elav-like family, namely HuD, HuC, Hel-N1, and HuR
(1-5). HuD, HuC, and Hel-N1 are exclusively expressed in post-mitotic
neurons and in neuroendocrine tumors. (4, 6, 7). Elav was originally
defined in studies of mutants that were defective in neural function
(8, 9). In Elav mutant flies, neuroblasts fail to completely
differentiate into neurons and result in embryonic lethality. It is
thought that HuD, HuC, and Hel-N1 are also involved in neuronal
differentiation, since they are expressed on termination of the
neuroblast cell cycle (10-12). Indeed, recent evidence has shown that
HuD is necessary for neuronal
differentiation.1 Treatment
of PC12 cells with nerve growth factor leads to cessation of the cell
cycle and differentiation into neuron-like cells with neurite outgrowth
(14). Treatment of PC12 cells with antisense oligonucleotides directed
against HuD abrogated their response to nerve growth
factor.1 In complementary experiments, Wakamatsu and Weston
(10) showed that transfection of HuD into chick neuroblasts accelerated
their differentiation. Thus, the Elav-like proteins are required for neuronal differentiation, and it is important to establish their mechanism of action.
The Elav-like proteins are RNA-binding proteins and contain three RNA
recognition motifs of the RNP2/RNP1 type (15). The first and second of
these RNA recognition motifs are in tandem and are separated from the
third by a segment rich in basic amino acids. Many mRNAs contain
U-rich regulatory elements that direct their rapid turnover (16, 17).
The Elav-like proteins specifically bind to these elements, stabilize
the mRNA, and thereby increase its steady state level. (3, 5,
18-23).1 The current hypothesis is that the Elav-like
proteins promote neuronal differentiation by stabilizing specific
mRNAs. We know that that the Elav-like proteins regulate tau and
GAP-43 mRNAs, two gene products necessary for the later stages of
neuronal differentiation (24).1 However, the Elav-like
proteins are turned on very soon after the last S phase of the
precursor neuroblast (6, 11). This event occurs before the documented
changes in GAP-43 and tau expression. This suggested to us that the
Elav-like proteins may regulate other mRNAs. In particular, we
postulated that the Elav-like proteins may regulate mRNAs involved
in the termination of the neuroblast cell cycle. In nerve growth
factor-treated PC12 cells, it is thought that the cessation of the cell
cycle is primarily mediated by the induction of p21waf1
(25, 26). p21waf1 inhibits cyclin-dependent
kinases and thereby induces cell cycle arrest at G1/S
(27-29). It is known that p21waf1 mRNA has a short
half-life and can be regulated at the posttranscriptional level
(30-34). Thus p21waf1 may be the connection between the
Elav-like proteins and the termination of the neuroblast cell cycle. In
this paper we show that HuD binds to a conserved U-rich element within
the 3'-UTR2 of
p21waf1 mRNA.
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MATERIALS AND METHODS |
Preparation of Labeled RNA Transcripts--
pZL-waf1 plasmid DNA
was digested with the appropriate restriction enzymes and transcribed
with T7 RNA polymerase in the presence of
-[32P]uridine triphosphate (Amersham Pharmacia
Biotech), as described previously (35). p21waf1 was
linearized with SmaI, yielding a full-length transcript of 2121 nucleotides. p21waf1-PstI was linearized
with PstI, yielding a transcript of 383 nucleotides containing the 5'-UTR and part of the open reading framing.
p21waf1-BsrBI was linearized with
BsrBI, yielding a transcript of 622 nucleotides containing
the 5'-UTR, open reading framing, and 52 nucleotides of the 3'-UTR.
p21waf1-BstEII was linearized with
BstEII, yielding a transcript of 1116 nucleotides containing
the 5'-UTR, open reading framing, and 546 nucleotides of the 3'-UTR.
DNA templates for the p21waf1 fragments AC, A, B, C, and
BstEII-end were synthesized by polymerase chain reaction
using the following oligonucleotides. For subfragment AC, corresponding
to 3'-UTR nucleotides 636-1122, the oligonucleotides were (T7A5)
TAATACGACTCACTATAGGCCTTAGTCTCAGTTTG and (C3) GGTCACCCTGCCCAA. For
subfragment A, corresponding to 3'-UTR nucleotides 636-789, the
oligonucleotides were (T7A5) and (A3) ACTCTTAGGAACCTCTCA. For
subfragment B, corresponding to 3'-UTR nucleotides 767-893, the
oligonucleotides were (T7B5)
TCCTAATACGACTCACTATAGGGTTGAATGAGAGGTTCCT and (B3)
GTAGCTGGCATGAAGC. For subfragment C, corresponding to 3'-UTR
nucleotides 874-1122, the oligonucleotides were (T7C5) TCCTAATACGACTCACTATAGGGCCGGCTTCATGCCA and (C3). For subfragment BstEII-end, corresponding to 3'-UTR nucleotides 1114-2121,
the oligonucleotides were (T7BstEII5)
TCCTAATACGACTCACTATAGGAGGGTGACCCTGAAGT and (end3)
TTTTAAAGTCACTAAGAATCA. All polymerase chain reaction-synthesized templates were transcribed with T7 RNA polymerase in the presence of
-[32P]uridine triphosphate (Amersham) and gel-purified
as described previously (35).
Purification of GST-HuD Proteins--
An overnight culture of
Escherichia coli BL 21 transformed with the GST-HuD
construct (20) was diluted 1:50 in LB broth. At an
A600 of 0.4, the culture was induced
with isopropyl-1-thio-
-D-galactopyranoside (0.1 mM). After 4 h of further growth, cells were spun down
and resuspended in 10 ml of buffer A (50 mM Tris (pH 8.0),
200 mM NaCl, 1 mM EDTA). The cells were lysed
by adding lysozyme (0.2 mg/ml) and Triton X-100 (1%). The lysate was
centrifuged at 12,000 × g for 30 min. The resultant
supernatant was loaded onto a glutathione-agarose affinity column (13 mg of protein/ml of resin). After washing the column with buffer B (50 mM Tris (pH 8.0), 200 mM NaCl, 1 mM
EDTA, 1% Triton X-100), GST-HuD was eluted with 50 mM Tris (pH 8.0), 5 mM glutathione. The eluted material containing
a predominant band of 67 kDa was pooled and stored at
70 °C.
Assay of RNA·HuD Complex Formation--
Reaction mixtures (20 µl) contained 50 mM Tris (pH 7.0), 150 mM
NaCl, 0.25 mg/ml tRNA, 0.25 mg/ml bovine serum albumin, 10 fmol of
labeled RNA, and protein as indicated. Mixtures were incubated at
37 °C for 10 min. After incubation, 4 µl of a dye mixture (50% glycerol, 0.1% bromphenol blue, 0.1% xylene cyanol) was added. Five
µl of the reaction mixture was immediately loaded onto a 1% agarose
gel in TAE buffer (40 mM Tris acetate, 1 mM
EDTA) and electrophoresed at 40 V for 2.5 h. The gel was dried
onto DE81 paper (Whatman) backed with gel drying paper and exposed to
XAR5 film (Eastman Kodak Co.) for 4-5 h at
70 °C. All experiments were repeated at least twice with identical results.
RNase T1 Selection Assay--
Reaction mixtures (20 µl)
contained 50 mM Tris (pH 7.0), 150 mM NaCl,
0.25 mg/ml bovine serum albumin, 0.25 mg/ml tRNA, 20 fmol of
radiolabeled mRNA, purified HuD as indicated. After 10 min of
incubation at 37 °C, 0.5 units of RNase T1 was added to each
reaction and incubated at 37 °C for an additional 10 min. The
mixtures were diluted 1:6 with buffer F (20 mM Tris (pH
7.0), 150 mM NaCl, 0.05 mg/ml tRNA) and filtered through
nitrocellulose (BA85, Schleicher & Schuell). After washing the
nitrocellulose twice with buffer F, bound HuD·RNA complex was
extracted with phenol-chloroform and concentrated by ethanol
precipitation. The RNA pellet was dissolved in formamide buffer and
denatured at 65 °C for 2 min. Samples were analyzed by
electrophoresis on a 12% polyacrylamide, 50% urea gel. Gels were
fixed in 1:1:8 acetic acid:methanol:water, dried, and exposed to the
XAR5 film at
70 °C overnight.
Nitrocellulose Filter Binding Assay--
Reaction mixtures (20 µl) contained 50 mM Tris (pH 7.0), 150 mM
NaCl, 0.25 mg/ml bovine serum albumin, 0.25 mg/ml tRNA, 10 fmol of
radiolabeled mRNA, and purified HuD as indicated. After incubation
for 10 min at 37 °C, the mixtures were diluted 1:6 with buffer F and
filtered through nitrocellulose. Filters were washed twice with buffer
F, and bound radioactivity was determined by Cerenkov counting. All
data were reproducible within a fluctuation of 10%.
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RESULTS |
HuD Binds to p21waf1 mRNA--
The structure and
sequence of the full-length p21waf1 mRNA
(p21/SmaI, 2121 nucleotides) is shown in Fig.
1. In the forthcoming text we will refer
to the mRNA as only p21. This will simplify our references to the
various subsegments. The transcript encoding this message is too large
to assay using conventional gel retardation assays. Thus the RNase T1
selection assay (20) was used to ascertain whether the p21 message
contained a binding site for HuD. Radiolabeled transcript was incubated
with purified recombinant HuD protein and treated with RNase T1, a
specific endonuclease that cleaves after G residues. This reaction was
conducted with saturating RNase T1 levels, so that all G residues in
the transcript are hydrolyzed under the reaction conditions used. The
reaction mixture was then filtered through nitrocellulose. RNA/protein
complexes are absorbed to nitrocellulose (36). These complexes were
eluted from the nitrocellulose by phenol/chloroform extraction and
analyzed by gel electrophoresis in order identify the segments of the
transcript that bound HuD.

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Fig. 1.
The structure and sequence of p21 mRNA.
A, the structure of full-length p21 mRNA
(p21/SmaI) is shown, indicating the 5'-UTR followed by the
open reading frame (ORF) (open box), 3'-UTR, and
poly(A) tail. The dark box corresponds to transcript A
(nucleotides 636-789), which contains the major HuD binding site. The
individual transcripts used in these studies are shown along with the
location of relevant restriction sites. B, the sequence of
full-length p21 mRNA. The major HuD binding site (nucleotides
657-698) is boldface.
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Three fragments of 24, 16, and 15 nucleotides were selected from the
full-length transcript (p21/Sma, Fig. 1A) by HuD
(Fig. 2A, lane 2),
whereas no fragments were selected by a GST protein (lane
1), used as a negative control in this experiment. To map these
binding sites more precisely, we used this assay to examine two
truncated transcripts, p21/Pst and p21/BstEII
(Fig. 1A). The p21/BstEII transcript binds
fragments of the same size as does the full-length transcript
(p21/Sma), although the p21/PstI transcript fails
to bind any fragments (Fig. 2A, lanes 2,
5, and 8, respectively). These data suggest that
the HuD binding site lies between the PstI and
BstEII sites. To confirm this, we used the RNase T1
selection assay to analyze the three adjacent transcripts,
p21/BsrBI, p21/BsrBI-BstEII, and
p21 BstEII-Sma (Fig. 1A), that were
synthesized to span the entire length of the p21 message. Since only
the p21 BsrBI-BstEII transcript yielded the
selected fragments (Fig. 2B, lanes 4-6), the HuD
binding site was localized to a region in the 3'-UTR between the
BsrBI and BstEII sites.

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Fig. 2.
HuD binds to elements in the 3'-UTR of p21
mRNA. A, 32P-labeled p21/Sma
(lanes 1-2), p21/BstEII (lanes 4-5),
and p21 Pst (lanes 7-8) transcripts (4 fmol,
20,000 cpm/pmol of UTP) were incubated with 50 nM HuD or
GST as indicated at 37 °C for 10 min. After treatment with RNase T1
(5 units/reaction) at 37 °C for 10 min, the reaction mixtures were
filtered through nitrocellulose. Bound RNA fragments were extracted and
electrophoresed on a 12% acrylamide, 50% urea gel. Lanes
3, 6, and 9 show RNase T1 digestions of the
indicated transcripts before nitrocellulose filtration. Lane
M, size markers, X174/HinfI digest. B,
same as A, except with 32P-labeled
p21/BsrBI (lanes 1-3),
p21/BsrBI-BstEII (lanes 4-6), and
p21/BstEII-Sma (lanes 7-9)
transcripts (7 fmol, 45,000 cpm/pmol of UTP).
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Quantitative Determination of the Affinity of HuD for
p21waf1 mRNA--
To quantitate the relative binding
affinities of HuD to p21waf1 mRNA, we employed the
method originally used for the R17 coat protein (36). A low amount of
labeled RNA (350 pM) was incubated with HuD protein under
conditions of protein excess. The reactions were filtered through
nitrocellulose, and the bound radioactivity was measured. Fig.
3A shows that the formation of
p21/BsrBI-BstEII RNA·HuD complex is first
detectable at a HuD concentration of 0.15 nM and plateaus
at a concentration above 500 nM with about 72% of the
input RNA bound. In contrast, complex formation with p21/BsrBI was not detectable even at 1000 nM
HuD. A plot of the log of complex/free RNA versus the log of
HuD concentration is shown in Fig. 3B. From this plot, the
Kd of the
p21/BsrBI-BstEII·HuD complex was determined to
be 62.7 nM. Thus the affinity of HuD for p21 mRNA is
similar to what we determined for c-fos mRNA
(Kd = 19 nM) (20).

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Fig. 3.
Quantitation of the affinity of HuD for
regions of p21 mRNA 3'-UTR. RNA-protein complex formation was
assayed by nitrocellulose filtration. 32P-Labeled
transcripts as indicated (14 fmol, 45,000 cpm/pmol of UTP) were
incubated with the indicated concentrations of HuD at 37 °C for 10 min. A, a plot of the percentage of RNA bound
versus the log of HuD concentration. B, plot of
the log of complex/free RNA versus log of HuD concentration.
, p21/BsrBI-BstEII; , p21/BsrBI.
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Mapping of the HuD Binding Site--
We next examined the sequence
of the p21/BsrBI-BstEII transcript (nucleotides
636-1122, Fig. 1B) to delineate the likely location of the
15-, 16-, and 24-nucleotide binding sites. Based on this analysis, we
divided p21/ BsrBI-BstEII segment into three
contiguous transcripts: A, B, and C. Transcript A contains a
16-nucleotide RNase T1 fragment separated from a 24-nucleotide RNase T1
fragment by two nucleotides, whereas B contains two adjacent
15-nucleotide fragments, and C contains only a single 16-nucleotide
fragment. Transcripts A, B, C, and
p21 BsrBI-BstEII were then tested for HuD
binding by gel retardation analysis (Fig.
4A). Purified recombinant HuD
was incubated with labeled transcript and assayed for complex formation. HuD binds both p21/BsrBI-BstEII and A
with similar affinity (lanes 3-5 and 8-10,
respectively) and with a considerably lower affinity to B (lanes
13-15). No binding to C was observed within the range of HuD
concentrations tested (lanes 18-20). As expected, no
complex formation was observed with transcripts A, B, or C when HuD was
replaced by GST (lanes 2, 7, 12, and
17). These semiquantitative observations were confirmed by
nitrocellulose binding assays by which the Kd values
of p21/BsrBI-BstEII, A, B, and C were determined
to be 62.7, 85.6, 757, and 10,700 nM, respectively. On the
basis of these data, it is likely that transcript A contains the major
HuD binding site within the 3'-UTR of p21waf1 mRNA. A
minor HuD binding site appears to also be present in B, whereas no site
with significant affinity for HuD is observed in C.

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Fig. 4.
Fine mapping of the HuD site.
A, 32P-labeled
p21/BsrBI-BstEII (lanes 1-5), A
(lanes 6-10), B (lanes 11-15), and C
(lanes 16-20) transcripts (7 fmol, 45,000 cpm/pmol of UTP)
were incubated with the indicated concentrations of GST or HuD. After
incubation at 37 °C for 10 min, 20% of the reaction mixtures were
resolved by gel electrophoresis on a 1% agarose gel. B,
32P-labeled p21/BsrBI (lanes 1-2), A
(lanes 4-5), and B (lanes 7-8) transcripts (7 fmol, 45,000 cpm/pmol of UTP) were incubated with 50 nM HuD
or GST as indicated at 37 °C for 10 min. After treatment with RNase
T1 (5 units/reaction) at 37 °C for 10 min, the reaction mixtures
were filtered through nitrocellulose. Bound RNA fragments were
extracted and electrophoresed on a 12% acrylamide, 50% urea gel.
Lanes 3, 6, and 9 show RNase T1
digestions of the indicated transcripts before nitrocellulose
filtration. Lane M, size markers, X174/HinfI
digest. C, the selected fragments (A24, A16, and B15) corresponding to
the 24 and 16 nucleotide bands derived from transcript A and the 15 nucleotide band derived from transcript B, as observed in Fig. 7, were eluted from a preparative gel and
analyzed after further digestion with RNase T1, 0.5 units/reaction
(lanes 2, 4, and 6, respectively) and
with no further RNase T1 digestion (lanes 1, 3,
and 5, respectively). Unbound transcript B was treated with
RNase T1 (lane 8) or untreated (lane 7) under the
same conditions. Lane M, size markers,
X174/HinfI digest.
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To confirm the location of the HuD binding sites within
p21waf1 mRNA, transcripts A and B were further analyzed
by the RNase T1 selection assay (Fig. 4B). Fragments of 24 and 16 nucleotides in length were selected after incubation of
transcript A with HuD (lane 5), whereas a 15-nucleotide
fragment was the predominant species selected after the binding of
transcript B to HuD (lane 8). As expected, no fragments were
selected when HuD was incubated with p21/BsrBI (lane
2) or from any of the transcripts incubated with GST (lanes
1, 4, and 7). In the RNase T1 selection
assay, it is possible that a selected fragment could result from the protection by HuD of one or more G residues situated between two smaller fragments. The three selected fragments (24, 16, and 15 nucleotides) were gel-purified and incubated with RNase T1 to determine
whether they contain any protected G residues (Fig. 4C,
lanes 2, 4, and 6 and lanes
1, 3, and 5, respectively). No new digestion
products were observed, thus indicating that the observed fragments are
as predicted 24, 16, (transcript A), and 15 nucleotides (transcript B)
in length. To ensure that the Rnase T1 used in these experiments was
active, the entire transcript B was digested by RNase T1 as a positive
control (lane 8). Thus, based on the data
obtained by gel retardation, RNase T1 digestion, and nitrocellulose
binding assays, we conclude that the major binding site in
p21waf1 mRNA is localized to a 42-nucleotide region
(nucleotides 657-698) within the 3'-UTR (Fig. 1B,
boldface). This site is similar to other HuD binding sites
in that it contains U-rich tracts (19, 20, 24). However, unlike other
known HuD binding sites, two G residues are present within the site in
p21waf1, and we obtained two fragments in the selection
assay. If the localization of the 24- and 16-nucleotide fragments is
correct, we should be able to select a 42-nucleotide fragment from a
partial digest of p21 mRNA. Thus, we generated partial digests of
p21/A using limiting concentrations of RNase T1 (Fig.
5A, lanes 7-12). As before, HuD selected fragments of 24 and 16 nucleotides from complete digests (lanes 1-2). However, as predicted, HuD
selected a 42-nucleotide fragment from the first partial digest and
progressively larger fragments from less digested RNA (lanes
3-5). Concomitant with the selection of the 42-nucleotide
fragment was the disappearance of both the 24- and 16-nucleotide
fragments (lanes 3-4). At all concentrations of RNase T1
tested, HuD binding to these fragments was specific. This was shown by
the lack of selected fragments from similar partial digests of a
transcript that lacks a HuD binding site (Fig. 5B,
lanes 1-12). These data indicate that the minimal HuD
binding site is 42 nucleotides in length.

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Fig. 5.
The effect of RNase T1 concentration on the
selection of HuD binding sites. A, lanes 1-6,
32P-labeled p21/BsrBI-BstEII (7 fmol,
45,000 cpm/pmol of UTP) was incubated with 100 nM HuD or
GST as indicated at 37 °C for 10 min. After treatment with the
indicated amounts of RNase T1 at 37 °C for 10 min, the reaction
mixtures were filtered through nitrocellulose. Bound RNA fragments were
extracted from the filter. RNA (lanes 7-12) was also
incubated with the indicated amounts of RNase T1 at 37 °C for 10 min. 10% of the reaction mixtures were electrophoresed on a 12%
acrylamide, 50% urea gel. Lane M, size markers,
X174/HinfI digest. B, same as for
A, except with transcript p21/BsrBI, as a
negative control.
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The Site in p21 mRNA Is Similar in Structure to Other HuD Sites
and Is Well Conserved--
We have compared the sequence of the p21
mRNA binding site with those we have found in c-fos,
c-myc, tau, interleukin-3, and GAP-43 mRNAs (Fig.
6A). The p21 binding site is
similar to the others in that it contains U-rich tracts. We have shown
that these U-rich tracts are essential for the binding of HuD to the
c-fos element (20). The importance of the U tract sequences
in the p21 site is further underscored by their strict conservation
between human, mouse, and rat p21 mRNAs (Fig. 6B).

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Fig. 6.
The site in p21 mRNA is similar in
structure to other HuD sites and is well conserved. A,
the binding sites in c-fos, interleukin-3, c-myc,
tau, GAP-43, and p21 mRNAs are shown. U tracts are
underlined. B, the 42-nucleotide HuD binding site
in the human p21waf1 mRNA was aligned with the
corresponding regions in both mouse and rat p21waf1
mRNA. ClustalW Formatted Alignment analysis was carried out using
MacVector 6.0 (Oxford Molecular LTD).
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Next, we tested whether the HuD·p21mRNA complex would be
displaced by the c-fos binding site. The sequence
AUAUUUAUAUUUUUAUUUUAUUUUUUU, termed the c-fos element,
has been determined to be the minimal HuD binding site within
c-fos mRNA (20). Another oligonucleotide, a mutated
c-fos element with the sequence AUACGUAUAUCGCUAUGCUAUUUUUUU that fails to bind HuD, served as the negative control in these experiments. As shown in Fig. 7, the
addition of unlabeled c-fos element oligonucleotide in molar
excess displaced the HuD·p21/BsrBI-BstEII complex in a gel retardation assay (lanes 3-5). No complex
displacement was observed when the same molar amounts of the mutated
c-fos element was added (lanes 6-8). We can
therefore conclude that the binding site in p21 is similar to the one
in c-fos mRNA and indeed to sites in c-myc,
tau Il-3, and GAP-43 mRNAs.

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Fig. 7.
The HuD/p21 mRNA complex is displaced by
the c-fos site 32P-labeled
p21/BsrBI-BstEII RNA (7 fmol, 45,000 cpm/pmol
of UTP) was incubated with 200 nM HuD. The indicated
molar excesses of the synthetic oligonucleotides (c-fos
element AUAUUUAUAUUUUUAUUUUAUUUUUUU or mutant c-fos
element AUACGUAUAUCGCUAUGCUAUUUUUUU) were added to the reactions
(lanes 3-5 and lanes 6-8, respectively). After
incubation at 37 °C for 10 min, 20% of the reaction mixtures were
resolved on a 1% agarose gel.
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DISCUSSION |
The Elav-like proteins have emerged as an important class of
proteins that are pleotropic effectors of gene expression in mammalian
cells. There are four members of the human family HuD, HuC, Hel-N1, and
HuR (1-4). These RNA-binding proteins regulate gene expression via a
specific interaction with U-rich elements in mRNA (18,
20-23).These U-rich elements are found in a wide variety of mRNAs
and usually target them for rapid degradation (17). The current model
is that activation of the Elav-like proteins leads to the stabilization
of a specific subset of mRNAs. It is likely that this
mechanism is responsible for the induction of neuronal differentiation
by the neuronal-specific Elav-like proteins. Some of the target
mRNAs stabilized by the Elav-like proteins during neuronal
differentiation have been identified. Two targets, GAP-43 and
tau, are required for the later steps in neuronal differentiation
(24).1 The neuronal Elav-like proteins, however, are
expressed before the up-regulation of GAP-43 and tau mRNA. Thus we
have looked for new mRNAs targets that may directly affect the
neuroblast cell cycle. In nerve growth factor-treated PC12 cells, there
is compelling evidence to suggest that the induction of
p21waf1, an inhibitor of cyclin-cdk complexes as well as
proliferating cell nuclear antigen, plays a critical role in arresting
these cells at G1/S, a requisite early step in cell
differentiation (25, 26). Thus we examined the interaction between
p21waf1 mRNA and HuD. In this report, we have localized
a high affinity HuD binding site to a conserved 42-nucleotide region
within a U-rich tract in the 3'-UTR of p21waf1 mRNA.
Thus, given that the Elav-like proteins regulate other mRNAs with
similar sites, it is very likely that the Elav-like proteins will
regulate p21waf1 mRNA. With the detailed knowledge of
the p21waf1 binding site reported here, experiments to
directly test this possibility can now be carried out.
Surprisingly, possible changes in the half-life of p21waf1
mRNA during neurogenesis have not yet been examined. Recent
studies, however, in other cell systems have shown that the half-life
of p21waf1 is regulated by diverse signals. In these
systems, regulation cannot be due to binding of HuD or any of the other
neuronal-specific members of the family. Rather, it is more likely to
be mediated by the ubiquitously expressed member of the Elav-like
protein family, HuR, which has also been shown to bind to this region of the p21waf1 message (data not shown).
p21waf1 is up-regulated in p53-deficient promyelocytic
HL-60 cells induced to differentiate along the monocytic lineage by
phorbol ester or 1
,25-dihydroxyvitamin D3. (30). The
marked increases of both p21waf1 mRNA and protein
expression observed in these cells is due to enhanced mRNA
stability. Similar increases in p21waf1 mRNA stability
were observed in MCF-7 breast carcinoma cells (37) and in MDA-MB-468
cells (a p53-mutant derivative of MCF-7 cells) induced to differentiate
with the retinoid CD437 (31). Zeng and El-Deiry (32) found that okadaic
acid, a potent serine/threonine phosphatase inhibitor, induced
p53-independent expression of p21waf1, both at the
transcriptional and posttranscriptional levels in several human tumor
cell lines. It has recently been reported (13) that UVC irradiation
elevates p21waf1 mRNA expression in mouse embryonal
fibroblasts and human rectal carcinoma cells by significantly enhancing
the stability of the mRNA encoding p21waf1. They
provide experimental evidence indicating that this effect is mediated
by a tyrosine kinase/phosphatase regulatory system (possibly one
modulated by c-Jun NH2-terminal kinase) and, in contrast to
the systems already discussed, requires the presence of functional p53.
Thus it will be interesting to see if the binding of the Elav-like
proteins to p21waf1 mRNA is activated by any of these
signals.
The connection drawn here between mRNA stability, differentiation
and the cell cycle is a novel one. It has been tacitly assumed that
transcriptional control is the rate-limiting pathway that defines
commitment to differentiation. A distinctive feature of the mechanism
postulated here is that the Elav-like proteins are capable of
coordinately regulating a subclass of mRNAs. We have recently
observed that the Elav-like proteins also bind to p27 mRNA, another
negative regulator of the cell
cycle.3 These observations
raise the possibility that abrogation of Elav-like protein activity in
differentiated cells may coordinately destabilize mRNAs encoding
negative regulators of the cell cycle. Indeed an important future goal
will be to investigate whether this would lead to reentry into S phase
and cell proliferation.