Immediate early genes are rapidly and transiently expressed on
stimulation of cells by a wide variety of agents, including growth
factors, cytokines, and neurotransmitters(1) . Although such
mRNAs have diverse cellular functions, they share the unifying feature
of a very short half-life(2) . As a result of this short
half-life, they can be regulated by alteration of either the rate of
transcription or the rate of degradation. The signal transduction
pathway that results in the increase in transcription of these genes is
relatively well understood(3) . Alteration of the rate of
degradation is an equally important pathway. The mechanism underlying
this pathway is poorly understood. Many immediate early mRNA's
are targeted for rapid degradation by virtue of cis-acting elements in
their 3`-UTR(
)(2, 4) . Shaw and Kamen (5) were the first to identify a cis element responsible for
regulated mRNA degradation. They showed that a 51-nucleotide AU-rich
element (ARE) from the 3`-UTR of
granulocyte-macrophage-colony-stimulating factor could increase the
degradation rate of any mRNA(5) . Subsequent work has shown
that similar functional ARE elements can also be identified in other
mRNAs(6, 7, 8) . The ARE element in IL-3 mRNA
is of particular interest, since the induction of IL-3 expression in
stimulated PB-3C cells is solely the result of the inhibition of mRNA
degradation(9) . There is little primary sequence similarity
between AREs from different mRNAs. The unifying feature appears to be
AUUUA within an AU-rich background. Recent studies have shown that
monomers such as UUAUUUAUU can function as destablizing elements albeit
at lower efficiency than the complete ARE element(10) .
Comparison of a number of destabilizing elements, however, has shown
that the presence of AUUUA in an AU-rich background does not
necessarily confer instability(11, 12) . Moreover some
potent AU-rich destabilizing sequences do not contain AUUUA. Thus it is
likely that the determinant is a common structural feature not readily
discernible from the primary sequence.
The cellular factors that
bind to the ARE element and regulate gene expression are the subject of
much investigation. Using RNA binding assays several groups have
identified proteins that interact with ARE
elements(13, 14, 15, 16, 17, 18) .
These activities fall into two classes called AU-A and AU-B/C. The AU-A
class bind to the AU-rich sequences in
granulocyte-macrophage-colony-stimulating factor, IL-3, c-fos,
and c-myc mRNAs(13, 14, 16, 17, 19) .
This activity is located in the nucleus, although inhibition of
cellular transcription can result in redistribution to the cytoplasm (20) . In HeLa cells, the sequence specificity of this factor
correlates with the sequences that are required for Ad IVA2 mRNA
destabilization(17) . The AU-B/C class is distinct in that they
bind to the 3`-UTR of cytokine mRNAs but not to the ARE of c-myc mRNA. Their importance is underscored by the observation that
their activity increases in concert with cytokine mRNA induction after
T-cell stimulation(14) . Factors that may be required for
ARE-mediated degradation have also been identified using a cell-free
system(21) . Purification and cloning of one of these factors
(called AUF1) has revealed that it binds to AREs(22) .
The
Elav-like RNA-binding proteins (HuD, HuC, and Hel-N1) are also good
candidates for trans-acting factors involved in selective mRNA
degradation, since they bind to ARE elements with high affinity and
selectivity(23, 24, 25, 26) . The
Elav-like proteins are only expressed in brain. ARE-mediated
degradation occurs in all cell types. Thus we decided to look for
Elav-like homologues which are expressed in non-neuronal cells. Using
degenerate oligonucleotides based on the Elav-like protein RNA binding
domain, we have cloned a new Elav-like gene that is expressed in all
tissues and whose protein product (HuR) binds to AREs with high
specificity and affinity.
EXPERIMENTAL PROCEDURES
Materials
HeLa cell total RNA was prepared from
a cytoplasmic extract kindly provided by Dr. J. Hurwitz (Program in
Molecular Biology, Memorial Sloan Kettering Cancer Center. Total brain,
thymus, muscle, and liver RNAs were purchased from Clontech. RNase T1
was obtained from Calbiochem.
Primers
The primers used were as follows: DG1,
5`-TGGTG(CT)AT(ACT)TT(CT)GT(ATCG)TA(CT)AA-3`; DG2,
5`-AC(GA)AA(ATGC)CC(GA)AA(ATGC)CC(TC)TT(GA)CA-3`; HuR5,
5`-TCGCAGCTGTACCACTCGCCAG-3`, HuR3, 5`-CCAAACATCTGCCAGAGGATC-3`.
PCR Cloning of HuR
Two degenerate primers were
designed that spanned residues 263-309 of HuD. This segment was
chosen, since it contained two cysteine residues that are peculiar to
the Elav-like class of RNA binding proteins. HeLa RNA was converted to
cDNA using reverse transcriptase. PCR was carried out with the
following Temp profile: Program, 2 h at 93 °C; 1 min at 33 °C;
2 min at 72 °C; 1 min at 92 °C (10 cycles); 1 min at 38 °C;
2 min at 72 °C; 1 min at 92 °C (30 cycles); 2 min at 38 °C;
10 min at 72 °C. An RT-dependent product of 140 nucleotides was
observed and was subcloned. The subclone pRNP49 was sequenced and found
to be similar but unique to HuD, HuC, and Hel-N1. This subclone was
identical in sequence to clone 33CC12, which was identified by Chris
Campbell. We used the insert of clone 33CC12 to isolate a full-length
cDNA. 33CC12 DNA was isolated and digested with the EcoRI. The
0.7-kilobase pair EcoRI insert was isolated and labeled by the
random hexamer priming method with [
-
P]dCTP
(Amersham Corp.). This labeled DNA was used to screen a HeLa cDNA
library obtained from Stratagene (La Jolla, CA). The recombinant phage
library was screened at a density of 1
10
plaque-forming unit on 150-mm plates of Escherichia coli strain BB4 (Stratagene). A single clone called pHuR9 was isolated,
purified, and converted to pBluescript plasmid by the phage rescue
protocol according to the manufacturer's instructions.
Restriction digestion of the plasmid DNA with EcoRI revealed
an insert of 2.5 kilobase pair. Double-stranded DNA was sequenced on
both strands using SK, KS, M13 universal, and reverse primers and
internal oligonucleotide primers. Sequences were merged and analyzed
for open reading frame and functional motifs with the MacVector
analysis software.
RT-PCR Analysis of HuR Expression
Total RNA (2
µg) from various human tissues (obtained from Clontech) was
incubated with 200 units of RT for 60 min at 37 °C in a total
reaction volume of 20 µl containing the following: 1 mM each dNTP, 28 units of RNase inhibitor, and 2.5 µM random hexamer primers. RT reactions were terminated by incubation
at 99 °C for 5 min and used directly for subsequent PCR
amplification of specific cDNAs. One-twentieth of the RT reaction
product was added to a PCR (20 µl final volume; which contained 50
mM Tris (pH 9.5), 1.5 mM MgCl
, 20 mM ammonium sulfate, 0.25 mM each dNTP, 5 µCi of
[
-
P]dCTP, 0.5 µM each HuR5 and
HuR3 primer and 0.5 unit of Taq polymerase (Perkin-Elmer). PCR
analyses were performed in an automated DNA clonal cycler (Ericomp)
with the following temperature profile: 3 min at 95 °C; 30 cycles
of 30 s at 65 °C, 30 s at 72 °C, and 30 s at 95 °C; 30 s at
65 °C; and 2 min at 72 °C. One-fifth of the PCR product was
electrophoresed on a 6% acrylamide gel, and the PCR products were
analyzed by autoradiography.
Construction and Purification of HuR-GST Fusion
Protein
A cDNA encoding residues 2-326 of HuR was
generated using PCR with BamHI-linked 5` primer and EcoRI-linked 3` primer. The resultant product was digested and
ligated into digested pGEX2T(27) . The resultant construct was
called pGEX-HuR. An overnight culture of E. coli BL 21,
transformed with pGEX-HuR, was diluted in 1:50 LB medium. At an A
of 0.4, the culture was induced with IPTG
(0.04 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 and Triton to a final concentration of 0.2 mg/ml and 1%,
respectively. 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), GST-HuR was
eluted with 50 mM Tris pH 8.0/5 mM Glutathione.
Protein concentration was measured using the Bradford assay. Thrombin
digestion of the purified fusion protein revealed a 26-kDa band
corresponding to GST and a 36-kDa band corresponding to HuR. Protein
fractions were pooled and stored at -70 °C.
Preparation of RNA Transcript
Plasmid DNAs were
digested with the appropriate restriction enzymes and transcribed in
the presence of [
P]uridine triphosphate
(Amersham) as described previously(23, 28) .
pNMUTR
was linearized with HindIII and transcribed with
T7 RNA polymerase to yield a transcript of 496 nucleotides of the
3`-UTR of N-myc mRNA. pNMUTR
was provided by Dr. Sue Cohn
(Northwestern). All transcripts are gel-purified as described
previously(28) . pIL-3 and pIL-3
AU were linearized with EcoRI and transcribed with T7 RNA polymerase to yield
transcripts of 419 and 203 nucleotides, respectively. pIL-3 and
pIL-3
AU were provided by Dr. Christoph Moroni(29) .
Ribonucleotides were chemically synthesized at a 0.2-µmol scale on
a 392 DNA/RNA synthesizer (Applied Biosystems, Foster City, CA) using
RNA phosphoramidites (Glen Research Corp., Sterling, VA).
Ribonucleotides were end-labeled using T4 kinase and
[
-
P] ATP to a specific activity of 1.5
10
cpm/pmol oligonucleotide and gel-purified.
RNA Complex Assay
Reaction mixtures (.02 ml)
contained 50 mM Tris (pH 7.0), 150 mM NaCl, 0.25
mg/ml tRNA, 0.25 mg/ml bovine serum albumin, 4 fmol of labeled RNA, and
protein as indicated. Mixtures were incubated at 37 °C for 10 min.
Following incubation, 5 µl of a dye mixture (50% glycerol, 0.1%
bromphenol blue, 0.1% xylene cyanol) was added, and 5 µl of the
mixture was immediately loaded on a 0.8% agarose gel in TAE buffer (40
mM Tris acetate, 1 mM EDTA). The gel was then
electrophoresed at 40 V for 2.5 hours. The gel was dried on DE-81 paper
(Whatman) with a backing of gel drying paper (Hudson City Paper, West
Caldwell, NJ) and exposed to XAR5 film (Eastman Kodak Co.) for 6 h at
-70 °C.
Nitrocellulose Filter Binding Assay
Reaction
mixtures (0.02 ml) contained 50 mM Tris (pH 7.0), 150 mM NaCl, 0.25 mg/ml bovine serum albumin, 0.25 mg/ml tRNA, 4 fmol of
radiolabeled mRNA, and purified HuR as indicated. After 10 min of
incubation at 37 °C, 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 filter twice with buffer F, bound radioactivity was
determined by Cerenkov counting.
RNase T1 Protection Assay
Reaction mixtures (0.02
ml) contained 50 mM Tris (pH 7.0), 150 mM NaCl, 0.25
mg/ml bovine serum albumin, 0.25 mg/ml tRNA, 4 fmol of radiolabeled
mRNA and purified HuR as indicated. After 10 min of incubation at 37
°C, RNase T1 was added and the reaction continued for a further 10
min. The mixtures were diluted 1:6 with buffer F and filtered through
nitrocellulose (BA85, Schleicher & Schuell). After washing the
nitrocellulose twice with buffer F, bound HuR-RNA complex on
nitrocellulose filter was eluted with buffer F and extracted with
phenol-chloroform. The resultant RNA was mixed with formamide buffer,
denatured at 65 °C for 3 min, and analyzed by 12%
polyacrylamide-urea gel electrophoresis. The gel was fixed with 1:1:8
acetic acid:methanol:water, dried on DE-81 paper with a backing of gel
drying paper, (Hudson City Paper) and exposed to the XAR5 film at
-70 °C overnight.
RESULTS
Isolation and Cloning of HuR
We sought to
establish whether there were other Elav-like family members that also
bind to ARE elements and are expressed in non-neuronal cell types. We
designed PCR primers that are derived from a conserved region adjacent
to RNA recognition motif III of HuD (Fig. 1A). RT-PCR
analysis of mRNA from HeLa cells revealed the presence of an
RT-dependent product of 140 nucleotides. This product was subcloned
(pRNP49), sequenced on both strands, and found to be highly related to,
but distinct from, HuD, HuC, and Hel-N1 sequences. We decided to call
this gene product HuR. An overlapping cDNA clone (p33CC12) was
fortuitously provided by C. Campbell who observed its similarity to the
Elav-like family. p33CC12 was independently isolated as part of a
subtraction screen for novel genes expressed in human kidney. The
sequence of p33CC12 confirmed that HuR was a new Elav-like family
member and indicated that it was the human homologue of the Xenopus gene elrA(30) . We then used p33CC12 to isolate a
full-length overlapping clone, which we called pHuR9. These three cDNA
clones comprise an open reading frame encoding a basic protein of 326
amino acids (predicted molecular mass of 36 kDa) starting with an AUG
at position 119 and terminating at position 1099 (Fig. 1B). Although no in-frame termination codon was
found preceding the first AUG at position 119, this AUG is probably the
true initiating AUG, since it satisfies the Kozak consensus rule and
encodes an N terminus with high homology to the N terminus of Xenopus elrA. Examination of the rare codon usage also
indicates that the sequences preceding the AUG at position 119 do not
code for protein. The predicted protein has an identical domain
arrangement as the other Elav-like family members (Fig. 2)(23, 25, 31, 32) . A
short N-terminal domain is followed by two RNA binding domains, a basic
linker domain, and a third RNA binding domain. The N-terminal segment
is the most diverse domain among the Elav-like proteins(23) .
The HuR N terminus has little homology to that of HuD, HuC, or Hel-N1,
but is almost identical to the Xenopus homologue
elrA(30) . The basic domains of HuD, Hel-N1, and HuC are
alternatively spliced yielding HuDpro, HuDmex, Hel-N2, and HuC
isoforms(23, 33) . The sequence of clones p33CC12 and
pHuR9 predict that HuR is not spliced in this domain and corresponds
most exactly with the HuDmex and Hel-N2 isoforms (Fig. 2). This
was confirmed by RT-PCR analysis of HuR mRNA (see below).
Figure 1:
A, organization of pRNP49, p33cc12 and
pHuR9 cDNA clones. The scale denotes nucleotide length in kilobase
pairs. The open box labeled open reading frame (ORF)
denotes the predicted open reading frame. The triplicate structure of
the putative RNA recognition motifs (RRM I, RRM II, and RRM III) is illustrated by the solid black boxes. B, nucleotide sequence and predicted open reading frame of
HuR. The accession number of this sequence is
U38175.
Figure 2:
Sequence alignment of HuC, Hel-N2, HuDmex, elrA, and HuR. Homology of the amino acid sequences was
analyzed by the Geneworks protein alignment program. Shaded residues indicate identity.
Expression of HuR
We have used the RT-PCR assay to
detect HuR mRNA in various human tissues. We designed primer pairs that
are specific for HuR and that spanned the putative alternative splice
site found in HuD and Hel-N1. With these primers, we not only measured
the expression of HuR mRNA, but also determined the splice site used. Fig. 3shows the RT-PCR analysis of HuR mRNA expression. A
reverse transcriptase-dependent product of 207 nucleotides was detected
using mRNA from all tissues assayed. The size of the RT-PCR product
indicates that the major form of HuR mRNA is the structure defined by
pHuR9 and p33CC12. Thus HuR most resembles HuDmex and Hel-N2.
Additionally, a larger product of 420 base pairs was also detected.
Further analysis of this product indicated that it was a PCR-generated
dimer of the 207-base pair product.
Figure 3:
Total RNA from various human tissues was
assayed for HuR mRNA by RT-PCR. The indicated tissue RNA was assayed in
the absence(-) and presence (+) of reverse transcriptase.
DNA markers (ØX174/HinfI digest) are shown in lane
M.
HuR Binds to the ARE of c-fos mRNA
The open
reading frame from pHuR9 was subcloned into the GST vector and the
fusion protein purified by affinity chromatography. By virtue of its
significant homology to other Elav-like proteins, we anticipated that
HuR would bind to AU-rich elements. In these studies we have used
transcripts derived from the 3`-UTR of c-fos mRNA, since its
ARE has been functionally defined by deletion analysis(8) . In
particular we have used a 214-nucleotide transcript (called AUFL) which
encompasses sequence from residues 568 to 781 downstream of the
c-fos stop codon. Fig. 4A shows that purified
HuR quantitatively converts the AUFL transcript to a stable protein-RNA
complex that migrates slowly on agarose gel electrophoresis. Complex
formation was proportional to the concentration of HuR and was not
apparent with high concentrations (480 nM) of GST. The complex
formed by HuR migrated faster than the comparable complex formed by
HuD. This difference may be due to differences in molecular weight or
charge. Significantly less complex formation was observed with a
transcript in which the ARE had been deleted (AU1/2) (Fig. 4B, lanes 4-6). Thus like HuD, HuR
specifically binds to the ARE of c-fos mRNA(26) . The
binding of HuR within the ARE was further analyzed by T1 selection
analysis. Fig. 5shows that HuR binds to three fragments of 43,
35, and 20 nucleotides, respectively, with the AUFL transcript. No
fragments were selected on incubation with the ARE deletion transcript.
Subsequent T1 digestion of the isolated selected fragments revealed
that HuR binds predominantly to the 35-nucleotide T1 fragment within
the c-fos ARE (Fig. 5B). The 35-nucleotide T1
fragment contains AUUUA, AUUUUA, and the AUUUUUA motifs. The minor
43-nucleotide fragment arises by the protection of the G residues
adjacent to the 35-nucleotide fragment in a minority of complexes. The
selection of the 20-nucleotide fragment at high concentrations of HuR
reveals the presence of a second independent low affinity binding site.
We next determined whether the HuR-AUFL complex would be displaced by a
molar excess of the unlabeled 35 nucleotide T1 fragment. A 35-mer, a
23-mer corresponding to the low affinity site, and an irrelevant 21-mer
(5`-CUAGAGUUCAUCGCAAUUGCA-3`) were synthesized. HuR-AUFL complex
formation was inhibited by a molar excess of the 35-mer, but no
inhibition was observed with the 23-mer (the low affinity site) or the
irrelevant 21-mer (Fig. 6). From these experiments it appeared
that the 35-nucleotide fragment could be the minimal binding site.
Accordingly we investigated the binding of HuR to the 35-nucleotide
fragment and also to a set of 3` deletions. HuR bound to the
35-nucleotide fragment with similar affinity (apparent K
is 3 nM) as that displayed with the AUFL transcript
(apparent K
is 2) (compare Fig. 7A with Fig. 8C). Comparable reactivity was also
noted with the 27 nucleotide fragment, but substantially less was
observed with the 20- and 13-nucleotide fragments. Thus we concluded
that the 27-nucleotide fragment is the minimal binding site. The most
obvious feature of the 27-nucleotide fragment is the presence of three
AU motifs, namely AUUUA, AUUUUA, and AUUUUUA. We tested the requirement
of these motifs by synthesizing mutant fragments in which the Us were
changed to Gs and Cs (Fig. 7B). Alteration of the AUUUA
motif had only a modest effect on binding (the apparent K
of the 27-mer is 6 nM, whereas the
apparent K
of 27-2 is 12 nM). Mutation,
however, of either the AUUUUA or AUUUUUA motifs had a significant
effect (27-3 and 27-4 exhibited only 29% binding at 100 nM of
HuR). Mutation of any two of the AU motifs further decreased binding,
and mutation of all three AU motifs completely eliminated binding. Thus
we conclude that all three AU motifs are required for maximal binding,
but that the AUUUUA and AUUUUUA motifs play the most important role.
Figure 4:
HuR
binds to the ARE of c-fos mRNA. A,
P-labeled AUFL RNA (3.7 fmol, 2.0
10
cpm/pmol of UTP) was incubated with the indicated concentration
of GST, HuR, or HuD. Following incubation at 37 °C for 10 min, 25%
of the reaction mixture was resolved by gel electrophoresis in a 0.8%
agarose gel. B,
P-labeled AUFL and AU1/2 RNA (3.7
fmol, 2.0
10
cpm/pmol of UTP) was incubated with
the indicated concentration of GST or HuR as described
above.
Figure 5:
RNase
T1 mapping of the HuR-ARE complex. A,
P labeled AU1/2 (lanes 1-3) or AUFL (lanes
5-7) RNA (3.8 fmol, 9
10
cpm/pmol of
UTP) were incubated with the indicated concentration of HuR at 37
°C for 10 min. After treating the reaction mixture with RNase T1,
(0.5 unit/reaction), the reaction mixtures were filtered through
nitrocellulose. Bound RNA fragments were extracted and electrophoresed
in a 12% acrylamide, 8 M urea gel. DNA size markers are shown
in lane M. Lane 8 shown a T1 digest of AUFL RNA prior to
selection. B, the 20 (lane 1), 35 (lane 2),
43 (lanes 3 and 4) nucleotide fragments were isolated
from a preparative gel and redigested with T1 (100 units/ml). C, the sequence of the AUFL RNA and the location of the
fragments bound by HuR. The sequence of the ARE is in bold,
and the predominant 35-nucleotide binding site is underlined.
The minor 20-nucleotide binding site is lightly underlined.
The protected G residue that results in the 43-nucleotide fragment is
indicated by a solid dot.
Figure 6:
The HuR-c-fos complex is
displaced by the 35-nucleotide recognition sequence. 4 fmol (9
10
cpm/pmol of UTP) of
P-labeled AUFL RNA was
incubated with 10 nM recombinant HuR protein. The indicated
molar excess of the 35-mer, 23-mer, and irrelevant oligonucleotides
were added into the reaction. After 10 min of incubation at 37 °C,
25% of reaction mixtures were resolved on 0.8% agarose
gel.
Figure 7:
Mutational analysis of the HuR binding
site. The indicated
P end-labeled RNAs (4 fmol, 1.5
10
cpm/pmol) were incubated with HuR at 37 °C
for 10 min and filtered through nitrocellulose. The bound radioactivity
was determined by Cerenkov counting. A, deletion mutations of
the c-fos ARE. B, point mutation in the c-fos ARE. C, sequences of deletions. D, sequences of
point mutations.
Figure 8:
HuR binds to c-myc,
N-myc, and IL-3 mRNAs. A and B, the
indicated
P-labeled RNA (4 fmol, 1.6
10
cpm/pmol of UTP) was incubated with the indicated concentration
of GST or HuR. Following incubation at 37 °C for 10 min, 25% of the
reaction mixture was resolved by gel electrophoresis in a 0.8% agarose
gel. C, following incubation as described above the reaction
mixture was filtered through nitrocellulose and the bound radioactivity
determined by Cerenkov counting.
HuR Binds to N-myc, c-myc, and IL-3 mRNAs
The
current classification of AU-binding proteins distinguish those
activities that solely bind to cytokine AREs versus those that
bind to all AREs. Fig. 8(A and B) shows that
HuR forms a stable complex with the 3` UTR of N-myc,
c-myc, and IL-3 mRNAs. No detectable complex formation was
detected with an irrelevant globin transcript or an IL-3 transcript
(IL-3
AU) in which the ARE element had been deleted. Quantitative
analysis using a nitrocellulose binding assay confirmed that HuR
displays a similar high affinity (K
= 2
nM) with the ARE-containing mRNAs, but significantly less
affinity with ARE deletions (K
for AU1/2 =
70 nM; only 10% binding to IL-3
AU at 100 nM) or
the irrelevant globin transcript (no detectable binding at 100
nM). A significant difference between the ARE of cytokine and
oncogene AREs is the arrangement of the AUUUA motifs. The IL-3 ARE
contains eight AUUUA motifs (labeled I-VIII, Fig. 9B) in a complicated arrangement. Fig. 9A shows that two major T1 fragments were selected
by HuR. No fragments were selected from an IL-3
AU transcript. In
addition, no T1 fragments were selected from a partial digest of
IL-3
AU. This confirms the specificity of HuR binding, in that it
does not simply select large T1 fragments. The major selected T1
fragments correspond to AUUUA motifs I and VI, VII, VIII. Mutational
analysis has shown that motifs VI, VII, and VIII are essential for the
destabilizing activity of the IL-3 ARE(34) .
Figure 9:
RNase T1 mapping of the HuR-IL-3 mRNA
complex. A,
P-labeled IL-3 mRNA (lanes
1-6) or IL-3
AU (lanes 8-12) RNA (6 fmol,
6
10
cpm/pmol of UTP) were incubated with HuR or
GST at 37 °C for 10 min. After treating the reaction mixture with
the indicated amount of RNase T1, the reaction mixtures were filtered
through nitrocellulose. Bound RNA fragments were extracted and
electrophoresed in a 12% acrylamide, 8 M urea gel. DNA size
markers are shown in lane M. Lanes 3, 6, 10, and 12 show the T1 digest of the indicated RNAs prior to selection. B, the sequence of the IL-3 ARE and the location of the
fragments bound by HuR. The sequence of the ARE is in bold,
and the AU motifs are labeled I-VIII. The predominant
binding sites are heavily underlined. The minor 21-nucleotide
binding site is lightly underlined and the protected G residue
indicated by a solid dot.
DISCUSSION
It is clear that the destabilization of mRNA by cis-acting
AREs is an important control point in gene expression. The mechanism
underlying this regulation is not completely understood. In the absence
of a cell-free system that reconstitutes this reaction, one approach is
to study the trans-acting factors that bind to AREs. Previously we have
shown that the Elav-like protein HuD binds specifically and avidly to
AREs(23, 26) . This protein is only expressed in
neurons(31, 32) . In this paper we have cloned and
characterized a more distant Elav-like family member called HuR, which
we have found is expressed in a wide variety of cell types. HuR
contains three RNA recognition motifs in an identical arrangement to
the other Elav-like proteins. We have found that the RNA binding
specificity of HuR is very similar to HuD and Hel-N1. This suggests
that HuR, HuD, Hel-N1, and HuC perform a similar function, albeit in
different cell types. The marked diversity of Elav-like proteins in
neuronal cells may reflect the important role of transient gene
expression in these cells.
We have shown that HuR binds to the ARE
of c-fos mRNA, but not to the adjacent elements AU1 and AU2.
Although AU1 and AU2 are well conserved in different species of cfos
mRNA their deletion from c-fos mRNA does not affect its
turnover(8) . Similarly HuR binds specifically to the ARE of
IL-3 mRNA. Thus the binding of HuR correlates with its proposed role in
selective mRNA turnover(8) . RNase T1 selection analysis has
revealed that HuR binds to one major site within the c-fos ARE. This high affinity site is comprised of AUUUA, AUUUUA, and
AUUUUUA motifs. All three AU motifs are required for maximal binding.
These results suggest that HuR does not recognize primary sequence, but
recognizes a higher order structure. The preponderance of U residues in
the target sequence indicate that such a structure may be stabilized by
U turns(35) . This idea is also reinforced by our analysis of
the IL-3 binding sites. The IL-3 ARE contains eight AUUUA motifs.
Although there is no discernible difference between these motifs, HuR
binds predominantly to two T1 fragments that encompass AU motifs I and
VI, VII, VIII. Motifs VI, VII, and VIII are essential for the
destabilizing activity of the IL-3 ARE.
Two other AU binding
activities have been cloned and characterized. AUF1 was identified by
its requirement in a cell-free mRNA decay system (22) and AUH
was affinity-purified using an (AUUUA)
affinity
column(29) . HuR does not resemble either of these gene
products. AUH does not contain any known RNA binding motif and exhibits
enoyl-CoA hydratase activity. Although AUF1 does contain a single RNA
recognition motif, there is no significant homology between it and any
of the three HuR RNA recognition motifs. The sequences within the ARE
that are bound by AUF1 and AUH have not been elucidated. It is possible
that they bind to a different subset than HuR and that all three
proteins are required for the assembly of the complex that degrades
mRNA. It is also possible that AUF1, AUH, and HuR respond to different
signal transduction pathways.
A number of groups have identified AU
binding proteins using gel retardation and UV cross-linking
assays(13, 14, 15, 16, 17, 18) .
HuR most resembles the activity identified by Vakalopoulou et
al.(17) . There is close agreement in molecular weight (32 versus 36 kDa), and both activities require a central AU motif
and flanking U residues. In this respect HuR may also correspond to the
32-kDa AU binding activity that binds to herpes virus saimiri small
RNAs(36) . HuR binds to oncogene mRNAs and to cytokine mRNAs.
Thus HuR would appear to belong to the AU-A class of RNA binding
proteins(13) . However in contrast to the previously described
AU-A activities, it binds to AREs with high affinity (the apparent K
is 2 nM). This high affinity is
comparable with the affinity of the previously described AU-B activity
for AUUUA multimers(14) . Although we have shown that a basal
level of HuR is expressed in all cells, it will be interesting to
investigate whether it is induced by factors that regulate cytokine
mRNA stabilization.
In summary the properties of HuR indicate that
it plays a role in ARE-dependent mRNA degradation. As we have found
with other Elav-like members, HuR does not have any detectable specific
endonuclease activity. Thus it is likely that the binding of HuR to
mRNA may facilitate the binding of other factors that degrade mRNA. It
is also equally possible that HuR may inhibit the interaction of
ARE-containing mRNAs with such degradation factors. An in vitro system capable of reconstituting ARE-dependent degradation will be
required to distinguish these possibilities. The HuR protein described
here may facilitate the generation of such an in vitro system
and permit the purification of other factors required for ARE-dependent
mRNA degradation.