(Received for publication, January 31, 1996; and in revised form, February 29, 1996)
From the
Rapid degradation of many labile mRNAs is regulated in part by
an A+U-rich element (ARE) in their 3`-untranslated regions.
Extensive mutational analyses of various AREs have identified important
components of the ARE, such as the nonamer motif UUAUUUAUU, two copies
of which serve as a potent mRNA destabilizer. To investigate the roles
of trans-acting factors in ARE-directed mRNA degradation, we
previously purified and molecularly cloned the RNA-binding protein AUF1
and demonstrated that both cellular and recombinant AUF1 bind
specifically to AREs as shown by UV cross-linking assays in
vitro. In the present work, we have examined the in vitro RNA-binding properties of AUF1 using gel mobility shift assays
with purified recombinant His-AUF1 fusion protein. We find
that ARE binding affinities of AUF1 correlate with the potency of an
ARE to direct degradation of a heterologous mRNA. These results support
a role for AUF1 in ARE-directed mRNA decay that is based upon its
affinity for different AREs.
Control of mRNA stability is an important component of
eukaryotic gene expression and involves cis-acting elements
that can be found in the coding region and/or UTRs ()of
mRNAs (reviewed in (1, 2, 3, 4, 5, 6) ).
One type of cis-acting instability element is comprised of the
AREs found in the 3`-UTRs of many unstable mRNAs (reviewed in (7) ). Many ARE-containing mRNAs are degraded by a sequential
pathway involving removal of the poly(A) tract followed by degradation
of the mRNA body(8, 9, 10) . In most cases
the poly(A) tract is thought to protect the mRNA from ribonuclease
attack so that its removal permits degradation of the mRNA body
(reviewed in (11) ). While it has been known for almost a
decade that AREs are important for mRNA
instability(12, 13, 14, 15) , the
mechanism(s) by which they mediate mRNA turnover is still unknown.
Despite the presence of AREs in many different mRNAs, there is no single evolutionarily conserved A+U-rich instability sequence. Typically AREs contain multiple copies of the pentanucleotide AUUUA, often in conjunction with one or more U-rich regions(14) . In addition, transfection studies indicate that as the number of tandemly repeated AUUU motifs is increased in a reporter mRNA, its instability increases. Likewise, two copies of the nonameric motif UUAUUUAUU act as a more potent destabilizer than a single nonameric motif(16, 17) . Together, these analyses suggest that potent destabilizing AREs are high affinity binding sites for a mRNA decay factor(s).
In order to investigate how AREs function in mRNA
turnover, we utilized a cell-free mRNA decay system to identify
proteins that may be relevant to ARE-directed mRNA
decay(8, 18, 19) . To this end, we previously
reported the purification, molecular cloning, and characterization of
the ARE-binding protein AUF1(20) . Cellular AUF1 purified from
cytoplasmic extracts of K562 human erythroid leukemia cells consists of
a 37- and a 40-kDa isoform. Cloning of the 37-kDa isoform,
p37, revealed two nonidentical RNA recognition motifs (21) and a short glutamine-rich region in the predicted amino
acid sequence. Cloning of murine cDNAs suggests that the 40-kDa isoform
may also contain 19 additional amino acids N-terminal to RNA
recognition motif 1(22) . Both cellular and recombinant
p37
(hereafter referred to as AUF1) bind the AREs
present in the c-fos and c-myc proto-oncogene mRNAs
and the granulocyte-macrophage colony-stimulating factor cytokine mRNA
as shown by UV cross-linking assays in vitro.
The potential
influence of AUF1 on ARE-directed mRNA decay extends beyond the control
of cytokine and proto-oncogene expression, however. Many mRNAs encoding
components of G protein-coupled receptors, such as -adrenergic
receptors (
-ARs), contain AREs. Moreover, receptor levels are
frequently subject to regulatory control. For example, exposure of
smooth muscle cells to agonist down-regulates
-AR mRNA
levels by inducing degradation of the mRNA(23) . Similarly,
agonist-mediated destabilization of the human
-AR mRNA
appears to be dependent upon an ARE(24) , and for both the
human
-AR and hamster
-AR mRNAs decay
occurs concomitantly with an increase in the cytoplasmic levels of
AUF1(25) . Since both cellular and bacterially expressed AUF1
bind the
-AR ARE(25) , the reciprocal
relationship between the half-life of
-AR mRNA and the
abundance of AUF1 suggests that the half-lives of ARE-containing mRNAs
may be dependent in part upon ARE-specific RNA binding affinity of
AUF1.
Here, we test the hypothesis that the binding affinity of AUF1
for an ARE should reflect the potency of that ARE as a mRNA
destabilizer. Using purified recombinant His-AUF1 fusion
protein, we find a direct relationship between the apparent K
for ARE binding by AUF1 and the potency
of the ARE to direct mRNA decay. These results support a role for AUF1
in ARE-directed mRNA decay that is based upon its affinity for
different AREs.
All enzymes and plasmid vectors were obtained from Promega Corp. (Madison, WI) unless otherwise noted. All plasmid constructions were confirmed by both restriction enzyme analyses and dideoxy sequencing with Sequenase (version 2.0, U.S. Biochemical Corp.).
Plasmid
p19R
+AT
4 was synthesized by first annealing the
complementary oligonucleotides 5`-CTAGATTTATTTATTTATTTAGCTTTAG-3` and
5`-TCGACTAAAGCTAAATAAATAAATAAAT-3` and then ligating to the
3.1-kilobase vector fragment derived from XbaI-SalI
digested p
19R
+AT
5.
The U sequence
was created as a BamHI-BglII fragment by annealing
the complementary oligonucleotides 5`-GATCCT
A-3` and
5-GATCTA
G-3`. BglII-digested p
19R
was
ligated to the annealed U
sequence to create plasmid
p
19R
+U32.
The XbaI-SalI fragment of
p19R
+AT
5 (described above) containing
(ATTT)
was removed, and a BglII site was created
using a synthetic linker, creating plasmid p
19R
AU.
Purified recombinant His-AUF1 fusion protein was
incubated with 1 fmol of
P-labeled RNA probe in a final
volume of 10 µl containing 10 mM Tris-HCl (pH 7.5), 5
mM magnesium acetate, 100 mM potassium acetate, 2
mM dithiothreitol, 0.1 mM spermine, 0.1 µg/µl
bovine serum albumin, 8 units of RNasin, 0.2 µg/µl tRNA, 5
µg/µl heparin, and 0.1 µg/µl poly(C). Reaction mixtures
were incubated on ice for 10 min. Complexes were resolved by
electrophoresis through nondenaturing 6% polyacrylamide gels
(acrylamide/bisacrylamide ratio of 60:1) in 45 mM Tris borate
(pH 8.3), 1 mM EDTA. Gels were prerun for 30 min at 13 V/cm
prior to sample loading. Gels were then run at 13 V/cm for 2-3 h,
dried, and visualized on a PhosphorImager (Molecular Dynamics). In some
experiments there was a loss of shifted products during
electrophoresis. This was observed as smears migrating between the
bound and free RNA bands. Thus, free RNA bands were routinely used for
quantitation using ImageQuant image analysis software (Molecular
Dynamics). Free probe concentration was plotted versus His
-AUF1 concentration, and apparent K
values were determined as the protein concentration at which 50%
of the RNA was bound(27) . For each RNA substrate tested,
binding assays were performed in triplicate, and the average apparent K
and standard deviation were determined. In
addition, the Newman-Kuels analysis of variance test was applied to
each set of apparent K
values to identify
significant differences (p < 0.05) between K
values for binding to the various RNA substrates.
Figure 1:
Characterization of purified
recombinant His-AUF1 protein. Left panel,
Coomassie Blue staining of His
-AUF1 protein. One microgram
of purified recombinant protein was fractionated in an SDS, 10%
polyacrylamide gel. The gel was then stained for protein visualization.
The apparent molecular mass of full-length His
-AUF1 is 51
kDa (arrow, lane 2). Lane 1 shows prestained
molecular mass markers. Right panel, UV cross-linking to
c-fos ARE. A binding reaction containing 25 ng of purified
recombinant protein and 40 fmol of radiolabeled c-fos ARE RNA
was treated with UV light and digested with RNase A as described under
``Materials and Methods.'' The reaction was fractionated in
an SDS, 10% polyacrylamide gel, and the protein bound to RNA was
detected by autoradiography. The 51-kDa fusion protein is the major
cross-linked species (arrow).
RNA-binding activity of His-AUF1 was assayed
by nondenaturing gel mobility shift assays using radiolabeled
substrates containing either one AUUUA motif or from two to five tandem
repeats of AUUU. (See Fig. 2for RNA sequences.) AUUUA motifs
are present in the AREs of most unstable proto-oncogene and cytokine
mRNAs, and the potency of an AUUU-containing sequence to act as a mRNA
destabilizer is proportional to the number of tandemly repeated AUUU
motifs(16, 17, 26) . Radiolabeled RNA
substrates were incubated with increasing concentrations of
His
-AUF1, and the products were resolved by native
polyacrylamide gel electrophoresis. Shown in Fig. 3are
representative gels along with plots of free RNA concentration versus fusion protein concentration. His
-AUF1 ARE
binding affinities increase as the number of tandemly repeated AUUU
motifs is increased (Table 1), with lowest affinity binding to
R
+AUUUA (apparent K
= 210
± 50 nM) and highest affinity binding to
R
+(AUUU)
(apparent K
= 19 ± 7 nM). The control RNA substrate
R
AU, which has all AUUU repeats deleted, is bound by
His
-AUF1 with low affinity (apparent K
= 660 ± 90 nM (data not shown)).
Figure 2:
Sequences of RNA substrates used in
mobility shift assays with His-AUF1. With the exception of
the c-myc ARE, each complete RNA substrate contained the last
80 nucleotides of the rabbit
-globin coding region including the
UGA termination codon and begins with an EcoRI site just 3` to
the T3 or SP6 bacteriophage promoter in each construct. A, the
R
AU substrate is depicted with the rabbit
-globin
sequence, shown as an open box, followed by nucleotides
immediately 3` of the UGA termination codon. Also shown is the site of
insertion of AUUUA or AUUU repeats in the R
+AUUUA and
R
+(AUUU)
substrates, respectively. The
UUAUUUAUU nonamer motifs are underlined in these sequences. B, the c-fos substrates are depicted with the rabbit
-globin sequence described above (open box) followed
immediately by either the c-fos ARE or the mutant ARE3 (hatched box). Nucleotide sequences of the c-fos ARE
and the ARE3 mutant are shown. U-to-A substitutions in the c-fos ARE3 mutant are underlined. C, the sequence of the
c-myc ARE is shown.
Figure 3:
His-AUF1 binding affinity for
AUUU-containing sequences increases as the number of tandemly repeated
AUUU motifs is increased. His
-AUF1 binding to RNA
substrates containing AUUUA or tandem repeats of AUUU (see Fig. 2) was analyzed by electrophoretic mobility shift assays
and apparent K
for His
-AUF1
binding to each RNA was determined as described under ``Materials
and Methods.'' Representative binding reactions using
His
-AUF1 and radiolabeled RNA substrates are shown at the top of each panel, and plots of
[RNA
] versus [His
-AUF1] are shown at the bottom of each panel. A, R
+AUUUA; B,
R
+(AUUU)
; C,
R
+(AUUU)
. For each RNA substrate, the K
value shown is the average of three
separate experiments.
Since
multiple tandem repeats of AUUU constitute primarily U-rich sequence,
the increasing binding affinity by His-AUF1 observed with
increasing AUUU copy number could result from an increase in the number
of uridylate residues. To test His
-AUF1 binding affinity
for U-rich RNA, binding to a substrate containing U
was
performed, since this sequence has been tested for mRNA destabilizing
activity(17) . The apparent K
for
His
-AUF1 binding to the R
+U
substrate is >500 nM (Fig. 4). While low
affinity binding occurs to the R
+U
substrate, no
binding is detected to the control
-globin substrate lacking the
U
sequence (Fig. 4). Nonetheless the estimated
binding affinity to R
+U
(>500 nM) is
at least 20-fold lower than that for binding to
R
+(AUUU)
(K
= 19
± 7 nM). Likewise, heterologous mRNAs containing
poly(U) tracts in their 3`-UTRs are
stable(16, 17, 28) . We conclude that
His
-AUF1 binding affinities for the AUUU-containing RNAs
parallel their potencies as mRNA destabilizers. The low affinity
binding to the U
substrate is also consistent with its
inability to promote mRNA degradation.
Figure 4:
R+U
is not a high
affinity His
-AUF1 binding substrate. RNA substrates
contained the last 80 nucleotides of the rabbit
-globin coding
region alone (R
) or linked to U
(R
+U
). Binding affinity of
His
-AUF1 for the R
+U
or R
substrates was determined by electrophoretic mobility shift assays as
described under ``Materials and Methods.'' Representative
plots of [RNA
] versus [His
-AUF1] for R
+U
(triangles) and R
(open circles) are
shown. The apparent K
for binding to
R
+U
was determined from three separate
experiments to be >500 nM, which was the highest protein
concentration used. No binding to R
was
detected.
Figure 5:
High affinity binding of
His-AUF1 to c-fos ARE substrates.
His
-AUF1 binding to RNA substrates containing the wild-type
c-fos ARE or the ARE3 mutant (see Fig. 2) was analyzed
by electrophoretic mobility shift assays, and apparent K
values for His
-AUF1 binding to each RNA were
determined as described under ``Materials and Methods.''
Representative binding reactions using His
-AUF1 and
radiolabeled RNA substrates are shown at the top of each panel, and plots of [RNA
] versus [His
-AUF1] are shown at the bottom of each panel. A, R
+fos ARE; B, R
+fos ARE3. For each RNA substrate, the K
value shown is the average of three
separate experiments.
The importance of AREs for mRNA turnover was first realized
in 1986(12, 14) , yet it is still unclear how AREs
function in mRNA decay. We have utilized biochemical approaches to
identify trans-acting factors that bind AREs in order to
relate such RNA binding to mRNA degradation. We previously purified,
characterized, and molecularly cloned the ARE-binding protein AUF1, and
in the present study we have examined binding affinities of a
His-AUF1 fusion protein for A+U-rich sequences with
defined relative potencies as mRNA destabilizers. Here, by determining
apparent K
values for His
-AUF1
binding, we demonstrate that AUF1-ARE binding affinity is directly
related to the potency with which an ARE destabilizes a heterologous
mRNA (Table 1). Additionally, the affinity of
His
-AUF1 for the most potent destabilizing AREs is within
the average range (10
M) of affinities
exhibited by several other RNA-binding proteins that recognize specific
sequences or structures(29) .
Certain A+U-rich
sequences are more potent mRNA destabilizers than others, suggesting
that the potencies of destabilizers are proportional to the binding
affinities of a cellular decay factor(s). For example, when placed in
the context of a heterologous, normally stable mRNA, AUUUA and
(AUUU) are relatively ineffective as destabilizing
elements; (AUUU)
has a modest destabilizing effect;
(AUUU)
increases the decay rate further; and (AUUU)
is the most potent destabilizer of the
five(16, 17, 26) . In fact, (AUUU)
increases the degradation rate of a reporter mRNA to about the
same extent as does the c-fos ARE(17) . Likewise,
His
-AUF1 binds the c-fos ARE (K
= 7.8 ± 0.4 nM) and the (AUUU)
substrate (K
= 19 ± 7
nM) with similar affinities. (The differences are not
statistically significant (p > 0.05).) Statistical analyses
were used to determine significant differences (i.e. p <
0.05) between K
values for His
-AUF1
binding to various RNA substrates, and as a result the RNA sequences
used in this study can be grouped into three general classes: (i) RNAs
that are either not bound or bound with low affinity by AUF1 and are
not mRNA destabilizers (
-globin, U
, and AUUUA); (ii)
RNAs that are bound with gradually increasing, moderate affinities by
AUF1 and have a gradually increasing, partial destabilizing effect
((AUUU)
< (AUUU)
< (AUUU)
);
and (iii) RNAs that are bound with the highest affinity by AUF1 and are
potent mRNA destabilizers (c-fos and c-myc AREs and
(AUUU)
). Based upon these ranges of AUF1 binding affinities
for various RNA substrates (low, moderate, high) and the relationship
of high affinity binding to mRNA decay, the affinity of AUF1 for a mRNA
may dictate the rate at which it is degraded. Therefore, cellular AUF1
concentration may be one determinant of mRNA half-life. In this regard
we found that by comparing Western blots of K562 cytoplasmic extracts
with known amounts of purified recombinant p37
(the
isoform used in these studies) that there are approximately 3.2
10
cytoplasmic molecules of p37
/cell (data
not shown). Assuming a diameter of 20 µm for K562 cells and 50% of
the cell volume as cytoplasm(30) , the concentration of
p37
is approximately 25 nM. This value is
comparable with the apparent K
for binding to the
c-myc ARE. Thus low cellular concentrations of active AUF1 may
be sufficient for binding to a mRNA that contains a high affinity
AUF1-binding site such as the c-myc and c-fos AREs.
Based upon our results, such mRNAs should have very short half-lives.
Likewise, mRNAs with AREs bound with lower affinities by AUF1 might
require a higher concentration of active AUF1 for binding; these mRNAs
should be degraded at a slower rate than those with high affinity
binding sites. Thus, the availability of active AUF1 for ARE binding is
a potential mechanism by which cells could control mRNA turnover rates
and one in which the decay of multiple mRNAs could be differentially
regulated by AUF1 concentration. Support for this hypothesis is the
relationship between AUF1 levels and ARE-directed mRNA destabilization
observed in DDT1-MF2 hamster smooth muscle cells treated
with(-)isoproterenol. In this case,(-)isoproterenol induces
an increase in cellular AUF1 protein and mRNA levels. This increase in
turn correlates with a faster decay rate for
-adrenergic receptor mRNA, which contains an AUF1
binding site(s) in the 3`-UTR(25) .
Recently, two groups
reported that the functional sequence within an ARE appears to be the
nonamer sequence UUAUUUAUU(16, 17) . One copy of the
sequence UUAUUUAUU in the 3`-UTR of a normally stable mRNA can increase
its degradation rate compared with the wild-type mRNA, while two copies
of the nonamer motif act as a very potent mRNA destabilizer. As
depicted in Fig. 2, (AUUU) contains one copy of the
nonamer (underlined in Fig. 2); (AUUU)
contains two overlapping copies; and (AUUU)
contains
two copies that overlap by a single nucleotide. Consistent with the
potencies of two copies or one copy of the nonamer as destabilizers,
binding affinity of His
-AUF1 for (AUUU)
is
3-fold and 5-fold greater than the binding affinities for (AUUU)
or (AUUU)
, respectively (Table 1). Thus, AUF1
may function in part via recognition of the nonamer motif.
Despite
the potential importance of the nonamer motif UUAUUUAUU in ARE-directed
mRNA decay, it is important to note that not all AREs found in unstable
mRNAs contain this motif. For example, the portion of the c-myc ARE that functions as a very potent mRNA destabilizer does not
contain this motif(28) . While the c-myc ARE does
contain noncontiguous AUUUA motifs, destabilizing AREs that contain no
AUUUA motifs have also been identified. In addition, the presence of
one or more AUUUA motifs in an ARE may not be sufficient for effective
mRNA destabilization(28) . Moreover, analysis of a c-fos ARE mutant, ARE3, with single U-to-A substitutions in all three
AUUUA motifs showed that intact AUUUA motifs are not required for rapid
mRNA deadenylation but are important for rapid degradation of the mRNA
body(9) . The apparent K for
His
-AUF1 binding to the c-fos ARE and the ARE3
mutant were not statistically different (7.8 ± 0.4 nM and 20 ± 4 nM, respectively; p >
0.05). Likewise, His
-AUF1 bound a mutant c-myc ARE
with single U-to-A mutations in both AUUUA motifs with affinity similar
to wild-type c-myc ARE (data not shown). Thus, intact AUUUA
motifs are not required for high affinity binding of
His
-AUF1 to the c-fos and c-myc AREs.
Therefore, although the study of AUUUA and UUAUUUAUU motifs has
contributed greatly to understanding of ARE-directed mRNA decay, it is
evident that these motifs may constitute only a subset of important
motifs within various AREs.
In conclusion, our results suggest that the affinity of AUF1 for particular ARE sequences is related to their potency as mRNA destabilizers. Future experiments will utilize AUF1 as a tool to define multiple classes of AREs and to define specific nucleotide requirements for AUF1 binding by selection of high affinity binding substrates from combinatorial libraries of RNA sequences (e.g., SELEX; reviewed in (31) ).