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INTRODUCTION |
In eukaryotes, the cytoplasmic concentration of an mRNA is a
critical determinant of its potential for translation and hence the
production rate of the encoded gene product. As such, the rate of
cytoplasmic mRNA decay influences both the timing and level of
expression of many gene products, involving either defined constitutive
mRNA decay rates or modulation of mRNA turnover rates in
response to external stimuli (reviewed in Refs. 1 and 2).
Many mammalian mRNAs encoding oncoproteins, cytokines/lymphokines,
inflammatory mediators, and G protein-coupled receptors are unstable,
owing to the presence of A+U-rich elements
(AREs)1 in their
3'-untranslated regions (3-6). AREs comprise a diverse family of
U-rich RNA sequences varying in length from 40 to 150 bases and
frequently containing one or more motifs of the form AUUUA, which may
be overlapping or dispersed (7). In general, mRNA turnover mediated
by AREs is characterized by rapid shortening of the poly(A) tail
followed by degradation of the mRNA body (8-10). Although several
proteins have been identified that may bind A+U-rich RNA sequences
(reviewed in Ref. 11), the mechanisms linking association of
trans-acting factors with accelerated mRNA decay remain
largely unknown.
The ARE-binding protein that has been most extensively characterized is
AUF1. The AUF1 gene encodes a family of four protein isoforms generated
by alternative pre-mRNA splicing that are denoted by their apparent
molecular weights as p37AUF1, p40AUF1,
p42AUF1, and p45AUF1 (12). Normally, the p42
and p45 isoforms appear exclusively nuclear (13, 14), likely owing to
an isoform-specific protein sequence that binds scaffold attachment
factor-B, a protein associated with the nuclear matrix (14). However,
several lines of evidence demonstrate that association of cytoplasmic
p37AUF1 and/or p40AUF1 with an ARE is
associated with acceleration of mRNA turnover. First, a protein
complex containing cytoplasmic p37AUF1 and
p40AUF1 is sufficient to destabilize polysomal
c-myc mRNA in vitro (15). Second, AUF1
associates directly with A+U-rich RNA sequences, and the affinity of
recombinant p37AUF1 for an ARE closely correlates with the
potential of the ARE to destabilize an mRNA in cis (16).
Third, inhibition of the p38 mitogen-activated protein kinase
pathway stabilizes ARE-containing mRNAs (17-19), concomitant with
the loss of an ARE binding activity containing AUF1 (19). Fourth, the
efficiency of ARE-directed mRNA turnover is compromised in cells
expressing low levels of endogenous p37AUF1 and
p40AUF1 (20, 21) or following sequestration of AUF1 by
treatment with hemin (22). Conversely, an increase in AUF1 protein
levels during congestive heart failure accompanies a decrease in levels of
1-adrenergic receptor mRNA (23, 24). The
1-adrenergic receptor transcript also contains an ARE in
its 3'-untranslated region that associates with AUF1 (24).
In previous studies, we demonstrated that the association of
recombinant AUF1 with an ARE in vitro proceeded by
sequential binding of AUF1 dimers involving protein-protein and
protein-RNA interactions (25), generating an oligomeric AUF1 complex on the ARE (25, 26). Based on observations that cellular AUF1 is
associated with other cellular factors (13) including the translation
initiation factor eIF4G, poly(A)-binding protein, and the heat shock
proteins Hsp70 and Hsc70 (27), we proposed that the oligomerization of
AUF1 on an ARE nucleates the assembly of a trans-acting,
mRNA-destabilizing complex on the RNA (11, 25). As such, the
potential of an ARE to associate with AUF1 and promote protein
oligomerization may be a critical determinant of mRNA turnover
efficiency. In this work, we have continued to define molecular
mechanisms contributing to the recognition of AREs by AUF1. Using a
fluorescence anisotropy-based assay for RNA-protein binding, we
observed that the association of AUF1 to the ARE from tumor necrosis
factor
(TNF
) mRNA was inhibited by magnesium ions, whereas
AUF1 binding to a similarly sized polyuridylate sequence was largely
unaffected. We present evidence that the TNF
ARE exhibits a
conformational change upon association with Mg2+ with the
net result of restricting RNA flexibility and shortening the distance
between the 5'- and 3'-termini of the RNA substrate. Based on
independent assessments of AUF1-ARE and Mg2+-ARE binding
equilibria, we have constructed a model for inhibition of AUF1 binding
and oligomerization to the TNF
ARE in the presence of
Mg2+. To our knowledge, this represents the first
indication that higher order RNA structures may regulate the
association of trans-acting factors with an ARE.
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EXPERIMENTAL PROCEDURES |
RNA Substrates--
The sequences and fluorophore positions of
RNA substrates (2'-hydroxyl) used in this study are listed in Table I.
All substrates containing 5'-fluorescein (Fl) or 5'-cyanine 3 (Cy3)
groups were synthesized by Dharmacon Research (Boulder, CO), then
2'-O-deprotected according to the manufacturer's
instructions (28) and evaporated to dryness in a Speed-Vac. Deprotected
RNA substrates were dissolved in 10 mM Tris·HCl (pH 8.0)
and quantified by A260, with estimates of
RNA extinction coefficients calculated as described (29). For
fluorescein-conjugated RNA probes, A260 was
corrected by quantitation of the fluorescein moiety at 493 nm with
493, Fl = 74,600 M
1·cm
1 and
260, Fl = 26,000 M
1·cm
1 as described (30). For
Cy3-conjugated RNAs, A260 was similarly corrected by quantitation of the Cy3 moiety at 552 nm using
552, Cy3 = 150,000 M
1·cm
1 and
260, Cy3 = 12,000 M
1·cm
1 (extinction
coefficients for Cy3 provided by Amersham Pharmacia Biotech).
The RNA substrates TNF
ARE and P-TNF3' were synthesized lacking
fluorescent labels (Dharmacon) and then 2'-O-deprotected and
quantified by A260 as described above. P-TNF3'
contains a phosphate group conjugated at the 5'-end. Fluorescein labels
were added to the 3'-ends of these RNAs by periodate oxidation and conjugation with Alexa Fluor 488 hydrazide (Molecular Probes, Eugene,
OR) as described (31) to generate TNF
ARE-Fl and P-TNF3'-Fl. Unconjugated fluorophore was removed from the labeled RNA preparations by passage through two Quick Spin G-25 spin columns (Roche Molecular Biologicals). The yields of RNA and conjugated fluorophore were monitored by absorbance as described above, with fluorophore coupling efficiencies typically >95%.
The double-labeled RNA oligonucleotide Cy-TNF-Fl was constructed by
ligation of RNA substrates Cy-TNF5' and P-TNF3'-Fl using T4 DNA ligase
and a bridging antisense DNA oligonucleotide as described (32).
Following ligation, the reaction was heated to 95 °C for 5 min and
quickly cooled on ice to denature DNA-RNA hybrids. The bridging DNA
oligonucleotide was then digested with RQ1-DNaseI. Ligated RNA
molecules were purified by denaturing polyacrylamide gel
electrophoresis and were recovered as described (33). The yield of
Cy-TNF-Fl RNA was estimated by measurement of total fluorescence
intensity of the Cy3 moiety (
ex = 535 nm;
em = 580 nm) and comparison with a standard curve of
Cy-TNF
ARE RNA. This assumes that the fluorescence quantum yield of
the 5'-Cy3 group was not significantly affected by the addition of the
fluorescein moiety to the 3'-end of the RNA, because placement of the
fluorophores on opposite ends of the RNA substrate and the overall
hydrophilicity of RNA make it unlikely that the 3'-Fl could
substantially alter the chemical environment of the 5'-Cy group.
Preparation of Recombinant
His6-p37AUF1--
The complete coding sequence
of p37AUF1 was excised from pTrcHisB-p37AUF1
(16) by digestion with Acc65I + HindIII and subcloned into similarly digested pBAD/HisB (InVitrogen, Carlsbad, CA) to generate pBAD/HisB-p37AUF1. Recombinant
His6-p37AUF1 was expressed from this plasmid in
Escherichia coli TOP10 cells induced with 0.0002% arabinose
for 4 h. Cells were disrupted by four rounds of sonication as
described (34). However, inclusion of polyethylene glycol-6000 in the
lysis buffer (5% final) abrogated the need for freeze-thaw cycles.
Purification of His6-p37AUF1 was performed by
Ni2+ affinity chromatography using a HiTrap Chelating
affinity column (Amersham Pharmacia Biotech). The column (1 ml) was
prerinsed at 120 ml/h with 8 column volumes of deionized water,
charged with 6 volumes of 20 mM NiCl2, rinsed
with 8 volumes of deionized water and 6 volumes of native imidazole
elution buffer (10 mM sodium phosphate, 500 mM
NaCl, 300 mM imidazole, pH 6.3), and equilibrated with 8 volumes buffer 1 (50 mM sodium phosphate, 500 mM NaCl, 20 mM imidazole, pH 8.0). The cleared
bacterial cell lysate was applied at 60 ml/h and washed with buffer 1 until A280 reached base line. His6 proteins
were eluted in native imidazole elution buffer at 30 ml/h with
fractions monitored by A280. Peak fractions were
pooled, buffer exchanged into 10 mM Tris·HCl (pH 7.5)
using HiTrap Desalting columns (Amersham Pharmacia Biotech) as
recommended by the manufacturer, and concentrated using a Centricon YM-30 concentrator (Millipore, Bedford, MA). Protein concentrations were determined by comparison of Coomassie Blue-stained
SDS-polyacrylamide gels containing recombinant
His6-p37AUF1 and a titration of bovine serum
albumin. Quantitation of band intensities was performed using the Kodak
EDAS 120 gel documentation system and accompanying one-dimensional
image analysis software (Eastman Kodak Co.).
Evaluation of RNA-Protein and RNA-Cation Binding Equilibria by
Fluorescence Anisotropy--
Association of recombinant
His6-p37AUF1 with fluorescent RNA substrates
was monitored by changes in the anisotropy of the fluorescein moiety
following protein binding. Anisotropy is related to the rotational
relaxation time of the fluorophore in solution (35, 36), which, for a
linear polymer like RNA, is dependent on gross molecular volume and/or
intramolecular segmental motion under conditions of constant
temperature and viscosity (35). Fluorescence anisotropy measurements
were made using the Beacon 2000 Variable Temperature Fluorescence
Polarization System (Panvera, Madison, WI) equipped with fluorescein
excitation (490 nm) and emission (535 nm) filters. Binding reactions
were performed with a range of His6-p37AUF1
protein concentrations and 0.2 nM fluorescein-labeled RNA
in a final volume of 100 µl containing 10 mM Tris·HCl
(pH 8.0), 100 mM potassium acetate, 2 mM
dithiolthreitol, 0.1 mM spermine, and 0.1 µg/µl
acetylated bovine serum albumin. Heparin (1 µg/µl) was included in
RNA-protein binding reactions to inhibit nonspecific RNA binding
activity. This concentration of heparin effectively prevented
association of His6-p37AUF1 (up to 250 nM protein dimer) with a fluorescent RNA substrate encoding
a fragment of the
-globin coding region (Ref. 25 and data not
shown). Magnesium acetate was used as a source of Mg2+ ions
where indicated. Samples lacking Mg2+ contained 0.5 mM EDTA unless otherwise noted. For equilibrium binding
experiments, the polarimeter was operated in static mode, with each
sample read as blank prior to addition of fluorescent RNA substrates.
Following probe addition, samples were incubated for 1 min at 25 °C
before anisotropy was measured. Preliminary on-rate analyses
demonstrated that anisotropic equilibrium was reached within 10-20 s
at this temperature for all binding equilibria described in this report
(data not shown). Data points represent the mean of 10 anisotropy
measurements for each binding reaction.
For evaluation of Mg2+ binding events involving RNA in the
absence of protein, Mg2+ titrations were monitored by
fluorescence ansiotropy as described above but in buffers lacking
heparin. Parallel titrations including heparin (1 µg/µl) yielded
consistent equilibrium constants for data sets between 0 and 5 mM Mg2+ but with small increases in absolute
anisotropy values (data not shown). At higher Mg2+
concentrations, however, more significant nonspecific increases in
anisotropy were observed in the presence of heparin, possibly because
of increases in sample viscosity resulting from the formation of
heparin·Mg2+ aggregates.
Concomitant with measurement of anisotropy values, total fluorescence
intensity was monitored for each sample to detect changes in the
fluorescence quantum yield of the fluorescein moiety as described (25).
For all experiments described in this work, total fluorescence
intensity did not vary significantly as a result of protein or cation
binding (see Fig. 1, A and B, and data not shown). Accordingly, the total measured fluorescence anisotropy (At) of a mixture of fluorescent species was
interpreted by simple additivity using Equation 1.
|
(Eq. 1)
|
Ai represents the intrinsic anisotropy of
each fluorescent species and fi its fractional
concentration (35, 37, 38). Using C-terminal deletion mutants of AUF1, a previous study demonstrated that AUF1 association with short U-rich
RNA substrates was well described by sequential binding of AUF1 dimers
(25). Applying this model to Equation 1, the total measured anisotropy
of AUF1-RNA binding reactions performed in the absence of
Mg2+ plotted against the concentration of added protein
dimer (P2) resolved to Equation 2 under conditions of
limiting [RNA] (i.e. [P2]free
[P2]total).
|
(Eq. 2)
|
Here, AR,
AP2R, and
AP4R represent the intrinsic
anisotropy values of the free RNA (R), AUF1 dimer-bound RNA
(P2R), and AUF1 tetramer-bound RNA (P4R), respectively. K1 is the constant describing the
R + P2
P2R equilibrium, whereas
K2 describes the P2R+ P2
P4R equilibrium (see Fig. 1E).
Additional algorithms employed in analysis of binding equilibria by
fluorescence anisotropy are described in the text where applicable. The
application of binding algorithms to experimental data was performed by
nonlinear regression using PRISM, version 2.0 (GraphPad, San Diego,
CA). The validity of all mathematical models was evaluated by the
coefficient of determination (R2) and analysis
of residual plot nonrandomness to detect any bias for data subsets
(PRISM). Where necessary, pair-wise comparisons of sum-of-squares
deviations between mathematical models were performed using the
F test (PRISM), with differences exhibiting p < 0.05 considered significant.
Analysis of RNA Folding by Fluorescence Resonance Energy
Transfer--
Changes in the distance between the 5'- and 3'-termini
of double-labeled RNA substrates were monitored by fluorescence
resonance energy transfer (FRET), using a modification of the method of Walter et al. (39). Reactions were assembled as described
above for evaluation of cation binding events involving RNA in the
absence of protein. All measurements of fluorescence intensity were
made using the Beacon 2000 fluorescence polarimeter (Panvera) operating in direct fluorescence mode. Prior to addition of fluorescent RNA
substrates, background fluorescence emission (
ex = 490 nm) was measured for each sample at 535 and 580 nm, given by
B535 and B580,
respectively. Following probe addition, samples were incubated for 1 min at 25 °C prior to measurement of total fluorescence intensity at
the same wavelengths, giving F535 and
F580. FRET efficiency in this system was
approximated by the ratio Q = (F580
B580)/(F535
B535). For analysis of cation-RNA binding
equilibria, Q was normalized to an approximation of the FRET
efficiency in the absence of cation (Q0), to
give the relative FRET efficiency (Q
Q0)/Q0.
Gel Filtration Chromatography--
For size fractionation of RNA
substrates, a 0.9 × 30-cm column loaded with Superdex 75 (Amersham Pharmacia Biotech) was washed with 0.1 M NaOH for
2 h prior to equilibration with diethylpyrocarbonate-treated gel
filtration buffer (10 mM Tris·HCl, 100 mM
potassium acetate, 2 mM dithiolthreitol, pH 8.0) containing
or lacking 5 mM magnesium acetate. RNA samples (1 nmol)
were loaded in 0.2 ml of gel filtration buffer containing 2% dextrose
and either 0.5 mM EDTA or 5 mM magnesium acetate. Elution of RNA was monitored by A254.
The column was calibrated by monitoring the elution of protein
standards (Sigma) by A280: soybean trypsin
inhibitor (20 kDa), cytochrome c (12.4 kDa), and aprotinin
(6.4 kDa). Column void volume was determined using blue dextran.
 |
RESULTS |
Association of Recombinant AUF1 Protein with the TNF
ARE Is
Inhibited by Mg2+--
Previously, we demonstrated that a
recombinant p37AUF1 deletion mutant lacking the C-terminal
29 amino acid residues (His6-p37AUF1-(1-257))
interacts with the core ARE sequence from TNF
mRNA, encoded by
the RNA substrate Fl-TNF
ARE (Table
I), by monitoring changes in the
anisotropy of the fluorescein moiety of the RNA resulting from protein
binding (25). This RNA sequence is a potent mRNA-destabilizing
element (40) and contributes to the extreme instability of TNF
mRNA in vivo (41, 42). Like the full-length
His6-p37AUF1,
His6-p37AUF1 (1) contains an N-terminal
dimerization domain (26) and exists as a dimer in solution (25). In the
presence of an RNA substrate containing the TNF
ARE or the
polyuridylate sequence contained within Fl-U32 (Table I),
His6-p37AUF1- (1) dimers associate
sequentially to form a tetrameric protein complex on the RNA (25).
Neither the dimerization (26) nor the RNA-binding activities (25) of
AUF1 proteins are affected by the presence of the N-terminal
His6 tag.
The association of recombinant His6-p37AUF1
with the RNA substrates Fl-TNF
ARE and Fl-U32 was
analyzed by measurement of the total fluorescence intensity and
fluorescence anisotropy of binding reactions containing the fluorescent
RNA substrates and titrations of recombinant protein (Fig.
1). For both the Fl-TNF
ARE and Fl-U32 substrates, increasing the concentration of
His6-p37AUF1 had little effect on total
fluorescence intensity (Fig. 1, A and B),
indicating that the fluorescence quantum yield was not significantly
affected by the presence of His6-p37AUF1. As
such, the contribution of each fluorescent species (RNA or RNA·protein complexes) to the total measured anisotropy
(At) could be defined as the product of its
intrinsic anisotropy (Ai) and its fractional
concentration (fi), described in Equation 1 (see
"Experimental Procedures"). Adapting this algorithm to the
RNA-dependent formation of AUF1 tetramers by sequential
association of protein dimers (Fig. 1E) yields Equation 2
(25). In the absence of Mg2+, the association of
His6-p37AUF1 with both Fl-TNF
ARE and
Fl-U32 were well described by sequential dimer binding
(Fig. 1, C and D, solid circles). This
was affirmed by strong coefficients of determination
(R2 = 0.9967 for Fl-TNF
ARE;
R2 = 0.9941 for Fl-U32) and random
positioning of residuals across the entire range of protein
concentrations tested. The presence of the second binding phase,
defined by K2 (Fig. 1E), was further indicated by poor regression of each data set using a binary binding model described by Equation 3 (data not shown).
|
(Eq. 3)
|
where AR-free and
AR-bound represent the intrinsic anisotropy
values of a fluorescent RNA substrate in the unbound and
protein-associated states, respectively, defined by a single
association constant K. Comparisons between the binary and
sequential dimer binding models using the F test supported
the two-stage formation of protein tetramers described by Equation 2
(p < 0.0001 for Fl-TNF
ARE and Fl-U32).
For both RNA substrates, solutions of the intrinsic anisotropy values
of the free RNA (AR), AUF1 dimer-bound RNA
(AP2R), and AUF1 tetramer-bound RNA
(AP4R) are presented in Table
II, along with solutions for equilibrium
constants K1 and K2. In
each case, the intrinsic anisotropy values increased with protein load (AR < AP2R < AP4R), consistent with the restriction of
fluorophore mobility by increasing molecular volume (35, 36). In
addition, the association of an AUF1 dimer with a free RNA substrate,
given by K1, exhibited much higher affinity than
the association of a subsequent protein dimer with a P2R
complex, given by K2, for both RNA
substrates.

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Fig. 1.
Evaluation of AUF1-RNA binding equilibria by
fluorescence anisotropy. Equilibrium binding reactions containing
fluorescent RNA substrates and titrations of recombinant
His6-p37AUF1 were assembled as described under
"Experimental Procedures." Binding reactions were performed in
either the absence (solid circles) or presence (open
circles) of 5 mM Mg2+. The total
fluorescence intensities ( ex = 490 nm; em = 535 nm) of Fl-TNF ARE (A) and Fl-U32
(B) substrates (0.2 nM) were monitored as a
function of AUF1 concentration to detect changes in the fluorescence
quantum yield of either probe in response to protein binding.
Fluorescence anisotropy was also measured for each binding reaction
(C and D). Anisotropy data sets for samples
lacking Mg2+ were resolved by nonlinear least squares
regression using Equation 2 with AR held
constant (Fl-TNF ARE, AR = 0.031;
Fl-U32, AR = 0.029) and
AP2R, AP4R,
K1, and K2 unfixed
(solid lines). Residual plots (bottom panels)
were prepared by subtraction of the regression-derived anisotropy value
(Ac) from the measured anisotropy value
(At) for each data point to detect any bias for
subsets of experimental data. Equation 2 was derived from a model for
association of AUF1 with U-rich RNA substrates by sequential binding of
AUF1 dimers (E), under conditions of constant fluorescence
quantum yield.
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Table II
Solution of anisotropy and equilibrium binding constants for
His6-p37AUF1 association with RNA substrates
(-Mg2+)
Solutions for the intrinsic anisotropy of the free RNA substrates
(AR) are based on measurements of anisotropy in the
absence of added protein (n = 9 for Fl-TNF ARE;
n = 4 for Fl-U32). The remaining anisotropy and
equilibrium binding constants (defined in the text) were derived from
triplicate AUF1 titration experiments performed in the absence of
Mg2+ as shown in Fig. 1. For each data set,
AR was held constant, and all other values were
determined by nonlinear regression using Equation 2 and a minimum of 30 data points. Regression solutions of parameters from individual data
sets were then averaged and are listed as ± n 1.
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|
The inclusion of 5 mM Mg2+ in the RNA-protein
binding reactions elicited different consequences in reactions
containing the Fl-TNF
ARE versus the Fl-U32
substrate. In binding reactions containing Fl-TNF
ARE,
Mg2+ repressed changes in anisotropy as a function of
protein concentration, indicating that AUF1 binding to this RNA
substrate was inhibited by the presence of the cation (Fig.
1C, open circles). By contrast, the presence of 5 mM Mg2+ induced little change in the anisotropy
of binding reactions containing Fl-U32 (Fig. 1D,
open circles). Because all binding reactions were performed
in a background of 100 mM potassium acetate, the observed
changes in anisotropy were unlikely to be due solely to the relatively
small change in ionic strength caused by inclusion of 5 mM
magnesium acetate.
In addition to variations in AUF1 binding, however, Mg2+
also significantly increased the intrinsic anisotropy of the Fl-TNF
ARE RNA substrate in the absence of AUF1 (cf. 0.031 ± 0.002, n = 9 in 0.5 mM EDTA
versus 0.047 ± 0.001, n = 6 in 5 mM Mg2+). The anisotropy of unconjugated
fluorescein was completely independent of Mg2+
concentration (cf. 0.0095 ± 0.0003, n = 4 in 0.5 mM EDTA versus 0.0092 ± 0.0003, n = 3 in 5 mM Mg2+), indicating
that the increase in anisotropy of the Fl-TNF
ARE substrate is
dependent on the presence of the RNA moiety. Although Mg2+
also increased the intrinsic anisotropy of Fl-U32, the
effect was less dramatic (cf. 0.029 ± 0.002, n = 4 in 0.5 mM EDTA versus 0.036 ± 0.002, n = 4 in 5 mM
Mg2+). The large increase in the intrinsic anisotropy of
Fl-TNF
ARE relative to Fl-U32 in the presence of
Mg2+ demonstrates that restriction of fluorescein mobility
in these substrates by Mg2+ is also sequence-specific for
the RNA. Because the anisotropy of a fluorescent RNA is dependent on
both its molecular volume and segmental motion when temperature and
viscosity are held constant (35, 36), these data suggest either that
the Fl-TNF
ARE substrate may form multimers in the presence of
Mg2+, or that RNA structures presenting limited flexibility
near the 5'-end may be induced or stabilized by the cation. The next
series of experiments was designed to distinguish between these possibilities.
Mg2+ Induces or Stabilizes Unimolecular Structural
Changes in the TNF
ARE--
Several experiments were performed to
characterize structural events that might contribute to the
Mg2+-induced decrease in fluorescein mobility observed with
the Fl-TNF
ARE RNA substrate. First, gel filtration chromatography
of RNA substrates through a Superdex 75 column demonstrated that the apparent molecular mass of Fl-TNF
ARE was not significantly
altered in the presence of 5 mM Mg2+ (Fig.
2). In both the presence and absence of
Mg2+, Fl-TNF
ARE eluted at ~13 to 14 kDa, close to its
calculated molecular mass of 12.5 kDa. By contrast, hybridization of
Fl-TNF
ARE to an antisense DNA oligonucleotide dramatically
decreased the elution volume, presenting an apparent molecular mass of
>40 kDa. This experiment indicated that Mg2+ did not
significantly affect the molecular volume of the Fl-TNF
ARE
substrate, making it unlikely that the Mg2+-induced
increase in fluorescein anisotropy was due to increased molecular
volume mediated by RNA multimerization.

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Fig. 2.
Analysis of RNA molecular volume by gel
filtration chromatography. RNA samples were fractionated through a
Superdex 75 gel filtration column as described under "Experimental
Procedures." The elution volumes of RNA samples
(Ve) were monitored by absorbance at 254 nm. The RNA
substrate Fl-TNF ARE was fractionated in both the absence
(solid line) and presence (dashed line) of 5 mM Mg2+. An additional sample was prepared in
which the Fl-TNF ARE RNA substrate was annealed to a complimentary
DNA oligonucleotide prior to gel filtration in the presence of 5 mM Mg2+ (dotted line). The column
void volume (Vo) and the elution volumes of protein
standards are indicated, with molecular masses given in kDa.
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In the absence of changes in molecular volume, an increase in the
anisotropy of the fluorescein moiety of Fl-TNF
ARE may thus be
reflective of restricted intramolecular segmental motion in the
presence of Mg2+. To test this hypothesis, a potential
interaction between Mg2+ and the RNA was considered using a
variation of a general binding scheme (43) involving the
Mg2+-dependent conversion of the RNA from a
more flexible state (no Mg2+, low At) to a less
flexible state (with Mg2+, high At) described by
a single association constant Kf.
|
(Eq. 4)
|
where x and y represent the stoichiometric
contributions of Mg2+ and the RNA, respectively, to the
RNA-cation complex. Because the fluorescence quantum yield of the
Fl-TNF
ARE substrate was unaffected by the presence of
Mg2+ (data not shown), this equilibrium binding
relationship may be applied to Equation 1, yielding Equation 5 under
conditions where [RNA] is limiting (i.e.
[Mg2+]free
[Mg2+]total).
|
(Eq. 5)
|
where AR and AR'
represent the intrinsic anisotropy values of the free and
cation-complexed fluorescein-labeled RNA substrates, respectively.
This model was first tested by measuring anisotropy values across a
titration of RNA concentrations, in either the absence or the presence
of Mg2+ (Fig. 3). Varying the
concentration of TNF
ARE RNA 5000-fold had no significant effect on
the anisotropy (At) of the Fl-TNF
ARE substrate
in reactions containing 0.5 mM EDTA, 1 mM Mg2+, or 5 mM Mg2+, indicating that
the Mg2+-induced changes in anisotropy were independent of
RNA concentration. By Equation 5, this only occurs when the binding
reaction is unimolecular with respect to RNA (i.e.
y = 1), further supporting the idea that
Mg2+ induces or stabilizes structural conformations of
TNF
ARE without forming RNA multimers.

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Fig. 3.
TNF
ARE-Mg2+ equilibrium is independent of RNA
concentration. Anisotropy values were measured for reactions
containing 0.2 nM Fl-TNF ARE supplemented with unlabeled
TNF ARE to generate a titration of RNA substrate concentrations
spanning 0.2-1000 nM. Measurements were taken for samples
containing 0.5 mM EDTA (open circles), 1 mM Mg2+ (solid triangles), or 5 mM Mg2+ (solid circles).
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Consequences of Mg2+ interaction with an RNA substrate
containing the TNF
ARE were also assessed by monitoring changes in
the mean distance between the 5'- and 3'-termini of the RNA (5'-3' distance) using FRET. The efficiency of FRET between a fluorescent donor and acceptor increases as the scalar distance between them decreases (44). In one experiment, FRET efficiency was estimated in the
absence of Mg2+ for probe combinations selected so that the
FRET donor (Fl) and acceptor (Cy) pairs were present either on
different molecules (Cy-TNF
ARE + TNF
ARE-Fl), on a single
molecule (Cy-TNF-Fl), or on a single molecule that was maintained in a
rigid form by hybridization to an unlabeled complimentary DNA
oligonucleotide (Cy-TNF-Fl:DNA duplex) (Fig.
4A). FRET efficiency was
significantly higher for the double-labeled RNA substrate (Cy-TNF-Fl)
relative to an equimolar mixture of singly labeled substrates
(Cy-TNF
ARE + TNF
ARE-Fl), indicating that the intramolecular
5'-3' distance of the probe Cy-TNF-Fl was well within the sensitivity
of this assay and that RNAs containing the TNF
ARE are unlikely to
multimerize in the absence of Mg2+. However, annealing the
Cy-TNF-Fl RNA substrate to a complimentary DNA molecule largely
abrogated this increase in FRET efficiency. Because the Cy-TNF-Fl RNA
substrate is retained as an elongated double helix in this hybrid, the
distance between the 5'- and 3'-termini of the RNA is maximized, thus
minimizing intramolecular FRET. This further indicated that the 5'-3'
distance of the single-stranded Cy-TNF-Fl probe was shorter compared
with a fully extended RNA molecule, supporting the proposition that the
TNF
ARE RNA substrates are inherently flexible in the absence of
Mg2+. Molecules exhibiting long tumbling rotations, such as
double-stranded nucleic acids, may be subject to FRET artifacts because
of linear polarization of the molecule (44). However, these artifacts are likely to be minimized in this case because each fluorophore is
linked to the RNA substrate by multiple single bonds (seven for the
5'-Cy3; two for the 3'-Fl), thus increasing the segmental rotational
freedom of each fluorophore independent of RNA-DNA hybridization. These
experiments demonstrated that the FRET assay could be used to detect
changes in the distance between the 5'- and 3'-termini of an RNA
substrate containing the TNF
ARE and thus would be useful in
evaluating changes in RNA conformation.

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Fig. 4.
Analysis of TNF
ARE-Mg2+ equilibrium by FRET. A, RNA
samples were assembled using the listed probe combinations
(left) as described under "Experimental Procedures"
without added Mg2+ (0.4 nM of each RNA). The
RNA-DNA duplex was generated in the presence of 100-fold excess DNA
oligonucleotide to maximize the hybridization efficiency of the RNA.
Bars show estimates of FRET efficiency (Q) for
triplicate reactions as ± n 1.
Parallel reactions containing 0.5 mM EDTA yielded similar
results (data not shown). B, the effect of Mg2+
on relative FRET efficiency ((Q Q0)/Q0) was assayed for
the bimolecular donor-acceptor pair Cy-TNF ARE + TNF ARE-Fl
(solid triangles), Cy-TNF-Fl (solid circles), and
the Cy-TNF-Fl:DNA duplex (open circles). The regression
function through the Cy-TNF-Fl data set (solid line) is
derived from Equation 6, relating the relative FRET efficiency as a
function of [Mg2+] for a bimolecular or noncooperative
interaction of Mg2+ and RNA.
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With the efficacy of this assay system established, changes in the
relative FRET efficiency of each combination of RNA substrates were
evaluated across a titration of Mg2+ concentrations. FRET
efficiency of the bimolecular donor-acceptor pair Cy-TNF
ARE + TNF
ARE-Fl was independent of Mg2+ concentration (Fig.
4B, solid triangles), further confirming that
Mg2+-induced RNA structural changes involving the TNF
ARE do not involve the formation of RNA multimers. By contrast,
Mg2+ significantly enhanced the FRET efficiency of the
unimolecular Cy-TNF-Fl probe in a dose-dependent manner
(Fig. 4B, solid circles), demonstrating that this
RNA substrate exhibits a cation-dependent change in
structure with the net effect of shortening the distance between the
5'- and 3'-termini of the RNA. Annealing the Cy-TNF-Fl substrate to a
complimentary DNA oligonucleotide abrogated the Mg2+-induced increase in FRET efficiency (Fig.
4B, open circles), suggesting that these
structural changes require base-specific contacts and/or that the RNA
substrate be inherently flexible. Taken together, the gel filtration
experiments, the RNA titration experiments, and the FRET studies
demonstrate that Mg2+ induces or stabilizes structural
changes in the TNF
ARE and that the structural changes are
unimolecular with respect to RNA.
Equilibrium binding of Mg2+ with the TNF
ARE--
Because Mg2+-induced folding of the Fl-TNF
ARE
substrate does not appear to involve the formation of RNA multimers,
Equation 5 was simplified by the solution of y = 1 to
yield Equation 6.
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(Eq. 6)
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To assess the stoichiometric contribution of Mg2+
(x) to this binding event, experiments were performed in
which the anisotropy of Fl-TNF
ARE was monitored across a titration
of Mg2+ concentrations (Fig.
5, solid circles). The
Mg2+-induced increase in the anisotropy of this RNA
substrate was well resolved by Equation 6 (Fig. 5, solid
line). Regression solutions of triplicate experiments yielded a
value of x = 0.9 ± 0.2. Resolution of this
constant to unity indicates that the RNA structural changes are
consistent with a transition between a more flexible RNA population (R)
to a less flexible one (R'), mediated by one or more noncooperatively binding magnesium ions. Using Equation 6 with x = 1, an
equilibrium constant describing the magnesium concentration effecting
half-maximal RNA folding was then solved as Kf = 7.0 ± 0.1 × 102 M
1
(n = 3). The association of Mg2+ with
Fl-TNF
ARE was also very dynamic, because restoration of anisotropic
equilibrium following addition of free Mg2+ or chelation of
Mg2+ with excess EDTA was complete within 10-20 s (data
not shown). As expected, the Fl-U32 substrate displayed
only modest changes in fluorescence anisotropy as the concentration of
Mg2+ increased (Fig. 5, open circles), further
confirming that the Mg2+-induced increase in the anisotropy
of the Fl-TNF
ARE substrate is dependent on the presence of the
TNF
ARE sequence.

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Fig. 5.
Anisotropic analysis of RNA-Mg2+
equilibria by Mg2+ titration. Equilibrium binding
reactions containing Fl-TNF ARE (solid circles) or
Fl-U32 (open circles) substrates (0.2 nM RNA) and a titration of Mg2+ in the absence
of protein were assembled as described under "Experimental
Procedures," with total fluorescein anisotropy ( ex = 490 nm; em = 535 nm) measured for each sample. EDTA was
not included in the 0 mM Mg2+ samples in this
experiment. However, anisotropy values for these reactions were not
significantly different than for similar reactions containing 0.5 mM EDTA (data not shown), indicating that contaminating
Mg2+ from other reaction components was negligible.
Regression of the Fl-TNF ARE data set was performed using Equation 6
with AR = 0.0279 and
AR', Kf, and
x unfixed (solid line).
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Characterization of TNF
ARE Structural Changes Induced or
Stabilized by Mg2+--
Additional experiments were
performed to elucidate details of RNA structures involving the TNF
ARE that were induced or stabilized by Mg2+. Anisotropy
experiments using the RNA substrate Fl-TNF
ARE-2 (Table I) were used
to determine whether sequences outside of the A+U residues within
Fl-TNF
ARE were required for RNA structural changes in the presence
of Mg2+. Similar changes in the anisotropy of both
Fl-TNF
ARE-2 and Fl-TNF
ARE substrates were observed across
titrations of Mg2+ (data not shown), indicating that A+U
residues within the TNF
ARE are sufficient for adoption of
Mg2+-induced RNA structures.
Next, a binary regression model analogous to Equation 6 was applied to
the changes in FRET efficiency of Cy-TNF-Fl as a function of
[Mg2+] (Fig. 4B, solid circles), to
determine whether Mg2+-induced changes in 5'-3' distance,
measured by FRET, and RNA flexibility, measured by anisotropy,
exhibited similar sensitivity with respect to the cation. Regression of
this data set using the binary model (Fig. 4B, solid
line) yielded an equilibrium constant K = 6 ± 1 × 102 M
1
(n = 2) that did not differ significantly from
Kf (7.0 ± 0.1 × 102
M
1), the anisotropic equilibrium constant
described above. These data suggest that shortening of the 5'-3'
distance of the TNF
ARE and restriction of its flexibility near the
5'-end may be consequences of the same Mg2+-induced or
-stabilized RNA folding event.
To evaluate whether restraint of RNA mobility was limited to the 5'-end
of the TNF
ARE, anisotropy of the 3'-fluorescein labeled RNA
substrate TNF
ARE-Fl (Table I) was also monitored across a titration
of Mg2+. Although this RNA substrate presented a value for
Kf comparable with Fl-TNF
ARE, (7 ± 2 × 102 M
1,
n = 2), a significant decrease in the overall change in
fluorescence anisotropy, given by
A = AR'
AR, was observed
(cf. 0.009 ± 0.002, n = 2 for TNF
ARE-Fl versus 0.021 ± 0.003, n = 3 for Fl-TNF
ARE). The decreased value of
A for TNF
ARE-Fl compared with Fl-TNF
ARE suggests that, although both ends of
the RNA are affected by structural changes following association of
Mg2+, the flexibility of the RNA appears more strongly
restricted close to the 5'-end.
Besides Mg2+, other divalent cations are also known to
stabilize higher order RNA structures (43, 45, 46). In anisotropy assays with Fl-TNF
ARE, both Ca2+ (Fig.
6A) and Mn2+ (Fig.
6B) increased the fluorescence anisotropy of the RNA
substrate in a dose-dependent manner. Regression solutions
using Equation 6 with x = 1 indicated that the affinity
of Fl-TNF
ARE for these cations was somewhat lower than for
Mg2+, however, giving equilibrium constants
(Kf) of 3.1 ± 0.4 × 102
M
1 and 4.0 ± 0.4 × 102 M
1 for Ca2+ and
Mn2+, respectively. These data demonstrate that other
divalent cations may also influence the higher order structure of the
TNF
ARE.

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Fig. 6.
Association of other divalent cations with
the TNF ARE. Anisotropy was measured for
equilibrium binding reactions containing Fl-TNF ARE (0.2 nM) and a titration of calcium chloride (A) or
manganese chloride (B). Regression of each data set was
performed using Equation 6 with AR = 0.0279 and
x = 1, and leaving AR' and
Kf unfixed (solid lines).
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In summary, the similarity in Mg2+-Fl-TNF
ARE binding
equilibria described by FRET and anisotropy and the effects of
Mg2+ on the anisotropy of the Fl-TNF
ARE-2 and TNF
ARE-Fl RNA substrates reveal that association of the TNF
ARE with
Mg2+ induces or stabilizes one or more spatially condensed,
A+U-dependent RNA structures. Furthermore, the flexibility
of the RNA appears to be restrained in this structure relative to the
Mg2+-free RNA, particularly toward the 5'-end. Promotion of
these RNA structures may not be specific for Mg2+, however,
because Ca2+ and Mn2+ may also inhibit
flexibility of the TNF
ARE near the 5'-end. Taken together with the
correlation between cation-induced RNA structural changes and
inhibition of AUF1 binding activity, these studies indicate that
alteration of RNA conformation and/or flexibility may play a direct
role in regulating the association of AUF1 with the TNF
ARE.
Mutually Exclusive Binding of AUF1 or Mg2+ to the
TNF
ARE Does Not Account for Inhibition of Protein Binding--
The
next series of experiments was designed to investigate the mechanisms
whereby Mg2+-induced conformational changes in RNA
substrates containing the TNF
ARE may inhibit the association of
AUF1. The simplest model of this inhibitory mechanism is based on
competition for free RNA between the reactions RNA + Mg2+
RNA·Mg2+, described by Kf, and
the reaction series RNA + P2
P2R and P2R + P2
P4R, described by
K1 and K2, respectively
(Fig. 7A). In essence, this
represents an extension of the binding scheme described in Fig.
1E, in which the free RNA pool is in equilibrium between
Mg2+-free (R) and Mg2+-bound (R') states and
where RNA conformational changes induced by association with
Mg2+ preclude binding of AUF1 dimers. Substitution of
equations describing the equilibrium binding constants
Kf, K1, and
K2 in terms of the concentrations of free RNA,
Mg2+, and AUF1 dimer into Equation 1 yields Equation 7.

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Fig. 7.
A model of Mg2+-induced
inhibition of AUF1 association by mutually exclusive RNA binding.
A, a schematic of an equilibrium binding model for
inhibition of AUF1-binding to the Fl-TNF ARE substrate by
Mg2+-dependent RNA sequestration (described in
text). B, AUF1-dependence of Fl-TNF ARE anisotropy in the
presence of Mg2+ (5 mM) (solid
circles; data set from Fig. 1C) and the solution of
Equation 7 (At versus [P2]
when [Mg2+] = 5 mM) using values for
intrinsic anisotropy and equilibrium constants from Table II
(AR, AP2R,
AP4R, K1, and
K2) and Fig. 5 (Kf), with
AR' unfixed (solid line).
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(Eq. 7)
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To test this model for inhibition of AUF1 binding to the Fl-TNF
ARE substrate by Mg2+, nonlinear regression of an
anisotropy data set generated by titration of
His6-p37AUF1 in the presence of 0.2 nM Fl-TNF
ARE and 5 mM Mg2+ was
applied to Equation 7 (Fig. 7B). Values of
AR, AP2R,
AP4R, K1, and
K2 were known from AUF1 titration experiments
performed with [Mg2+] = 0 (Fig. 1C and Table
II). Likewise, the value of Kf was established from Mg2+ titration experiments performed in the absence of
AUF1 ([P2] = 0; Fig. 5). Whereas a value for the
intrinsic anisotropy of the Mg2+·RNA complex
(AR') was also provided by solution of
Mg2+ titrations in the absence of protein using Equation 6
(Fig. 5), this constant was not fixed in subsequent regressions
involving protein titrations because of the use of heparin as a
competitor for nonspecific RNA-binding activity in these experiments.
Although the inclusion of heparin in Mg2+ titration
experiments up to 5 mM Mg2+ did not
significantly affect the equilibrium constant describing the
Mg2+-RNA interaction (Kf) (data not
shown), small increases observed in absolute anisotropy values could
potentially influence the solution of more global regression functions.
As shown in Fig. 7B, however, the regression function
described by Equation 7 (solid line) did not accurately
reflect the observed anisotropy data. This function did generate a
reasonable estimate for the intrinsic anisotropy of the
Mg2+·RNA complex (AR' = 0.048 ± 0.002), but the model overestimated anisotropy values for the global
equilibrium of His6-p37AUF1 association with
Fl-TNF
ARE in the presence of 5 mM Mg2+,
particularly for concentrations of protein dimer above 2 nM. Although no independent experimental evidence is
available to dismiss this binding model, extensive mathematical
simulations using these data sets reveal no situation in which the
association of AUF1 with the Fl-TNF
ARE RNA substrate in the
presence of 5 mM Mg2+ can be satisfied without
significant changes in the binding parameters defined by earlier
experiments (Table II and Fig. 5). The negative pressure on measured
anisotropy values as a function of protein concentration suggested the
existence of another fluorescent binding complex (i.e.
containing RNA) that exhibited low intrinsic anisotropy and/or was
restrictive of RNA-dependent protein oligomerization. Accordingly, an alternative model for inhibition of AUF1 binding to the
TNF
ARE by Mg2+ was explored.
A Convergent Model of AUF1-TNF
ARE-Mg2+ Binding
Equilibrium--
To accommodate the discrepancy between the
"mutually exclusive binding model" described above and the measured
anisotropy of the Fl-TNF
ARE substrate across a titration of AUF1 in
the presence of Mg2+, it was postulated that an additional
fluorescent species might exist, consisting of a ternary complex of
Fl-TNF
ARE, Mg2+, and an AUF1 dimer. This complex could
exert a negative influence on total measured anisotropy by exhibiting a
low intrinsic anisotropy, forming with poor affinity for AUF1 dimers,
forcing Mg2+ to be ejected prior to formation of AUF1
tetramers, or any combination of these mechanisms. The assembly of this
complex, denoted P2R', would proceed either by association
of an AUF1 dimer with a Mg2+-bound RNA, described by
K1', or by binding of Mg2+ to the
AUF1-dimer bound P2R complex, described by
Kf2 (Fig.
8A). Because this binding
pathway converges at the Mg2+-free P2R species
as a necessary prerequisite for AUF1 tetramer formation as
[P2] increases, regardless of [Mg2+], it
has been termed the "convergent binding model". Similar to the
derivation of Equation 7, substitution of equilibrium binding functions
describing the concentrations of all fluorescent species in
this model into Equation 1 allowed their solution by Equations 8 and
9.

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Fig. 8.
A convergent model for inhibition of AUF1
association on an ARE by Mg2+. A, schematic
of an equilibrium binding model for inhibition of AUF1 binding to the
Fl-TNF ARE substrate including a ternary complex containing
Mg2+, RNA, and a dimer of AUF1 (P2R'). Details
of the model are described in the text. B, regression
solution of Equation 8 (solid line) to the AUF1 titration
experiment performed in the presence of Fl-TNF ARE (0.2 nM) and Mg2+ (5 mM) presented in
Fig. 1C. Values for intrinsic anisotropy and equilibrium
constants from Table II (AR,
AP2R, AP4R,
K1, and K2) and Fig. 5
(Kf) were fixed in the regression, leaving
AR', AP2R', and
K1' unfixed. A residual plot (lower
panel) was prepared by subtraction of the regression-derived
anisotropy value (Ac) from the measured anisotropy
value (At) for each data point.
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(Eq. 8)
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(Eq. 9)
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This binding model was tested by fitting anisotropy values of
Fl-TNF
ARE from a His6-p37AUF1 titration
experiment in the presence of 5 mM Mg2+ by
nonlinear regression. Similar to the test of the mutually exclusive
binding model considered in Fig. 7, values of
AR, AP2R, AP4R, K1,
K2, and Kf were fixed in
Equation 8 based on prior experimentation (Table II and Fig. 5). Values
of AR', K1', and
AP2R' were left unfixed, generating the
regression solution presented in Fig. 8B (upper
panel, solid line). The utility of this model was
supported by a strong coefficient of determination
(R2 = 0.9930) and the random positioning of
residuals across the entire protein titration (Fig. 8B,
lower panel). Using the solution of
K1' from Equation 8, together with the
previously determined values of Kf and
K1, the remaining equilibrium constant
Kf2 was solved using Equation 9. The
values of all intrinsic anisotropy and equilibrium binding constants
describing the convergent binding model of AUF1-TNF
ARE-Mg2+ equilibrium are listed in Table
III. In addition, the rapid dynamics of
all RNA-Mg2+ and RNA-AUF1 interactions tested (Ref. 25 and
data not shown) indicates that the global equilibrium is quickly
re-established following changes in RNA structure or AUF1
concentrations.
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Table III
Solution of anisotropy and equilibrium binding constants for the
convergent binding model of AUF1-TNF ARE-Mg2+ equilibrium
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Although a binding species containing both an AUF1 tetramer and
Mg2+ may exist in this system, the precision of the
regression solution in the absence of terms describing an additional
complex indicate that its concentration is likely too low to
significantly contribute to total measured anisotropy. The implications
of the convergent binding model for regulating the formation of AUF1
multimers by changes in RNA structure and their potential effects on
mRNA turnover rates are described under "Discussion."
 |
DISCUSSION |
Application of the convergent binding model to the global
AUF1-TNF
ARE-Mg2+ equilibrium satisfies the experimental
data in many respects. The model includes the potential for independent
association of Mg2+ or protein with the RNA substrate. In
the absence of Mg2+, AUF1 tetramer assembly proceeds by
sequential binding of protein dimers described by
K1 and K2. In the absence
of protein, Mg2+ induces or stabilizes a conformational
change in the RNA described by Kf. However, in
the presence of both Mg2+ and AUF1, inhibition of protein
association with the RNA may be achieved by two primary mechanisms.
First, the binding affinity of a Mg2+-bound RNA
(K1') versus a Mg2+-free
RNA (K1) substrate for an AUF1 dimer is
decreased approximately 5-fold (Table III). The resulting diminution of
the change in free energy upon AUF1 binding when Mg2+ is
already bound to the RNA indicates that the folded RNA substrate presents reduced potential for intermolecular contacts with the protein. An appealing corollary of this premise is that the reiterative nature of AREs and the potential for multiple RNA-protein contact sites
involving a single AUF1 dimer (four RNA recognition
motifs/dimer) allow the possibility that RNA recognition motifs from
both AUF1 subunits may contribute to optimal binding affinity. Second,
the convergent binding model predicts that Mg2+ must be
ejected from the P2R' complex prior to tetramerization of
AUF1 on the RNA. Because this event is accompanied by an increase in
free energy, retention of RNA substrate in the P2R' state
requires higher concentrations of protein to force the equilibrium in
favor of AUF1 tetramers.
Although no physical evidence has been presented to unequivocally
demonstrate the existence of the ternary P2R' complex,
elucidated values of anisotropy and equilibrium binding constants
associated with its formation hint at some of its properties. For
example, although multiple regions of the TNF
ARE may be involved in
its association with Mg2+, the resulting RNA conformation
is likely more constrained toward its 5'-end in both the R' and
P2R' states. In the formation of the R' state, this is
evidenced by the decrease in
A observed for the
3'-labeled TNF
ARE-Fl substrate compared with the 5'-labeled Fl-TNF
ARE substrate upon Mg2+ binding. Because the
Mg2+-induced increase in anisotropy of these RNAs is
largely, if not solely, due to inhibition of segmental motion
accompanying changes in RNA conformation, restriction of bond rotations
close to the fluorophore would exert a greater influence on its
anisotropy than constraints on the mobility of more distal bonds. In
the formation of the P2R' state from the R' precursor, the
intrinsic anisotropy does not significantly change following protein
binding (Table III), indicating that addition of an AUF1 dimer has
little effect on probe anisotropy when Mg2+ is already
bound. This may reflect distal placement of the AUF1 dimer relative to
Mg2+ on the RNA or that the limitations on protein-RNA
contacts imposed by the Mg2+-induced RNA structure
(described above) may prevent significant changes in RNA segmental
motion following protein binding. The latter of these hypotheses is
further supported by the observation that the intrinsic anisotropy of
the Fl-TNF
ARE is dramatically increased in the high affinity
P2R state (AP2R) relative to the lower affinity P2R' complex (AP2R').
However, this model also implies that changes in molecular volume
following protein binding play a relatively small role in regulating
the mobility of the fluorescein moiety in this system.
To our knowledge, this work presents the first evidence that AREs may
adopt higher order RNA structures in the presence of Mg2+.
The generation or stabilization of RNA tertiary structures by Mg2+ has been well documented for many highly ordered RNA
systems, including ribozymes (39, 43, 47), RNA pseudoknots (48, 49),
RNA helical junctions (50), internal ribosome entry sites (51), and
transfer RNAs (52, 53). AREs, however, generally appear to lack any
significant potential for Watson-Crick base pairing, so contributions
of classical duplexed structures are likely to be minimal. However, the
observation that the Mg2+-induced increase in anisotropy of
Fl-TNF
ARE is significantly greater than that observed with a
similarly sized polyuridylate substrate (Fl-U32) suggests
that the interspersed adenosine residues that distinguish the TNF
ARE from poly(U) are likely involved. Future studies will focus on the
elucidation of binding determinants regulating the association of
Mg2+ with an ARE.
The implications of Mg2+-dependent RNA folding
on ARE function may be related to the differences in sequence
composition of AREs from different mRNAs (7). For example, AREs
from mRNAs encoding cytokines/lymphokines and inflammatory
mediators typically contain several overlapping repeats of AUUUA,
similar to the sequence of Fl-TNF
ARE. By contrast, these pentameric
repeats are generally dispersed or even absent altogether in AREs from
proto-oncogene mRNAs, which are largely divergent except for a high
proportion of uridylate residues. Because Mg2+-induced
changes in ARE conformation and subsequent association with AUF1 are
dependent on RNA sequence (this work), the activity of a given ARE to
promote rapid constitutive mRNA decay may be regulated by its
potential for adopting higher order RNA structures. For example, an
mRNA containing an ARE presenting strong structural constraints is
likely to be less accessible for AUF1 recognition and hence will be
more stable, because AUF1 binding activity closely correlates with the
ability of an ARE to destabilize mRNA (16, 19-22, 24). In the case
of the Fl-TNF
ARE substrate, the RNA-Mg2+ binding
equilibrium constant (Kf) of 7.0 × 10
2 M
1 equates to a
dissociation constant (Kd) of 1.4 mM.
This is near the physiological range of intracellular Mg2+
concentration (54), suggesting that structural variants of this ARE may
play a significant role in regulating the accessibility of
trans-acting factors in vivo. Similar mechanisms
are also likely to regulate protein-binding events involving other RNA
elements. A recent study demonstrated that Mg2+ inhibits
the association of insulin-like growth factor II mRNA-binding protein to the H19 RNA (55). Also, Mg2+-induced
tertiary structures are involved in the association of ribosomal
protein L11 with 23 S ribosomal RNA (56). In addition to potential
roles in regulating constitutive mRNA decay rates, however,
mechanisms involving ARE structures may also serve to modulate mRNA
decay in inducible systems. For example, association of some
"modulator" protein proximal to the ARE may act as an enhancer or
repressor of AUF1 binding activity by remodeling the local topology of
the RNA. In this manner, flanking RNA sequences may also serve as
cis-modifiers of mRNA turnover, by imparting some
thermodynamic or kinetic influence to an RNA structure involving the
ARE or by acting as target sites for the association of other RNA-binding proteins.