Folding of A+U-rich RNA Elements Modulates AUF1 Binding

POTENTIAL ROLES IN REGULATION OF mRNA TURNOVER*

Gerald M. WilsonDagger, Kristina Sutphen, Keng-yu Chuang, and Gary Brewer§

From the Department of Molecular Genetics and Microbiology, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854

Received for publication, October 27, 2000, and in revised form, December 18, 2000


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In mammals, A+U-rich elements (AREs) are potent cis-acting determinants of rapid cytoplasmic mRNA turnover. Recognition of these sequences by AUF1 is associated with acceleration of mRNA decay, likely involving recruitment or assembly of multi-subunit trans-acting complexes. Previously, we demonstrated that AUF1 deletion mutants formed tetramers on U-rich RNA substrates by sequential addition of protein dimers (Wilson, G. M., Sun, Y., Lu, H., and Brewer, G. (1999) J. Biol. Chem. 274, 33374-33381). Here, we show that binding of the full-length p37 isoform of AUF1 to these RNAs proceeds via a similar mechanism, allowing delineation of equilibrium binding constants for both stages of tetramer assembly. However, association of AUF1 with the ARE from tumor necrosis factor (TNFalpha ) mRNA was significantly inhibited by magnesium ions. Further fluorescence and hydrodynamic experiments indicated that Mg2+ induced or stabilized a conformational change in the TNFalpha ARE. Based on the solution of parameters describing both the protein-RNA and Mg2+-RNA equilibria, we present a dynamic, global equilibrium binding model describing the relationship between Mg2+ and AUF1 binding to the TNFalpha ARE. These studies provide the first evidence that some AREs may adopt higher order RNA structures that regulate their interaction with trans-acting factors and indicate that mRNA structural remodeling has the potential to modulate the turnover rates of some ARE-containing mRNAs.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 beta 1-adrenergic receptor mRNA (23, 24). The beta 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 alpha  (TNFalpha ) mRNA was inhibited by magnesium ions, whereas AUF1 binding to a similarly sized polyuridylate sequence was largely unaffected. We present evidence that the TNFalpha 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 TNFalpha 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.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 epsilon 493, Fl = 74,600 M-1·cm-1 and epsilon 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 epsilon 552, Cy3 = 150,000 M-1·cm-1 and epsilon 260, Cy3 = 12,000 M-1·cm-1 (extinction coefficients for Cy3 provided by Amersham Pharmacia Biotech).

The RNA substrates TNFalpha 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 TNFalpha 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 (lambda ex = 535 nm; lambda em = 580 nm) and comparison with a standard curve of Cy-TNFalpha 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 beta -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.
A<SUB>t</SUB>=<LIM><OP>∑</OP><LL>i</LL></LIM> A<SUB>i</SUB>f<SUB>i</SUB> (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 approx  [P2]total).
A<SUB>t</SUB>=<FR><NU>A<SUB><UP>R</UP></SUB>+A<SUB><UP>P<SUB>2</SUB>R</UP></SUB>K<SUB>1</SUB>[<UP>P</UP><SUB>2</SUB>]+A<SUB><UP>P<SUB>4</SUB>R</UP></SUB>K<SUB>1</SUB>K<SUB>2</SUB>[<UP>P</UP><SUB>2</SUB>]<SUP>2</SUP></NU><DE>1+K<SUB>1</SUB>[<UP>P</UP><SUB>2</SUB>]+K<SUB>1</SUB>K<SUB>2</SUB>[<UP>P</UP><SUB>2</SUB>]<SUP>2</SUP></DE></FR> (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 right-left-harpoons  P2R equilibrium, whereas K2 describes the P2R+ P2 right-left-harpoons  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 (lambda 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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Association of Recombinant AUF1 Protein with the TNFalpha 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 TNFalpha mRNA, encoded by the RNA substrate Fl-TNFalpha 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 TNFalpha 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 TNFalpha 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.

                              
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Table I
RNA substrates used in this study

The association of recombinant His6-p37AUF1 with the RNA substrates Fl-TNFalpha 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-TNFalpha 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-TNFalpha 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-TNFalpha 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).
A<SUB>t</SUB>=<FR><NU>A<SUB><UP>R-free</UP></SUB>+A<SUB><UP>R-bound</UP></SUB> · K[<UP>P</UP><SUB>2</SUB>]</NU><DE>1+K[<UP>P</UP><SUB>2</SUB>]</DE></FR> (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-TNFalpha 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 (lambda ex = 490 nm; lambda em = 535 nm) of Fl-TNFalpha 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-TNFalpha 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-TNFalpha 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 x ± sigma n-1.

The inclusion of 5 mM Mg2+ in the RNA-protein binding reactions elicited different consequences in reactions containing the Fl-TNFalpha ARE versus the Fl-U32 substrate. In binding reactions containing Fl-TNFalpha 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-TNFalpha 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-TNFalpha 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-TNFalpha 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-TNFalpha 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 TNFalpha ARE-- Several experiments were performed to characterize structural events that might contribute to the Mg2+-induced decrease in fluorescein mobility observed with the Fl-TNFalpha ARE RNA substrate. First, gel filtration chromatography of RNA substrates through a Superdex 75 column demonstrated that the apparent molecular mass of Fl-TNFalpha ARE was not significantly altered in the presence of 5 mM Mg2+ (Fig. 2). In both the presence and absence of Mg2+, Fl-TNFalpha ARE eluted at ~13 to 14 kDa, close to its calculated molecular mass of 12.5 kDa. By contrast, hybridization of Fl-TNFalpha 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-TNFalpha 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-TNFalpha 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-TNFalpha 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.

In the absence of changes in molecular volume, an increase in the anisotropy of the fluorescein moiety of Fl-TNFalpha 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.
x<UP>Mg</UP><SUP>2+</SUP>+y<UP>RNA</UP> ⇌ <UP>RNA</UP><SUB>y</SUB> · <UP>Mg</UP><SUP>2<UP>+</UP></SUP><SUB>x</SUB> (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-TNFalpha 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 approx  [Mg2+]total).
A<SUB>t</SUB>=<FR><NU>A<SUB><UP>R</UP></SUB>+A<SUB><UP>R</UP>′</SUB>K<SUB><UP>f</UP></SUB>[<UP>Mg<SUP>2+</SUP></UP>]<SUP>x</SUP>[<UP>RNA</UP>]<SUP>4−1</SUP></NU><DE>1+K<SUB><UP>f</UP></SUB>[<UP>Mg<SUP>2+</SUP></UP>]<SUP>x</SUP>[<UP>RNA</UP>]<SUP>4−1</SUP></DE></FR> (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 TNFalpha ARE RNA 5000-fold had no significant effect on the anisotropy (At) of the Fl-TNFalpha 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 TNFalpha ARE without forming RNA multimers.


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Fig. 3.   TNFalpha ARE-Mg2+ equilibrium is independent of RNA concentration. Anisotropy values were measured for reactions containing 0.2 nM Fl-TNFalpha ARE supplemented with unlabeled TNFalpha 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).

Consequences of Mg2+ interaction with an RNA substrate containing the TNFalpha 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-TNFalpha ARE + TNFalpha 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-TNFalpha ARE + TNFalpha 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 TNFalpha 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 TNFalpha 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 TNFalpha ARE and thus would be useful in evaluating changes in RNA conformation.


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Fig. 4.   Analysis of TNFalpha 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 x ± sigma 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-TNFalpha ARE + TNFalpha 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.

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-TNFalpha ARE + TNFalpha ARE-Fl was independent of Mg2+ concentration (Fig. 4B, solid triangles), further confirming that Mg2+-induced RNA structural changes involving the TNFalpha 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 TNFalpha ARE and that the structural changes are unimolecular with respect to RNA.

Equilibrium binding of Mg2+ with the TNFalpha ARE-- Because Mg2+-induced folding of the Fl-TNFalpha 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.


A<SUB>t</SUB>=<FR><NU>A<SUB><UP>R</UP></SUB>+A<SUB><UP>R</UP>′</SUB>K<SUB><UP>f</UP></SUB>[<UP>Mg<SUP>2+</SUP></UP>]<SUP>x</SUP></NU><DE>1+K<SUB><UP>f</UP></SUB>[<UP>Mg<SUP>2+</SUP></UP>]<SUP>x</SUP></DE></FR> (Eq. 6)
To assess the stoichiometric contribution of Mg2+ (x) to this binding event, experiments were performed in which the anisotropy of Fl-TNFalpha 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-TNFalpha 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-TNFalpha ARE substrate is dependent on the presence of the TNFalpha ARE sequence.


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Fig. 5.   Anisotropic analysis of RNA-Mg2+ equilibria by Mg2+ titration. Equilibrium binding reactions containing Fl-TNFalpha 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 (lambda ex = 490 nm; lambda 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-TNFalpha ARE data set was performed using Equation 6 with AR = 0.0279 and AR', Kf, and x unfixed (solid line).

Characterization of TNFalpha ARE Structural Changes Induced or Stabilized by Mg2+-- Additional experiments were performed to elucidate details of RNA structures involving the TNFalpha ARE that were induced or stabilized by Mg2+. Anisotropy experiments using the RNA substrate Fl-TNFalpha ARE-2 (Table I) were used to determine whether sequences outside of the A+U residues within Fl-TNFalpha ARE were required for RNA structural changes in the presence of Mg2+. Similar changes in the anisotropy of both Fl-TNFalpha ARE-2 and Fl-TNFalpha ARE substrates were observed across titrations of Mg2+ (data not shown), indicating that A+U residues within the TNFalpha 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 TNFalpha 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 TNFalpha ARE, anisotropy of the 3'-fluorescein labeled RNA substrate TNFalpha ARE-Fl (Table I) was also monitored across a titration of Mg2+. Although this RNA substrate presented a value for Kf comparable with Fl-TNFalpha ARE, (7 ± 2 × 102 M-1, n = 2), a significant decrease in the overall change in fluorescence anisotropy, given by Delta A = AR' - AR, was observed (cf. 0.009 ± 0.002, n = 2 for TNFalpha ARE-Fl versus 0.021 ± 0.003, n = 3 for Fl-TNFalpha ARE). The decreased value of Delta A for TNFalpha ARE-Fl compared with Fl-TNFalpha 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-TNFalpha 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-TNFalpha 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 TNFalpha ARE.


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Fig. 6.   Association of other divalent cations with the TNFalpha ARE. Anisotropy was measured for equilibrium binding reactions containing Fl-TNFalpha 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).

In summary, the similarity in Mg2+-Fl-TNFalpha ARE binding equilibria described by FRET and anisotropy and the effects of Mg2+ on the anisotropy of the Fl-TNFalpha ARE-2 and TNFalpha ARE-Fl RNA substrates reveal that association of the TNFalpha 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 TNFalpha 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 TNFalpha ARE.

Mutually Exclusive Binding of AUF1 or Mg2+ to the TNFalpha 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 TNFalpha 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+ right-left-harpoons  RNA·Mg2+, described by Kf, and the reaction series RNA + P2 right-left-harpoons  P2R and P2R + P2 right-left-harpoons  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-TNFalpha ARE substrate by Mg2+-dependent RNA sequestration (described in text). B, AUF1-dependence of Fl-TNFalpha 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).


A<SUB>t</SUB>=<FR><NU>A<SUB><UP>R</UP></SUB>+A<SUB><UP>R</UP></SUB>,K<SUB><UP>f</UP></SUB>[<UP>Mg<SUP>2+</SUP></UP>]+A<SUB><UP>P2R</UP></SUB>K<SUB>1</SUB>[<UP>P<SUB>2</SUB></UP>]+A<SUB><UP>P4R</UP></SUB>K<SUB>1</SUB>K<SUB>2</SUB>[<UP>P<SUB>2</SUB></UP>]<SUP><UP>2</UP></SUP></NU><DE><UP>1+K<SUB>f</SUB></UP>[<UP>Mg<SUP>2+</SUP></UP>]+K<SUB>1</SUB>[<UP>P<SUB>2</SUB></UP>]+K<SUB>1</SUB>K<SUB>2</SUB>[<UP>P<SUB>2</SUB></UP>]<SUP><UP>2</UP></SUP></DE></FR> (Eq. 7)
To test this model for inhibition of AUF1 binding to the Fl-TNFalpha 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-TNFalpha 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-TNFalpha 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-TNFalpha 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 TNFalpha ARE by Mg2+ was explored.

A Convergent Model of AUF1-TNFalpha ARE-Mg2+ Binding Equilibrium-- To accommodate the discrepancy between the "mutually exclusive binding model" described above and the measured anisotropy of the Fl-TNFalpha 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-TNFalpha 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-TNFalpha 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-TNFalpha 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.


A<SUB>t</SUB>=<FR><NU>A<SUB><UP>R</UP></SUB>+A<SUB><UP>R</UP>′</SUB>K<SUB><UP>f</UP></SUB>[<UP>Mg<SUP>2+</SUP></UP>]+A<SUB><UP>P<SUB>2</SUB>R</UP></SUB>K<SUB>1</SUB>′K<SUB><UP>f</UP></SUB>[<UP>Mg<SUP>2+</SUP></UP>][<UP>P</UP><SUB>2</SUB>]+A<SUB><UP>P<SUB>2</SUB>R</UP></SUB>K<SUB>1</SUB>[<UP>P</UP><SUB>2</SUB>]+A<SUB><UP>P<SUB>4</SUB>R</UP></SUB>K<SUB>1</SUB>K<SUB>2</SUB>[<UP>P</UP><SUB>2</SUB>]<SUP>2</SUP></NU><DE>1+K<SUB><UP>f</UP></SUB>[<UP>Mg<SUP>2+</SUP></UP>]+K<SUB>1</SUB>′K<SUB><UP>f</UP></SUB>[<UP>Mg<SUP>2+</SUP></UP>][<UP>P</UP><SUB>2</SUB>]+K<SUB>1</SUB>[<UP>P</UP><SUB>2</SUB>]+K<SUB>1</SUB>K<SUB>2</SUB>[<UP>P</UP><SUB>2</SUB>]<SUP>2</SUP></DE></FR> (Eq. 8)

K<SUB>1</SUB>′K<SUB><UP>f</UP></SUB>=K<SUB>1</SUB>K<SUB><UP>f</UP>2</SUB> (Eq. 9)
This binding model was tested by fitting anisotropy values of Fl-TNFalpha 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-TNFalpha 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-TNFalpha ARE-Mg2+ equilibrium

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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Application of the convergent binding model to the global AUF1-TNFalpha 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 TNFalpha 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 Delta A observed for the 3'-labeled TNFalpha ARE-Fl substrate compared with the 5'-labeled Fl-TNFalpha 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-TNFalpha 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-TNFalpha ARE is significantly greater than that observed with a similarly sized polyuridylate substrate (Fl-U32) suggests that the interspersed adenosine residues that distinguish the TNFalpha 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-TNFalpha 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-TNFalpha 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.

    ACKNOWLEDGEMENT

We thank Dr. Lara Izotova for helpful discussions regarding the rapid purification of recombinant proteins.

    FOOTNOTES

* This work was funded by Grant R01 CA 52443 from the National Institutes of Health and a grant from the Arthritis Foundation (to G. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence may be addressed: Dept. of Molecular Genetics and Microbiology, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway, NJ 08854. Tel.: 732-235-3379; Fax: 732-235-5223; E-mail: wilsongm@umdnj.edu.

§ To whom correspondence may be addressed: Dept. of Molecular Genetics and Microbiology, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway, NJ 08854. Tel.: 732-235-3473; Fax: 732-235-5223; E-mail: brewerga@umdnj.edu.

Published, JBC Papers in Press, December 21, 2000, DOI 10.1074/jbc.M009848200

    ABBREVIATIONS

The abbreviations used are: ARE, A+U-rich element; Cy3, cyanine 3; Fl, fluorescein; FRET, fluorescence resonance energy transfer; TNFalpha , tumor necrosis factor alpha .

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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