Alternatively Expressed Domains of AU-rich Element RNA-binding Protein 1 (AUF1) Regulate RNA-binding Affinity, RNA-induced Protein Oligomerization, and the Local Conformation of Bound RNA Ligands*

AU-rich element RNA-binding protein 1 (AUF1) binding to AU-rich elements (AREs) in the 3′-untranslated regions of mRNAs encoding many cytokines and other regulatory proteins modulates mRNA stability, thereby influencing protein expression. AUF1-mRNA association is a dynamic paradigm directed by various cellular signals, but many features of its function remain poorly described. There are four isoforms of AUF1 that result from alternative splicing of exons 2 and 7 from a common pre-mRNA. Preliminary evidence suggests that the different isoforms have varied functional characteristics, but no detailed quantitative analysis of the properties of each isoform has been reported despite their differential expression and regulation. Using purified recombinant forms of each AUF1 protein variant, we used chemical cross-linking and gel filtration chromatography to show that each exists as a dimer in solution. We then defined the association mechanisms of each AUF1 isoform for ARE-containing RNA substrates and quantified relevant binding affinities using electrophoretic mobility shift and fluorescence anisotropy assays. Although all AUF1 isoforms generated oligomeric complexes on ARE substrates by sequential dimer association, sequences encoded by exon 2 inhibited RNA-binding affinity. By contrast, the exon 7-encoded domain enhanced RNA-dependent protein oligomerization, even permitting cooperative RNA-binding activity in some contexts. Finally, fluorescence resonance energy transfer-based assays showed that the different AUF1 isoforms remodel bound RNA substrates into divergent structures as a function of protein:RNA stoichiometry. Together, these data describe isoform-specific characteristics among AUF1 ribonucleoprotein complexes, which likely constitute a mechanistic basis for differential functions and regulation among members of this protein family.

In complex organisms, individual cells must precisely control protein expression to rapidly respond to ever-changing stimuli. Protein expression can be regulated at multiple levels, including the rates of transcription, translation, and protein degradation. Messenger RNA stability represents another critical mechanism for regulating protein expression, because alterations in mRNA decay kinetics can modulate the cytoplasmic mRNA concentrations required to program protein synthesis independent of changes in the mRNA synthetic rate. Across the population of cellular mRNAs, decay rates can vary by 2 orders of magnitude or more. Furthermore, the stability of many mRNAs can be dramatically altered in response to selected stimuli (reviewed in Refs. 1,2).
Information directing the decay kinetics of specific transcripts is located within the mRNAs themselves. The best characterized of these cis-acting mRNA stability determinants are the AU-rich elements (AREs) 4 located in the 3Ј-untranslated regions (3ЈUTRs) of many labile mRNAs (3). In humans, AREs may be present in as many as 8% of all transcripts, including many that encode clinically significant factors like proto-oncogenes, cytokines, cell cycle regulators, and inflammatory mediators (4). Regulation of mRNA decay through AREs involves the association of cellular trans-acting factors, collectively known as ARE-binding proteins (reviewed in Refs. 5,6); however, more recently, selected microRNAs have been implicated as trans-regulators of ARE function (7,8). Some ARE-binding proteins, like members of the Hu family of proteins, act as stabilizing factors by preventing mRNA decay (9,10), whereas others, like tristetraprolin (11,12), KSRP (13,14), and AUF1 (15)(16)(17)(18), are generally associated with enhanced degradation of targeted transcripts. Some other AREbinding proteins, including TIA-1 and TIAR, do not appear to modulate mRNA decay kinetics directly but rather inhibit their translation (reviewed in Ref. 19).
AUF1, also known as heterogeneous nuclear ribonucleoprotein D (hnRNP D), is a family of four proteins derived from alternative splicing of a common pre-mRNA, named according to their apparent molecular masses as p37 AUF1 , p40 AUF1 , p42 AUF1 , and p45 AUF1 (20). All isoforms have two tandemly arranged RNA recognition motifs, which are essential for RNA binding, a C-terminal extension that includes a glutamine-rich domain, and an acidic N-terminal region that may contribute to protein dimerization (21,22). The two largest isoforms also include a 49-amino acid domain near the C terminus encoded by exon 7, whereas p40 AUF1 and p45 AUF1 both contain a 19-amino acid insert encoded by exon 2 that is contiguous with RRM1 (Fig. 1A). AUF1 was first identified as a cytoplasmic factor that stimulated the decay of c-myc mRNA in cell-free mRNA decay assays (23), and it is most frequently associated with acceleration of ARE-directed mRNA decay (described above). However, emerging data indicate that AUF1 may also enhance mRNA translational efficiency in some cases, possibly involving interaction with components of the translation machinery like eIF4G and poly(A)binding protein (24) or competition for ARE targets between AUF1 and the translational repressor TIAR (25).
Although studies assessing the function of AUF1 in posttranscriptional control of gene expression frequently do not discriminate among the family members, some reports indicate variations in the function and/or regulated expression of individual AUF1 isoforms. First, p42 AUF1 and p45 AUF1 are normally nuclear, whereas p37 AUF1 and p40 AUF1 are found within both nuclear and cytoplasmic compartments in many cell types (21, 26 -28). Second, distinct isoforms have been implicated in the regulated decay of selected ARE-containing mRNAs. For example, a reporter mRNA containing the ARE from interleukin-3 mRNA was stabilized by concomitant siRNA-directed suppression of p40 AUF1 and p45 AUF1 but not by coordinated depletion of p42 AUF1 and p45 AUF1 or all isoforms simultaneously (17). By contrast, specific overexpression of p37 AUF1 , and to some extent p40 AUF1 , destabilized a reporter mRNA containing the ARE from granulocyte-macrophage colony-stimulating factor mRNA, although p42 AUF1 and p45 AUF1 had no effect (18). Third, some stimuli have been reported to modulate the cellular levels of specific AUF1 isoforms. In sheep uterus, estradiol increased expression of p45 AUF1 but not other isoforms (29). Similarly, prostaglandin A 2 specifically increased levels of p45 AUF1 in H1299 nonsmall-cell lung carcinoma cells (30). Finally, the differentially expressed sequences that distinguish AUF1 family members provide opportunities for isoform-specific regulation through post-translational events. For example, in THP-1 monocytic leukemia cells, polysome-associated p40 AUF1 is phosphorylated at two sites encoded by exon 2, which are accordingly absent in p37 AUF1 and p42 AUF1 (28). Dephosphorylation of these sites is associated with stabilization of ARE-containing mRNAs coupled with condensation of local RNA structure (28,31).
Taken together, the potential for differential function and regulation among AUF1 isoforms suggests that each may exhibit unique biochemical characteristics that mediate distinctive RNA-binding paradigms. In this study, we have used biochemical and fluorescence spectroscopic approaches to compare the RNA-binding and RNA structural remodeling properties of all four AUF1 isoforms. Using purified, recombinant AUF1 proteins and two families of model AREs, we show that all AUF1 isoforms assemble into oligomeric protein complexes on ARE substrates through two distinct binding steps. However, the differentially expressed protein domains encoded by exons 2 and 7 each influence the RNA-binding activities of AUF1 proteins in distinct ways, based on variations in RNA-binding affinity, RNA-induced protein oligomerization, and the structural properties of bound RNA substrates. Together, these data are consistent with a model whereby alternative splicing of the AUF1 pre-mRNA generates a family of RNA-binding proteins with diverse biochemical properties, which may contribute to the heterogeneity of AUF1 function and complexity of its regulation.

EXPERIMENTAL PROCEDURES
RNA Substrates-Synthesis, deprotection of 2Ј-hydroxyl groups, and purification of all RNA oligonucleotides were performed by Integrated DNA Technologies or Dharmacon. Lyophilized RNA pellets were resuspended in deionized H 2 O. RNA yields and fluorophore labeling efficiencies were quantified by absorbance, incorporating fractional contributions of coupled fluorophores to A 260 for substrates containing fluorescein (Fl) and/or cyanine-3 (Cy3) moieties as described previously (32,33). Oligoribonucleotides designated ARE xx are variants of the core ARE sequence from TNF␣ mRNA ( Table  1). The RNA substrate "fos ARE domain I" encodes a 46-nucleotide segment of the ARE from human c-fos mRNA that is sufficient to accelerate decay of a reporter transcript in cis (34). The substrate "R␤" encodes a fragment of the coding sequence from rabbit ␤-globin mRNA. RNA substrate variants containing 5Ј-linked cyanine-3 (Cy3) or fluorescein (Fl) groups are indicated by relevant prefixes, where applicable. Similarly, RNA substrates containing 3Ј-fluorescein groups are suffixed by "Fl". As needed, 5Ј-hydroxyl RNA substrates were radiolabeled to specific activities of 3-5 ϫ 10 3 cpm/fmol using T4 polynucleotide kinase and [␥-32 P]ATP as described previously (35).
Preparation of Recombinant AUF1 Proteins-cDNAs encoding human p42 AUF1 and p45 AUF1 were inserted downstream of a His 6 tag motif in pBAD/HisB (Invitrogen) using standard subcloning techniques and verified by restriction digestion and automated DNA sequencing. Construction of pBAD/HisB-based expression vectors encoding p37 AUF1 and p40 AUF1 was described previously (31,36). All pBAD-HisB/ AUF1 plasmids were transformed into Escherichia coli Ro-  setta 2 cells (Novagen), and expression of recombinant His 6 -AUF1 proteins was induced with arabinose (0.02%). His 6 -AUF1 proteins were purified from bacterial cell lysates by Ni 2ϩ -affinity chromatography over a HiTrap chelating affinity column (GE Healthcare) essentially as described previously (36), except that after loading and washing the Ni 2ϩ -affinity resin, 6 column volumes of Triton wash buffer (50 mM sodium phosphate (pH 8.0) containing 500 mM NaCl, 20 mM imidazole, and 1% Triton X-100) was also applied prior to His 6 -AUF1 elution. This additional washing step improved the purity of the recovered AUF1 proteins. Proteins used for RNA-binding studies were buffer-exchanged into 10 mM Tris-HCl (pH 7.5) using a HiPrep 26/10 desalting column (GE Healthcare). For gel filtration or chemical cross-linking studies where primary amino groups must be excluded, His 6 -p37 AUF1 , -p40 AUF1 , and -p42 AUF1 were buffer-exchanged into 10 mM HEPES-KOH (pH 7.5). Because His 6 -p45 AUF1 formed significant aggregates in the HEPES buffer, a solubility screening test was performed essentially as described previously (37) to identify an optimal buffer for this isoform. 10 mM MOPS-NaOH (pH 7.5) was selected as the best buffer lacking primary amino groups among those screened based on minimization of high molecular mass complexes or aggregates as measured by dynamic light scattering using a Zetasizer nano series instrument (Malvern Instruments). All recombinant proteins were quantified by Coomassie Blue-stained SDS-PAGE against a titration of bovine serum albumin.
Gel Filtration Chromatography-A HiPrep 16/60 column (GE Healthcare) packed with Sephacryl S-200 high resolution resin was equilibrated with gel filtration buffer (10 mM HEPES-KOH (pH 7.5) containing 150 mM KCl for all isoforms except p45 AUF1 , for which 10 mM MOPS-NaOH (pH 7.5) containing 150 mM KCl was used). Protein samples (140 -180 g) were diluted in gel filtration buffer (0.5 ml final volume) and centrifuged at 16,100 ϫ g for 5 min to remove particulates before loading. Samples were fractionated at 30 ml/h with elution monitored by absorbance at 280 nm. Column void volume was determined using blue dextran. The column was calibrated by monitoring elution of the following protein standards (Sigma) by A 280 : alcohol dehydrogenase (150 kDa), bovine serum albumin (66 kDa), and carbonic anhydrase (29 kDa).
Measurements of RNA-Protein Binding-Association of recombinant AUF1 proteins with RNA substrates was qualitatively assessed using electrophoretic mobility shift assays (EMSA) essentially as described previously (38). Briefly, reactions containing limiting concentrations of 32 P-labeled RNA substrates (0.2 nM) and varying concentrations of protein were assembled in 10 mM Tris-HCl (pH 8) containing 50 mM KCl, 2 mM DTT, 0.5 mM EDTA, 0.1 g/l acetylated bovine serum albumin, 1 g/l heparin, and 10% glycerol (10 l final volume). After incubating for 15 min on ice, reactions were fractionated on a 6% native acrylamide gel at 4°C. Gels were then dried and products visualized using a PhosphorImager.
Quantitative measurement of protein-RNA-binding equilibria was performed using fluorescence anisotropy essentially as described previously (35,36). At constant temperature and viscosity, protein binding to a fluorophore-labeled RNA increases the rotational relaxation time of the fluorophore by increasing its molecular volume and decreasing intramolecular segmental motion of the RNA substrate (39,40). Binding reactions were assembled as described for EMSA, except that Fl-labeled RNA was employed in place of the radiolabeled substrate, total volume was 100 l, and glycerol was omitted. After a 1-min incubation at 25°C, fluorescence anisotropy of the Fl-RNA substrates was measured using a Beacon 2000 fluorescence polarization system (Panvera) equipped with fluorescein excitation (490 nm) and emission (535 nm) filters.
Preliminary on-rate studies demonstrated that binding equilibrium was achieved for all AUF1 isoforms within this time frame (Refs. 31, 35 and data not shown). Concomitant with fluorescence anisotropy, total fluorescence emission from each reaction was also measured to verify that protein binding did not significantly alter the fluorescence quantum yields of RNA substrates (data not shown).
Binding constants describing AUF1 association with RNA substrates were resolved by nonlinear regression of total measured anisotropy as a function of protein concentration using PRISM version 3.03 software (GraphPad). Based on our interpretation of all AUF1 isoforms as dimers in solution (described under "Results"), association of a single protein dimer (P 2 ) to a Fl-labeled RNA substrate under limiting RNA concentrations (i.e., where [P 2 ] free Ϸ [P 2 ] total ) and constant fluorescence quantum yield is described by Equation 1.
Here, A t is the total measured anisotropy; A R and A P2R represent the intrinsic anisotropy of the free RNA and dimerbound RNA substrate, respectively, and K is the apparent equilibrium association constant (K ϭ 1/K d ). Equation 1 can be expanded to describe sequential two-step association of AUF1 dimers with an RNA substrate yielding tetrameric protein complexes by incorporating terms representing the intrinsic anisotropy of the protein tetramer⅐Fl-RNA complex (A P4R ) and tandem apparent association constants K 1 and K 2 in Equation 2 (35,40).
Reactions best described by cooperative binding were resolved by a variant of the Hill model in Equation 3 where A PxR represents the intrinsic anisotropy of the saturated protein⅐Fl-RNA complex; [P 2 ]1 ⁄ 2 is the concentration of His 6 -AUF1 dimers giving half-maximal binding, and h represents the degree of cooperativity.
The appropriateness of each binding model was evaluated by the coefficient of determination (R 2 ) from individual experiments and analysis of residual plot nonrandomness to detect any bias for data subsets (PRISM). When multiple models were considered for a common data set, pairwise comparisons of sum-of-squares deviations were performed using the F test (PRISM), with differences exhibiting p Ͻ 0.05 considered significant. Macromolecular Binding Density Analysis-The apparent site size of AUF1 dimer binding on an Fl-RNA substrate and the fractional contributions of each binding step to changes in its fluorescence anisotropy were resolved using macromolecular binding density analysis (MBDA). This method quantifies the degree of binding (Α, defined as the population-averaged number of AUF1 dimers bound per Fl-labeled RNA substrate) and free AUF1 dimer concentration ([P 2 ] F ) without any prior knowledge or assumptions regarding their relationship to the observed anisotropy signal change (⌬A obs ). Described in greater detail elsewhere (41,42), MBDA presumes that every complex i containing an Fl-labeled RNA contributes to ⌬A obs at a given point in the titration, where ⌬A obs ϭ A i Ϫ A R , and A R is the experimentally determined anisotropy of Fl-RNA in the absence of protein. Thus, if each i complex has a maximal per mol contribution ⌬A i to the anisotropy, the experimentally observable signal ⌬A obs is related to Α by Equation 4.
In other words, the total signal is the population-weighted sum of contributions of the per mol anisotropy for each species i. Accordingly, the same value of ⌬A obs obtained at two different total RNA concentrations, [R] T1 and [R] T2 , must be in the same physical state with the same average degree of binding Α and free concentration of protein [P 2 ] F at the corresponding total protein concentrations, [P 2 ] T1 and [P 2 ] T2 . Therefore, the paired ([P 2 ] T , [R] T ) concentrations obtained at a point from each isotherm possessing the same ⌬A obs can be plotted to obtain the average binding density and free protein dimer concentration as the slope and vertical intercept, respectively, of [P 2 ] T versus [R] T by Equations 5 and 6, where x ϭ 1 or 2.
In this study, we analyzed isotherms across three RNA concentrations to obtain an estimate of the errors associated with Α and [P 2 ] F . To minimize the influence of a nonspecific binding component that contributes to ⌬A obs at high protein concentrations, MBDA was limited to binding data corre-sponding to ⌬A obs Ͻ70 -80% of the asymptotic maximum (⌬A sat ) for each anisotropy isotherm. The intersection of the linearly extrapolated limiting slopes of the two binding regimes observed for ⌬A obs versus Α was used to calculate Α int , the AUF1 dimer:RNA stoichiometry that demarks a clear transition in the average anisotropic contribution with increasing binding density. The apparent site size for this intermediate complex was then estimated as the ratio of the RNA nucleotide length (N) and the intermediate stoichiome- Assays of RNA Folding Using Steady State FRET-RNA remodeling by each AUF1 isoform was monitored by changes in the distance between 3Ј-Fl (donor) and 5Ј-Cy3 (acceptor) groups appended to the termini of RNA substrates using FRET, essentially as described previously (43). Briefly, FRET efficiency (E FRET ) varies inversely with the scalar distance (r) between a fluorescent donor (in this case Fl) and an appropriate acceptor (Cy3) by Equation 7.
The Förster distance (R 0 ) is calculable from fluorophore spectral data and represents the separation between donor and acceptor yielding E FRET ϭ 0.5 (44). For Fl and Cy3 dyes appended to single-stranded nucleic acids, R 0 has been calculated as 55.7 Å (45). Varying concentrations of specified AUF1 isoforms were incubated with RNA substrates ARE 38 -Fl (donor alone) or Cy3-ARE 38 -Fl (donor-acceptor pair) as described for anisotropy analyses (see above), except that final RNA concentrations were 2 nM. Fluorescence from the FRET donor (Fl) was measured using a Cary Eclipse fluorometer (Varian) equipped with a sub-microcell cuvette ( ex ϭ 485 nm, em ϭ 520 nm, 10-nm slit widths). Background fluorescence was quantified from samples lacking RNA substrates. Inner filter effects and photobleaching were insignificant in these experiments (data not shown). E FRET between donor and acceptor fluorophores was calculated using Equation 8 (derived from Ref. 43), where F Cy-Fl is the background-corrected fluorescence of the donor in the presence of the acceptor (measured from the Cy-ARE 38 -Fl substrate), F Fl is the background-corrected donor fluorescence from parallel reactions lacking the acceptor (measured using the ARE 38 -Fl substrate), and f DA is the fractional labeling of the Cy3 acceptor on the Cy-ARE 38 -Fl RNA.
Correlating AUF1-dependent changes in RNA conformation with formation of specific AUF1⅐RNA complexes required estimation of the relative concentration of each RNA⅐protein complex across titrations of protein. These fractional distributions were calculated using the sequential twostep association model (described above); however, the elevated concentrations of RNA substrates required for FRET measurements necessitated consideration of protein depletion, which becomes significant at low total protein concentrations. By conservation of mass, the two-step association model requires that the total AUF1 dimer ([P 2 ] T ) and RNA ([R] T ) concentrations be distributed between the free ([P 2 ] F , [R] F ), the single dimer-bound mode ([P 2 R]), or the tetramerbound mode ([P 4 R]) by Equations 9 and 10.
The concentration of each bound species is related to [P 2 ] F and [R] F through the known apparent binding constants K 1 and K 2 by Equations 11 and 12, whereas the concentration of free RNA substrate can be calculated using Equation 13.
However, solution of each function requires an explicit value for [P 2 ] F . This was calculated by combining Equations 9 -13 to generate Equation 14. The

RESULTS
All AUF1 Isoforms Form Complexes Consistent with Dimers in the Absence of RNA-All recombinant His 6 -AUF1 proteins produced using the pBAD/His vector system and purified by Ni 2ϩ -affinity chromatography yielded proteins of expected molecular weights and Ͼ95% purity by SDS-PAGE (Fig. 1B). Previous gel filtration and analytical ultracentrifugation studies showed that p37 AUF1 is a dimer in solution and suggested that N-terminal sequences common to all isoforms were responsible for AUF1 dimerization (22). Chemical cross-linking similarly demonstrated that p37 AUF1 formed dimers in the absence of high affinity RNA substrates (35). Subsequently, glutathione S-transferase pulldown experiments indicated that recombinant versions of all AUF1 isoforms could form protein-protein interactions with one another (46), although the overall size of these complexes was not defined.
To determine whether all AUF1 isoforms formed dimers in solution similar to those described for p37 AUF1 , two experiments were performed. First, recombinant forms of each AUF1 isoform were individually fractionated on a size-exclusion column. No distinct peaks were observed at elution volumes predicted for monomeric proteins (Fig. 1C). The major elution peaks for both His 6 -p40 AUF1 and His 6 -p45 AUF1 corresponded to apparent molecular weights twice those of each monomer, consistent with these proteins eluting as dimers ( Table 2). The apparent molecular weights of His 6 -p37 AUF1 and His 6 -p42 AUF1 were significantly greater than those of monomers, although somewhat less than those calculated for protein dimers. Because His 6 -p37 AUF1 had already been characterized as a dimer by analytical ultracentrifugation (22) and chemical cross-linking (35), our data suggest that His 6 - p37 AUF1 and His 6 -p42 AUF1 are likely eluting as higher mobility dimers. It is possible that the absence of exon 2-encoded sequences in these isoforms confers distinct conformational features that alter their hydrodynamic properties. No larger oligomeric forms were detected for any AUF1 isoform in the absence of RNA. In a second series of experiments, AUF1 dimerization was tested using the chemical cross-linker DSP. All DSP-treated His 6 -AUF1 proteins yielded products consistent with both intramolecular (Fig. 1D, bottom bands) and intermolecular (Fig. 1D, top bands) cross-links, the latter migrating with apparent molecular weights predicted for dimeric proteins. The efficiency of intermolecular cross-linking could not be directly assessed in these experiments because DSP treatment significantly weakened binding of all recombinant AUF1 proteins to anti-AUF1 antibodies or Coomassie Blue-based stains (data not shown). However, the comparable distributions of cross-linked products observed for His 6 -p40 AUF1 , His 6 -p42 AUF1 , and His 6 -p45 AUF1 relative to His 6 -p37 AUF1 , which was previously characterized as a dimer, suggest that all AUF1 isoforms are similarly dimeric in solution.
Alternatively Expressed Domains of AUF1 Regulate Both RNA-binding Affinity and RNA-induced Protein Oligomerization-Previously, we showed that two dimers of p37 AUF1 can sequentially bind the ARE from TNF␣ mRNA ( Fig. 2A) to first form a protein dimer⅐RNA (P 2 R) complex, followed by a protein tetramer⅐RNA (P 4 R) complex (35,36). Using EMSAs, we observed that the other AUF1 isoforms also bind this RNA substrate in two stages, consistent with the p37 AUF1 model (Fig. 2B). The faster migrating complex (complex I) is observed at lower protein concentrations, consistent with the initial protein dimer binding event (P 2 R), whereas a slower mobility band appearing at higher AUF1 concentrations (complex II) is consistent with the P 4 R complex. These binding events are RNA sequence-specific, because no complexes were detected in EMSAs using the R␤ RNA substrate, which lacks AU-rich sequences (data not shown). Comparing the protein concentration dependence of RNA binding among all isoforms yielded two interesting observations. First, formation of complex I required higher protein concentrations for AUF1 isoforms containing sequences encoded by exon 2 relative to their respective exon 2-deficient isoforms (Fig. 2B, cf. His 6 -p40 AUF1 versus His 6 -p37 AUF1 and His 6 -p45 AUF1 versus His 6 -p42 AUF1 ), suggesting that the inclusion of exon 2-encoded sequences inhibits formation of the initial AUF1⅐RNA P 2 R complex. Second, complex II assembly was observed at lower protein concentrations for AUF1 isoforms that contained sequences encoded by exon 7 (Fig. 2B, cf. His 6 -p42 AUF1 versus His 6 -p37 AUF1 and His 6 -p45 AUF1 versus His 6 -p40 AUF1 ), suggesting that the second binding step (i.e. leading to formation of the P 4 R complex) might be enhanced by the exon 7-encoded domain.
To quantitatively assess recombinant His 6 -AUF1 binding to RNA targets, we measured the change in fluorescence anisotropy of the Fl-ARE 38 substrate as a function of protein concentration. As reported previously (31,36), His 6 -p37 AUF1 (Fig.  3A) and His 6 -p40 AUF1 (data not shown) binding to this RNA target was well described by the two-step sequential binding model of Equation 2, permitting explicit solutions of binding constants describing both stages of RNP assembly ( Table 3). Appropriateness of the two-step binding model was supported by random distributions of residuals (e.g. Fig. 3A, bottom) and high coefficients of determination (R 2 Ͼ 0.98) across FIGURE 2. Evaluation of His 6 -AUF1 binding to the ARE 38 RNA substrate by EMSA. A, sequential dimer association model for AUF1 binding to an RNA substrate. B, binding reactions containing a 5Ј-32 P-labeled ARE 38 RNA substrate, and titrations of each recombinant His 6 -AUF1 isoform were assembled as described under "Experimental Procedures" prior to fractionation on nondenaturing gels. Bands corresponding to distinct RNA⅐protein complexes formed on the 32 P-ARE 38 substrate are indicated as complexes I and II. Unbound probe is designated as free RNA. because of redundancy in fitting the A P2R and K 2 parameters. This frequently occurs for data sets where K 1 and K 2 vary by less than 5-fold and prevents explicit solution of binding constants using this approach when the value of A P2R is unknown (40). However, the protein concentration dependence of His 6 -p42 AUF1 (Fig. 3B) and His 6 -p45 AUF1 (data not shown) binding to the Fl-ARE 38 substrate was well described using the cooperative binding model of Equation 3. Resolution of h significantly greater than 1 for His 6 -p42 AUF1 binding to this RNA substrate (Table 3) suggests that binding may be cooperative, with the second dimer binding more strongly than the first. This possibility is dependent on the intrinsic anisotropy of the protein dimer-bound RNA complex (A P2R from Equation 2) and is addressed further below. Analyses of His 6 -p45 AUF1 association with the Fl-ARE 38 substrate using Equation 3 resolved h Ϸ 1, indicating that both protein binding steps are likely to be thermodynamically similar.
Comparing the binding constants describing the association of each His 6 -AUF1 isoform with the Fl-ARE 38 substrate supports independent roles for domains encoded by alternatively spliced exons of AUF1. First, constants describing His 6 -p37 AUF1 and His 6 -p40 AUF1 binding to the Fl-ARE 38 substrate show that the presence of exon 2-encoded sequences in His 6 -p40 AUF1 inhibits the first RNA-binding event by nearly 6-fold and the second binding event by ϳ2-fold relative to the p37 isoform (Table 3). Although EMSAs from this (Fig. 2B) and previous studies (20) indicated that p40 AUF1 binds ARE substrates more weakly than p37 AUF1 , the anisotropy-based experiments show that this distinction is principally manifested at the initial contact with the RNA substrate. However, the anisotropy-based binding assays also revealed a novel effect of exon 7-encoded sequences on the assembly of AUF1 RNPs, involving dramatic enhancement of the second binding step. Although a His 6 -p37 AUF1 dimer binds to the P 2 R complex on the Fl-ARE 38 substrate with a modest apparent K d of 74 nM based on the two-step binding model (Equation 2), the potential cooperativity of His 6 -p42 AUF1 binding to this substrate shown by Hill analysis (Equation 3) indicates that the affinity of the second binding step is near or better than the [P 2 ]1 ⁄ 2 value of 4.4 nM (Table 3). Similarly, the second His 6 -p40 AUF1 dimer is recruited very weakly with an apparent K d of 160 nM, although inclusion of exon 7-encoded sequences (yielding His 6 -p45 AUF1 ) improves the affinity of this step to ϳ14 nM, the [P 2 ]1 ⁄ 2 value for His 6 -p45 AUF1 binding to the Fl-ARE 38 substrate. Together with the protein concentration dependence of binding described by EMSAs (Fig. 2B), our anisotropybased binding studies show that the AUF1 protein domain encoded by exon 7 enhances formation of AUF1 oligomers on the TNF␣ ARE by improving the affinity of the second dimer binding event, although sequences encoded by exon 2 can inhibit AUF1 recruitment to the ARE substrate by weakening the affinity of the first binding step.

Enhanced ARE-induced AUF1 Oligomerization Regulated by Exon 7-encoded Sequences Does Not Result from Variations in Binding Site
Size-Although our binding studies show that the exon 7-encoded sequences near the C terminus of His 6 -p42 AUF1 and His 6 -p45 AUF1 enhance assembly of protein tetramers on the TNF␣ ARE substrate, it is not known how this domain promotes binding of the second AUF1 dimer. Conceivably, this domain may participate in protein-protein contacts or may direct conformational events that enhance the second binding step. Alternatively, the RNA-binding site for AUF1 isoforms lacking exon 7-encoded sequences may be too large to permit multiple high affinity binding events on the 38-nucleotide TNF␣ ARE substrate. In the latter case, high affinity recruitment of the second His 6 -p37 AUF1 or His 6 -p40 AUF1 dimer might be precluded simply because an insufficient length of RNA is presented to the protein.  Table 3. For comparison, optimal fits to single-site binding models (Equation 1) are also shown for each dataset (dotted lines). Residual plots show random deviation of the preferred binding models (A calc ) from observed data (A obs ) (lower panels).

TABLE 3 Affinity of recombinant AUF1 isoforms for the Fl-ARE 38 RNA substrate
Apparent dissociation constants were solved for each binding step of His 6 -p37 AUF1 and His 6 -p40 AUF1 on the Fl-ARE 38 substrate by resolving association constants K 1 and K 2 from anisotropy isotherms (e.g. Fig. 3) using Equation 2, which were then converted using K d ϭ 1/K. For His 6 -p42 AUF1 and His 6 -p45 AUF1 , the cooperative binding model of Equation 3 resolved the protein dimer concentrations yielding 50% binding (͓P 2 ͔ 1/2 ) and the Hill coefficients (h). All values represent the mean Ϯ standard deviation of n independent experiments.

JOURNAL OF BIOLOGICAL CHEMISTRY 39133
To distinguish these possibilities, we assessed the influence of the exon 7-encoded domain of AUF1 on apparent RNAbinding site size by performing macromolecular binding density analysis (MBDA; described in Refs. 41, 47) of His 6 -p37 AUF1 and His 6 -p42 AUF1 on the Fl-ARE 38 RNA substrate. Fluorescence anisotropy isotherms were collected across titrations of His 6 -p37 AUF1 and His 6 -p42 AUF1 using three different RNA substrate concentrations. The isotherms shift to the right with increasing RNA concentration because more protein is required to reach an equivalent degree of RNA saturation, reflected by the change in anisotropy (Fig. 4A). MBDA was performed using these anisotropy isotherms as described under "Experimental Procedures." For both His 6 -p37 AUF1 and His 6 -p42 AUF1 , changes in Fl-ARE 38 anisotropy as a function of the population-averaged number of AUF1 dimers bound per Fl-ARE 38 RNA substrate (Α) clearly show nonlinear behavior, indicating the presence of at least two binding phases (Fig.  4B). The intersection obtained by linearly extrapolating the limiting slopes of the two binding regimes quantifies the AUF1:Fl-ARE 38 stoichiometry where the transition occurs (Fig. 4B, dotted lines). This binding transition occurs at Α ϳ1 for both His 6 -p37 AUF1 and His 6 -p42 AUF1 , suggesting that the low binding density phase reflects the progress of one AUF1 dimer binding to Fl-ARE 38 , whereas the second binding re-gime describes subsequent AUF1 oligomerization processes. Based on the diversity of RNA sequences targeted by AUF1 (25, 48 -51) and the repetitive AU-rich motifs within the Fl-ARE 38 substrate (Table 1), we considered elements targeted by AUF1 within this RNA as a quasi-homogeneous lattice of sites. Accordingly, the binding density at the transition between binding regimes provides an estimate of the RNA site size for the low binding density complex (52) based on n ϭ 38/Α for the 38-nt Fl-ARE 38 RNA, yielding apparent site sizes of 34 Ϯ 2 nt for His 6 -p37 AUF1 and 33 Ϯ 2 nt for His 6 -p42 AUF1 on this RNA substrate.
The apparent RNA site sizes for His 6 -p37 AUF1 and His 6 -p42 AUF1 predicted by MBDA were independently confirmed through equilibrium binding experiments with ARE substrates of varying lengths (Table 1). Expanding the RNA target to the 44-nt Fl-ARE 44 substrate yielded no significant changes in the affinity of His 6 -p37 AUF1 (Table 4) or His 6 -p42 AUF1 (Table 5), despite a 16% increase (ϩ6 nt) in RNA length. For His 6 -p37 AUF1 , the apparent binding affinity of the first protein dimer (K d1, app ) was also unaffected by truncation of the RNA substrate to 34 nt (Table 4, cf. Fl-ARE 34 and Fl-ARE 35 versus Fl-ARE 38 ). However, the initial protein binding event was inhibited by over 2-fold for the 30-nt Fl-ARE substrate versus the Fl-ARE 34 target and much more severely as the RNA sub-  35 3.2 Ϯ 0.3 130 Ϯ 20 3 Fl-ARE 34 3.0 Ϯ 0.3 117 Ϯ 6 3 Fl-ARE 30 7.0 Ϯ 0.4 Ͼ600 3 Fl-ARE 24 26 Ϯ 2 N A b 3 Fl-ARE 20 33 Ϯ 3 N A 3 Fl-ARE 18 69 Ϯ 12 NA 4 a Apparent equilibrium dissociation constants describing the first (K d1, app ) and second (K d2, app ) stages of His 6 -p37 AUF1 dimer binding to Fl-labeled RNA substrates were resolved from anisotropy plots using the two-step binding model of Equation 2 as described in Table 3. For RNA substrates Յ24 bases in length, the second protein dimer binding event was not detectable. In these cases, simplification of Equation 2 with K d2, app ϭ 0 yields Equation 1, giving the apparent bimolecular dissociation constant for the initial binding event only. All constants are expressed as the mean Ϯ S.D. for n independent experiments. b NA means not applicable.  34 11.5 Ϯ 0.2 1 3 Fl-ARE 30 14 Ϯ 2 1 4 Fl-ARE 24 28 Ϯ 3 1 3 Fl-ARE 20 33 Ϯ 4 1 3 Fl-ARE 18 106 Ϯ 22 1 4 a The concentration of His 6 -p42 AUF1 dimers yielding half-maximal binding (͓P 2 ͔1 ⁄ 2 ) to Fl-labeled RNA substrates and associated Hill coefficients (h) were calculated from A t versus ͓His 6 -p42 AUF1 dimer͔ anisotropy plots (e.g. Fig. 3B) using Equation 3. For RNA substrates Յ34 bases in length, the Hill coefficient did not significantly differ from unity. In these cases, solution of Equation 3 with h ϭ 1 yielded apparent bimolecular dissociation constants (K d, app ). All constants are expressed as the mean Ϯ S.D. across n independent experiments. strate was further truncated. These data indicate that 30 -34 nt of ARE sequence are required for maximal His 6 -p37 AUF1 binding activity, agreeing closely with the MBDA-resolved site size estimate (see above). Interestingly, the apparent affinity of the second His 6 -p37 AUF1 dimer binding step (K d2, app ) was similarly sensitive to the length of the RNA substrate, because this binding event was extremely weak or undetectable for ARE substrates of 30 nt or less. For His 6 -p42 AUF1 , potential cooperativity for some RNA substrates precluded explicit solutions for individual binding constants (discussed above). However, comparison of the protein dimer concentrations required for half-maximal binding ([P 2 ]1 ⁄ 2 ) revealed a modest decrease in affinity between the 38-and 35-mer RNA substrates (Table 5), but more pronounced diminution in His 6 -p42 AUF1 binding activity for substrates of 34 nt or less.
The effects of ARE length on the RNA-binding affinities of His 6 -p37 AUF1 and His 6 -p42 AUF1 resolved by these binding studies are completely consistent with apparent RNA site sizes resolved by MBDA (see above) and do not significantly differ for His 6 -p37 AUF1 versus His 6 -p42 AUF1 . As such, isoform-dependent variations in the binding site size of the AUF1 dimer⅐RNA (P 2 R) complex cannot account for the enhanced RNA-dependent protein oligomerization activity observed in AUF1 isoforms containing the exon 7-encoded domain.
Finally, MBDA was used to solve explicit solutions for apparent dissociation constants describing the initial (K d1, app ) and secondary (K d2, app ) His 6 -p42 AUF1 dimer binding events on the Fl-ARE 38 substrate, thus permitting quantitative comparisons of each stage of AUF1 RNP assembly. MBDA allowed individual binding constants to be resolved for His 6 -p42 AUF1 because the slope of the low binding regime (⌬F 1 ϭ ∂⌬A/∂Α) is a quantitative measure of the average intrinsic anisotropy change resulting from a single AUF1 dimer binding to the RNA. Applied to the sequential dimer binding model of Equation 2, ⌬F 1 thus provides an independent measure of the A P2R parameter (⌬F 1 ϭ A P2R Ϫ A R ). For example, the MBDA-determined ⌬F 1 for His 6 -p37 AUF1 dimer binding to the Fl-ARE 38 substrate was 0.050 Ϯ 0.001, equivalent to 54% of the maximal protein-induced change in anisotropy of this substrate based on ⌬A max ϭ 0.0928 (Fig. 4). This value is indistinguishable from the average A P2R obtained from nonlinear least squares fitting of His 6 -p37 AUF1 titrations using the sequential dimer binding model of Equation 2, where ⌬A P2R (ϭA P2R Ϫ A R ) was 52 Ϯ 4% of ⌬A max across six independent protein titrations (e.g. Figs. 3A and 4A). For His 6 -p42 AUF1 binding the Fl-ARE 38 substrate, MBDA resolved ⌬F 1 ϭ 81 Ϯ 1 mA, equivalent to 60% of ⌬A max . Applying this value as the fractional amplitude of the first binding event in the sequential dimer binding model of Equation 2 revealed that His 6 -p42 AUF1 dimers bind this RNA target with K d1, app ϭ 6.6 Ϯ 1.1 nM and K d2, app ϭ 3.8 Ϯ 0.9 nM (n ϭ 5). The significantly enhanced affinity of the second dimer binding event over the first (p ϭ 0.0022 versus K d1, app ) affirms that His 6 -p42 AUF1 dimers bind cooperatively to the Fl-ARE 38 substrate. Furthermore, comparing the resolved K d2, app values for His 6 -p37 AUF1 (Table 3) versus His 6 -p42 AUF1 (above) binding to this RNA target indicates that the presence of the exon 7-encoded do-main enhances the affinity of the second AUF1 dimer binding step by almost 20-fold.
To this point, all RNA-binding experiments were performed using the ARE from TNF␣ mRNA or truncated versions thereof. To determine whether exon 7-encoded sequences enhanced AUF1 oligomerization on an independent RNA substrate, we also compared the binding properties of His 6 -p37 AUF1 and His 6 -p42 AUF1 on the 46-nt ARE domain I fragment from c-fos mRNA (34). EMSAs showed that both isoforms formed two complexes on this RNA substrate in a protein concentration-dependent manner (Fig. 5A). Furthermore, assembly of the larger P 4 R RNP (complex II) was observed at lower protein concentrations for His 6 -p42 AUF1 than for His 6 -p37 AUF1 , consistent with the preferential formation of P 4 R complexes previously described for the p42 isoform on the TNF␣-based ARE 38 substrate (Fig. 2B). Quantitative anisotropy-based binding experiments showed that the first His 6 -p37 AUF1 dimer bound strongly to the Fl-fos ARE domain I substrate (Fig. 5B, left; K d1, app ϭ 3.8 Ϯ 0.5 nM; n ϭ 4), whereas the second dimer bound much more weakly (K d2, app ϭ 120 Ϯ 20 nM), a relationship similar to that observed for His 6 -p37 AUF1 complex assembly on the TNF␣ ARE. By contrast, resolution of His 6 -p42 AUF1 binding to the Fl-fos ARE domain I substrate required the cooperative binding model, and yielded [P 2 ]1 ⁄ 2 ϭ 5.6 Ϯ 0.8 nM with h ϭ 0.96 Ϯ 0.04 (n ϭ 3) (Fig. 5B, right), consistent with similar binding affinities for both stages of P 4 R assembly. Together, these data show that enhancement of the second AUF1 binding step by exon 7-encoded sequences is not specific for the TNF␣ ARE, and may apply to all AUF1 substrate RNAs.
AUF1 Isoform-dependent Effects on Local Structural Remodeling of an RNA Substrate-Previously, we used FRET to show that both His 6 -p37 AUF1 and His 6 -p40 AUF1 structurally condense bound RNA substrates, based on protein-induced diminution of the distance between their 5Ј and 3Ј termini (31,32). In this study, we showed that AUF1 sequences encoded by exon 7 dramatically enhance the affinity of the second protein dimer binding step that leads to formation of AUF1 tetrameric complexes on RNA targets. To determine whether exon 7-encoded sequences direct unique structural consequences on AUF1-bound RNA, either promoting or as a consequence of this second binding step, we used FRET to compare protein-dependent changes in the global structure of the Cy3-ARE 38 -Fl substrate across all AUF1 isoforms.
In the absence of protein, FRET efficiency (E FRET ) between the 3Ј-Fl and 5Ј-Cy3 moieties of the Cy3-ARE 38 -Fl substrate was typically between 0.40 and 0.45 under the reaction conditions employed. Titration of His 6 -p37 AUF1 led to an increase in E FRET to values of 0.70 to 0.75 (Fig. 5A, black line), indicating that protein binding brings the termini of the RNA substrate closer in space. Comparing the protein concentration dependence of E FRET with the fractional concentration of each fluorescent species shows that this condensation of RNA structure correlates with the initial protein dimer binding event that assembles the P 2 R complex (Fig. 5A, gray lines). No significant change in E FRET was observed accompanying the P 2 R 3 P 4 R transition for His 6 -p37 AUF1 , consistent with previous findings (32). Because the affinity of the ARE 38 substrate for His 6 -p40 AUF1 is weaker than for His 6 -p37 AUF1 (Table 3), higher protein concentrations are required for the p40 isoform to drive complex assembly. However, the consequences of His 6 -p40 AUF1 binding on E FRET between the 5Ј and 3Ј termini of the Cy-ARE 38 -Fl substrate (Fig. 6B) were very similar to those observed with His 6 -p37 AUF1 and favor adoption of a condensed RNA conformation.
AUF1 isoforms containing the exon 7-encoded domain induced dramatically different conformations on associated RNA targets. For His 6 -p42 AUF1 , binding of a single protein dimer to the Cy3-ARE 38 -Fl substrate was associated with increasing E FRET (Fig. 5C), similar to that described for the p37 isoform. However, formation of the P 4 R complex coincided with a decrease in E FRET , indicating that the RNA adopts an increasingly extended conformation as the P 2 R population shifts toward P 4 R. In fact, within the His 6 -p42 AUF1 P 4 R complex the distance between the termini of the Cy3-ARE 38 -Fl substrate is similar to that experienced by the unbound RNA, based on the similarities in E FRET values. Finally, the protein concentration dependence of E FRET for His 6 -p45 AUF1 binding to the Cy3-ARE 38 -Fl substrate resembled that described for His 6 -p42 AUF1 binding, although the degree of RNA extension observed at high protein concentrations was much less dramatic (Fig. 6D). These data indicate that the RNA remodeling activities of His 6 -p45 AUF1 may be intermediate to those of His 6 -p40 AUF1 and His 6 -p42 AUF1 and suggest that the domains encoded by exons 2 and 7 may exert distinct and possibly additive effects on the assembly of RNP complexes and the conformational fate of targeted RNA substrates.

DISCUSSION
Several features of the AUF1 family of RNA-binding proteins support the hypothesis that the individual isoforms generated by alternative pre-mRNA splicing perform distinct but possibly overlapping functions. Although all AUF1 isoforms display ARE binding activity, variations have been reported in their subcellular distribution, regulated expression, and mRNA-destabilizing roles (described under Introduction). In this study, we have quantitatively compared the RNA-binding properties of each isoform using a variety of biophysical approaches, and we have identified isoform-specific differences in RNA recognition and remodeling that may contribute to the functional and regulatory heterogeneity of these proteins.
For all AUF1 isoforms, association with model ARE substrates was consistent with sequential binding of protein Solid circles represent the mean Ϯ S.E. for at least three independent experiments. Black lines highlight protein concentration-dependent trends in E FRET and do not denote any physical meaning. For titrations of His 6 -p37 AUF1 (A) and His 6 -p42 AUF1 (C), the right vertical axis quantifies the fractional concentrations of free RNA (solid gray lines), P 2 R complexes (dashed gray lines), and P 4 R complexes (dotted gray lines) as a function of His 6 -p37 AUF1 or His 6 -p42 AUF1 concentration using binding constants resolved by anisotropy experiments and fit to the sequential dimer binding model with consideration of protein depletion (Equations [11][12][13][14]. dimers to generate a protein tetramer⅐RNA (P 4 R) complex. Inclusion of sequences encoded by exon 2 (in p40 AUF1 and p45 AUF1 ) weakened the affinity of these binding events, in agreement with semi-quantitative EMSA-based experiments using extended ARE-containing RNA substrates (20) and single-stranded telomeric DNA sequences (51). Because the exon 2-encoded domain is contiguous with RRM1, its inclusion may alter the conformation of this RNA-binding pocket or its access to nucleic acid substrates. Although exon 2-encoded sequences significantly inhibited RNA-binding affinity, FRET experiments of RNA folding suggested that this domain likely had little effect on the conformation of bound RNA substrates. By contrast, the domain encoded by exon 7 exerted profound effects on both the energetics of AUF1 RNP assembly and the structural consequences of the associated RNA. Initial binding of all AUF1 isoforms with the ARE 38 substrate yielded a P 2 R complex containing a conformationally condensed RNA (Fig. 7). However, protein isoforms containing exon 7-encoded sequences (p42 AUF1 and p45 AUF1 ) displayed much stronger affinity at the second binding step (K 2 ), which in the case of p42 AUF1 resulted in cooperative binding to the ARE 38 substrate. Furthermore, the ARE substrate within p42 AUF1 -or p45 AUF1 -containing P 4 R complexes presented a less compact conformation than RNPs containing exon 7-deficient AUF1 isoforms (Fig. 6).
Although the mechanistic basis for enhanced protein oligomerization on ARE substrates by p42 AUF1 or p45 AUF1 remains unknown, several observations suggest that the exon 7-encoded domain directs unique conformational events during RNP assembly. First, AUF1 isoforms containing exon 7-encoded sequences exert greater rotational restriction on the 5Ј-Fl groups of RNA substrates in the P 4 R complex than AUF1 proteins lacking this domain. For example, formation of the P 4 R complex with His 6 -p37 AUF1 increased the fluorescence anisotropy of the Fl group on the 5Ј-end of the ARE 38 substrate by 0.093 Ϯ 0.002 relative to the free RNA (Fig. 3A). By contrast, the His 6 -p42 AUF1 P 4 R complex increased the anisotropy of this fluorophore by 0.134 Ϯ 0.001 (Fig. 3B). Similar differences were observed comparing His 6 -p40 AUF1 versus His 6 -p45 AUF1 binding to the Fl-ARE 38 substrate (data not shown) and among His 6 -p37 AUF1 versus His 6 -p42 AUF1 P 4 R complexes on the Fl-fos ARE domain I RNA substrate (Fig.  5B). Second, the apparent RNA site size of an AUF1 dimer is unaffected by the presence of the exon 7-encoded domain ( Fig. 4 and associated text), indicating that ARE substrates do not present more high affinity sites to AUF1 isoforms containing sequences encoded by exon 7. Finally, the exon 7 domain-dependent effects of AUF1 on RNA substrate conformation in the P 4 R complex (Fig. 6) indicate a direct consequence of this alternatively expressed domain on RNP architecture. Intriguingly, a recent report indicates that the splicing repressor hnRNP A1 can also bind cooperatively to RNA substrates (53). Although hnRNP A1 and AUF1 share many common features, there is no sequence in hnRNP A1 orthologous to the exon 7-encoded domain of AUF1 (supplemental Fig. 1), suggesting that a distinct biochemical mechanism is likely involved in cooperative assembly of hnRNP A1⅐RNA complexes.
The AUF1 isoform-dependent conformational remodeling of RNA substrates demonstrated in this work highlights an emerging theme of reciprocal interplay between RNA structure and protein binding. Many previous reports have described how local RNA structure can impact recruitment of trans-acting proteins. For example, the iron-responsive RNA-binding protein IRP and nucleolin both show preferential binding to structured RNA targets (54,55), whereas the ARE binding activities of AUF1 and Hsp70 are inhibited when single-stranded RNA domains are occluded (43). More recently, however, models are developing where protein binding impacts local RNA structure, similar to that proposed for AUF1 in this work and previously (32). For example, under hypoxic stress hnRNP L binds to a translational regulatory domain within the VEGF mRNA 3ЈUTR, which dramatically alters RNA folding in this region and ultimately enhances translation (56). For both p37 AUF1 and p42 AUF1 , the first RNA-binding step was associated with diminution of the distance between the 5Ј and 3Ј termini of an associated RNA substrate (Fig. 6). It is appealing to speculate that the large apparent RNA site sizes observed for P 2 R complexes involving p37 AUF1 or p42 AUF1 (ϳ33-34 nt) may be coupled to this local condensation of RNA structure, possibly involving recognition of disparate sequence motifs and steric occlusion of intervening substrate nucleotides. One logical functional consequence of protein-dependent changes in local RNA structure is the potential to control accessibility for ancillary RNA-binding factors, a theme that is also gaining recognition. For example, AUF1 and HuR coordinately enhance association of each other with p16 INK4 mRNA and also assist in recruiting RISC complexes to this transcript (57). Interplay between AUF1 and HuR binding may be a common theme among ARE-containing mRNAs, as gene array studies have identified many transcripts that can bind both proteins simultaneously (50). Protein-dependent changes in local RNA structure may also impact microRNA accessibility, given the contributions of base pair complementarity to the specificity of miRISC⅐mRNA complex formation. For example, HuR can FIGURE 7. AUF1 isoform-specific control of protein oligomerization and local RNA structure. A schematic depicting the effects of exon 7-encoded sequences on RNP assembly by AUF1 and on the relative conformation of bound RNA substrates (described in green). Initial contact between any AUF1 isoform and an RNA substrate induces a condensed RNA conformation; however, sequences encoded by exon 2 (in p40 AUF1 and p45 AUF1 ) weaken this interaction (K 1 ). For AUF1 isoforms lacking exon 7-encoded sequences (p37 AUF1 and p40 AUF1 ), the second binding step forming the tetrameric protein⅐RNA complex (K 2 ) is relatively weak and maintains the bound RNA in a condensed conformation. By contrast, the second binding step is much stronger for AUF1 isoforms containing sequences encoded by exon 7 (p42 AUF1 and p45 AUF1 ) and confers a relatively open conformation on the bound RNA substrate. enhance recruitment of the microRNA let-7 to c-myc mRNA (58), but it can also serve as a negative regulator of microRNA function by blocking miR-122-directed repression of CAT-1 mRNA translation (59). Another RNA-binding protein, Drd1, binds within the 3ЈUTR of p27 mRNA but blocks miR-221 binding to proximal sites (60).
Based on the distinctive biochemical properties displayed by the different AUF1 isoforms in this work, we anticipate that modulating the expression or activity of individual AUF1 isoforms will enhance the selectivity of post-transcriptional gene regulation. In this manner, the induction of p45 AUF1 by prostaglandin A 2 in lung carcinoma cells (30) could elicit different physiological consequences than those expected from any other AUF1 isoform, based on the unique p45 AUF1 concentration dependence on RNP composition and local RNA conformation. However, the complexity of post-transcriptional regulatory control by AUF1 isoforms may be further expanded by the potential for AUF1 proteins to form heterodimers (46) and by post-translational modifications of specific isoforms. For example, p40 AUF1 phosphorylated within the exon 2-encoded domain at Ser-83 and Ser-87 retains ARE substrates in elongated conformations independent of RNP stoichiometry (31), unlike any unmodified AUF1 isoform studied here (Fig. 6). Because p37 AUF1 lacks these residues, activation of signaling pathways directing p40 AUF1 phosphorylation at these sites could thus selectively regulate the function of p40 AUF1 , without altering the other major cytoplasmic isoform. Cellular radiolabeling, phosphoamino acid-specific antibodies, and two-dimensional Western analyses further indicate that other AUF1 isoforms can also be post-translationally modified on Ser, Thr, and Tyr residues (21,28,(61)(62)(63), hinting at a geometrically expanding array of regulatory possibilities.
Finally, the isoform-specific models of AUF1 RNP assembly described in this study may help explain some conflicting literature reports of AUF1 function, particularly in cases where individual AUF1 isoforms are overexpressed in cells. Suppression of AUF1 using siRNA-based approaches is most frequently associated with stabilization of mRNA substrates, consistent with a general mRNA-destabilizing role for at least some AUF1 proteins (17,64,65). However, ectopic overexpression of AUF1 proteins has prompted much more variable conclusions, associated with both mRNA-destabilizing (18) and -stabilizing (66) roles. This dichotomy was most clearly presented in a recent work showing that both siRNA-directed suppression and ectopic overexpression of AUF1 stabilized a reporter mRNA containing the ARE from IL-6 mRNA (16). Although some of these apparent contradictions may reflect differences in mRNA targets or cell types, we suggest that the mRNA metabolic consequences of modulating AUF1 levels may be strongly influenced by isoform-dependent effects on the sensitivity of RNP size and conformation to changes in protein concentrations. For example, the weak K 2 values resolved for p37 AUF1 and p40 AUF1 binding to ARE substrates (Table 3) indicates that RNPs assembled from these isoforms would exist principally as P 2 R complexes across a wide range of protein concentrations (modeled in Fig. 6A for p37 AUF1 ). It is therefore possible that the P 2 R complex represents the ma-jor cellular p37 AUF1 or p40 AUF1 RNP under normal physiological conditions. However, dramatic overexpression of these isoforms would be expected to shift the distribution of their cognate RNPs in favor of P 4 R complexes, which may direct very different functional consequences on targeted RNA substrates.