Assembly of AUF1 Oligomers on U-rich RNA Targets by Sequential Dimer Association

Many labile mammalian mRNAs are targeted for rapid cytoplasmic turnover by the presence of A + U-rich elements (AREs) within their 3′-untranslated regions. These elements are selectively recognized by AUF1, a component of a multisubunit complex that may participate in the initiation of mRNA decay. In this study, we have investigated the recognition of AREs by AUF1 in vitro using oligoribonucleotide substrates. Gel mobility shift assays demonstrated that U-rich RNA targets were specifically bound by AUF1, generating two distinct RNA-protein complexes in a concentration-dependent manner. Chemical cross-linking revealed the interaction of AUF1 dimers to form tetrameric structures involving protein-protein interactions in the presence of high affinity RNA targets. From these data, a model of AUF1 association with AREs involving sequential dimer binding was developed. Using fluorescent RNA substrates, binding parameters of AUF1 dimer-ARE and tetramer-ARE equilibria were evaluated in solution by fluorescence anisotropy measurements. Using two AUF1 deletion mutants, sequences C-terminal to the RNA recognition motifs are shown to contribute to the formation of the AUF1 tetramer·ARE complex but are not obligate for RNA binding activity. Kinetic studies demonstrated rapid turnover of AUF1·ARE complexes in solution, suggesting that these interactions are very dynamic in character. Taken together, these data support a model where ARE-dependent oligomerization of AUF1 may function to nucleate the formation of a trans-acting, RNA-destabilizing complex in vivo. Many labile mammalian mRNAs are targeted for rapid cytoplasmic turnover by the presence of A + U-rich elements (AREs) within their 3′-untranslated regions. These elements are selectively recognized by AUF1, a component of a multisubunit complex that may participate in the initiation of mRNA decay. In this study, we have investigated the recognition of AREs by AUF1 in vitro using oligoribonucleotide substrates. Gel mobility shift assays demonstrated that U-rich RNA targets were specifically bound by AUF1, generating two distinct RNA-protein complexes in a concentration-dependent manner. Chemical cross-linking revealed the interaction of AUF1 dimers to form tetrameric structures involving protein-protein interactions in the presence of high affinity RNA targets. From these data, a model of AUF1 association with AREs involving sequential dimer binding was developed. Using fluorescent RNA substrates, binding parameters of AUF1 dimer-ARE and tetramer-ARE equilibria were evaluated in solution by fluorescence anisotropy measurements. Using two AUF1 deletion mutants, sequences C-terminal to the RNA recognition motifs are shown to contribute to the formation of the AUF1 tetramer·ARE complex but are not obligate for RNA binding activity. Kinetic studies demonstrated rapid turnover of AUF1·ARE complexes in solution, suggesting that these interactions are very dynamic in character. Taken together, these data support a model where ARE-dependent oligomerization of AUF1 may function to nucleate the formation of a trans-acting, RNA-destabilizing complex in vivo. A + U-rich element dithio-bis(succinimidyl propionate) RNA recognition motif tumor necrosis factor α untranslated region polyacrylamide gel electrophoresis The control of cytoplasmic mRNA turnover plays a major role in regulating both the level and timing of expression of many gene products in eukaryotes (reviewed in Refs. 1Ross J. Microbiol. Rev. 1995; 59: 423-450Crossref PubMed Google Scholar and 2Peltz S.W. Brewer G. Bernstein P. Hart P.A. Ross J. Stein G.S. Stein J.L. Lian J.B. Critical Reviews in Eukaryotic Gene Expression. CRC Press, Inc., Boca Raton, FL1991: 99-126Google Scholar). In many cases, sequence elements within individual mRNAs function ascis-acting determinants of their stability, either constitutively or in response to external stimuli. Conceptually, modulation of mRNA turnover rates may be envisioned as altering the activity or accessibility of one or more ribonucleases toward a specific transcript. At present, however, few mechanistic details are available linking cis-acting RNA sequence elements with the decay machinery necessary for hydrolysis of the target mRNA. AREs1 are potentcis-acting determinants of rapid cytoplasmic mRNA turnover in mammalian cells. They generally consist of one or more overlapping AUUUA pentamers contained within or near a U-rich tract (3Chen C.-Y.A. Shyu A.-B. Trends Biochem. Sci. 1995; 20: 465-470Abstract Full Text PDF PubMed Scopus (1688) Google Scholar, 4Zubiaga A.M. Belasco J.G. Greenberg M.E. Mol. Cell. Biol. 1995; 15: 2219-2230Crossref PubMed Scopus (472) Google Scholar, 5Lagnado C.A. Brown C.Y. Goodall G.J. Mol. Cell. Biol. 1994; 14: 7984-7995Crossref PubMed Scopus (310) Google Scholar). These elements are present in the 3′-untranslated regions (3′-UTRs) of many labile mRNAs, including several encoding inflammatory mediators, cytokines, oncoproteins, and G protein-coupled receptors (3Chen C.-Y.A. Shyu A.-B. Trends Biochem. Sci. 1995; 20: 465-470Abstract Full Text PDF PubMed Scopus (1688) Google Scholar, 6Shaw G. Kamen R. Cell. 1986; 46: 659-667Abstract Full Text PDF PubMed Scopus (3124) Google Scholar, 7Caput D. Beutler B. Hartog K. Thayer R. Brown-Shimer S. Cerami A. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 1670-1674Crossref PubMed Scopus (1218) Google Scholar, 8Tholanikunnel B.G. Malbon C.C. J. Biol. Chem. 1997; 272: 11471-11478Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 9Stoecklin G. Hahn S. Moroni C. J. Biol. Chem. 1994; 269: 28591-28597Abstract Full Text PDF PubMed Google Scholar, 10Peppel K. Vinci J.M. Baglioni C. J. Exp. Med. 1991; 173: 349-355Crossref PubMed Scopus (122) Google Scholar). mRNA turnover mediated by AREs is usually characterized by rapid 3′ to 5′ shortening of the poly(A) tract followed by decay of the mRNA body (11Wilson T. Treisman R. Nature. 1988; 336: 396-399Crossref PubMed Scopus (506) Google Scholar, 12Xu N. Chen C.-Y.A. Shyu A.-B. Mol. Cell. Biol. 1997; 17: 4611-4621Crossref PubMed Scopus (308) Google Scholar, 13Brewer G. Ross J. Mol. Cell. Biol. 1988; 8: 1697-1708Crossref PubMed Scopus (215) Google Scholar, 14Brewer G. J. Biol. Chem. 1998; 273: 34770-34774Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 15Ford L.P. Watson J. Keene J.D. Wilusz J. Genes Dev. 1999; 13: 188-201Crossref PubMed Scopus (219) Google Scholar). In addition, ARE-directed mRNA decay is dependent upon active translation of the mRNA in many cellular systems (16Aharon T. Schneider R.J. Mol. Cell. Biol. 1993; 13: 1971-1980Crossref PubMed Scopus (123) Google Scholar, 17Savant-Bhonsale S. Cleveland D.W. Genes Dev. 1992; 6: 1927-1939Crossref PubMed Scopus (148) Google Scholar, 18Winstall E. Gamache M. Raymond V. Mol. Cell. Biol. 1995; 15: 3796-3804Crossref PubMed Scopus (48) Google Scholar, 19Curatola A.M. Nadal M.S. Schneider R.J. Mol. Cell. Biol. 1995; 15: 6331-6340Crossref PubMed Scopus (69) Google Scholar, 20Jacobson A. Peltz S.W. Annu. Rev. Biochem. 1996; 65: 693-739Crossref PubMed Scopus (577) Google Scholar), suggesting that some link also exists between protein synthesis and mRNA decay mechanisms. AUF1 is an RNA-binding protein that exhibits many characteristics of atrans-acting factor participating in ARE-directed mRNA turnover (reviewed in Ref. 21Wilson G.M. Brewer G. Prog. Nucleic Acids Res. Mol. Biol. 1999; 62: 257-291Crossref PubMed Scopus (123) Google Scholar). Current evidence indicates that AUF1 may function as a targeting system for AREs, either recruiting or promoting the assembly of multisubunit trans-acting complexes at these sites (22Brewer G. Mol. Cell. Biol. 1991; 11: 2460-2466Crossref PubMed Scopus (406) Google Scholar, 23DeMaria C.T. Brewer G. J. Biol. Chem. 1996; 271: 12179-12184Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 24DeMaria C.T. Sun Y. Long L. Wagner B.J. Brewer G. J. Biol. Chem. 1997; 272: 27635-27643Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Several cytoplasmic proteins co-immunoprecipitate with AUF1, indicating that AUF1 associates with additional factors in vivo (25Zhang W. Wagner B.J. Ehrenman K. Schaefer A.W. DeMaria C.T. Crater D. DeHaven K. Long L. Brewer G. Mol. Cell. Biol. 1993; 13: 7652-7665Crossref PubMed Scopus (497) Google Scholar). Some of these have been identified immunologically as the translation initiation factor eIF4G, poly(A)-binding protein, heat shock protein 70, and the 70-kDa heat shock cognate protein (26Laroia G. Cuesta R. Brewer G. Schneider R.J. Science. 1999; 284: 499-502Crossref PubMed Scopus (348) Google Scholar). The identification of these associated proteins is evidence of a physical link between AUF1 and factors involved in translation and mRNA turnover. Elucidation of the mechanisms contributing to rapid mRNA turnover by AREs will require further understanding of both the molecular architecture of the trans-acting complex(es) as well as the molecular events involved in recognition of AREs by these factors. In particular, the direct interaction of AUF1 with target RNA sequences may serve to nucleate factor binding or transduce some signal to activate pre-assembled complexes. In this study, we have investigated the interaction of AUF1 in vitro with the following two U-rich oligoribonucleotides: the core ARE from tumor necrosis factor α (TNFα) mRNA, and a uridylate homopolymer. We present evidence that recognition of U-rich RNA sequences by AUF1 initiates the assembly of AUF1 multimers involving both RNA-protein and protein-protein interactions by sequential binding of AUF1 dimers. The application of fluorescence anisotropy to the study of RNA:AUF1 solution equilibria allowed estimation of the equilibrium constants for both initial and secondary binding events as well as an assessment of complex dynamics by off-rate analyses. Finally, we discuss potential functional consequences of RNA-dependent AUF1 oligomerization in the control of cytoplasmic mRNA turnover. All RNA oligonucleotides (2′-hydroxyl) were synthesized by Dharmacon Research (Boulder, CO). The sequence of each RNA probe is listed in Fig. 1 A. Following 2′-O-deprotection according to the manufacturer's instructions (27Scaringe S.A. Wincott F.E. Caruthers M.H. J. Am. Chem. Soc. 1998; 120: 11820-11821Crossref Scopus (217) Google Scholar), RNA oligonucleotides were quantified by absorbance at 260 nm. Estimates of the extinction coefficients for each RNA probe at 260 nm were calculated as described (28.Google Scholar). For fluorescein-tagged probes, absorbance at 260 nm was corrected by quantitation of the fluorescein moiety at 495 nm as described (29Heyduk T. Ma Y. Tang H. Ebright R.H. Methods Enzymol. 1996; 274: 492-503Crossref PubMed Scopus (139) Google Scholar). The substrate "TNFα ARE" corresponds to the core ARE from the 3′-UTR of human TNFα mRNA. The RNA substrate "U32" contains a uridylate homopolymeric sequence, and "Rβ" encodes a fragment of the rabbit β-globin coding region. Duplicate oligoribonucleotides containing 5′-fluorescein labels were also synthesized and are designated Fl-TNFα ARE, Fl-U32, and Fl-Rβ, respectively. For gel mobility shift assays, TNFα ARE, U32, and Rβ substrates were radiolabeled using T4 polynucleotide kinase (Promega, Madison, WI) and [γ-32P]ATP (4500 Ci/mmol) (ICN Biomedicals, Costa Mesa, CA) to specific activities of 3–5 × 103 cpm/fmol. Unincorporated radiolabel was removed by spin column chromatography using G-25 Quick Spin columns (Roche Molecular Biochemicals). Probe-specific activity was determined by liquid scintillation counting, and RNA integrity was verified by denaturing polyacrylamide gel electrophoresis and autoradiography. 32P-Labeled RNA probes were detected as single bands (data not shown) indicating that they were predominantly (>99%) full length. The construction of plasmids pTrcHisB-AUF1-(1–257) and pTrcHisB-AUF1-(1–229) was described previously (24DeMaria C.T. Sun Y. Long L. Wagner B.J. Brewer G. J. Biol. Chem. 1997; 272: 27635-27643Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). pTrcHisB-AUF1-(1–257) encodes a stable, N-terminal His6-tagged mutant of human p37AUF1lacking 30 amino acid residues from the C terminus but showing comparable ARE binding activity to full-length p37AUF1(24DeMaria C.T. Sun Y. Long L. Wagner B.J. Brewer G. J. Biol. Chem. 1997; 272: 27635-27643Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). pTrcHisB-AUF1-(1–229) encodes a truncation mutant of p37AUF1 lacking all sequences C-terminal of the RNA recognition motifs (RRMs). Recombinant His6-AUF1 mutant proteins were expressed and purified as described (30Wilson G.M. Brewer G. Methods Compan. Methods Enzymol. 1999; 17: 74-83Crossref Scopus (55) Google Scholar) and were judged to be >95% pure by SDS-PAGE. For protein cross-linking studies, His6 proteins were dialyzed against 10 mmHEPES·KOH (pH 7.5) prior to concentration. Where indicated, a 3500-Da N-terminal fragment containing the His6 tag was removed from His6-p37AUF1-(1–257) using the recombinant enterokinase kit (Novagen, Madison, WI) according to the manufacturer's instructions. A mock-digested reaction was also assembled to control for changes in protein activity resulting from prolonged incubation at room temperature (2 h). All recombinant proteins were quantified by the method of Bradford (31Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar) using bovine serum albumin as standard. Protein concentrations were also evaluated by comparison of Coomassie Blue-stained SDS-PAGE gels containing recombinant proteins and a titration of bovine serum albumin. Determination of protein concentrations by both methods yielded estimates within 10%. Binding reactions for gel mobility shift assays were performed with a range of His6-AUF1 fusion protein concentrations and 0.15 nm32P-labeled RNA in a final volume of 10 μl containing 10 mm Tris·HCl (pH 7.5), 100 mmpotassium acetate, 5 mm magnesium acetate, 2 mmdithiothreitol, 0.1 mm spermine, 0.1 μg/μl acetylated bovine serum albumin, 8 units of RNasin (Promega), 33% glycerol, and 1 μg/μl heparin. Reactions were incubated for 10 min at room temperature and immediately fractionated through 6% (40:1 acrylamide:bisacrylamide) non-denaturing gels as described (30Wilson G.M. Brewer G. Methods Compan. Methods Enzymol. 1999; 17: 74-83Crossref Scopus (55) Google Scholar). Reaction products were visualized by PhosphorImager scan (Molecular Dynamics, Sunnyvale, CA). Dithio-bis(succinimidyl propionate) (DSP)-mediated protein cross-linking was performed in 10-μl reactions containing 10 mm HEPES·KOH (pH 7.5), 100 mm potassium acetate, and 5 mm magnesium acetate. In this buffer system, HEPES-dialyzed His6-p37AUF1-(1–257) or His6-p37AUF1-(1–229) was diluted to 5 μm in the presence or absence of 1.5 μmRNA. DSP (Pierce) was then added to a final concentration of 2.5 mm, and reactions were allowed to proceed for 10 min at room temperature. Cross-linking was then quenched by addition of Tris·HCl (pH 7.5) to 1 m final concentration and incubation for a further 15 min. Reaction products were fractionated by SDS-PAGE in the absence of reducing agents. Complexes containing AUF1 were identified by probing immunoblots with anti-AUF1 antiserum (25Zhang W. Wagner B.J. Ehrenman K. Schaefer A.W. DeMaria C.T. Crater D. DeHaven K. Long L. Brewer G. Mol. Cell. Biol. 1993; 13: 7652-7665Crossref PubMed Scopus (497) Google Scholar). Secondary antibody detection was performed using the SuperSignal Chemiluminescent Detection Kit (Pierce) and exposure to x-ray film. 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 assembled as described for gel mobility shift assays (above) except that no glycerol was added, and the final volume was 100 μl. For equilibrium binding experiments, the polarimeter was operated in static mode, with each sample read as blank prior to addition of fluorescein-labeled RNA probes. Following probe addition, samples were incubated for 1 min before anisotropy was measured. Preliminary on-rate analyses demonstrated that anisotropic equilibrium was reached within 10 s (data not shown). Data points represent the mean of 10 measurements for each binding reaction. Samples for off-rate analyses were similarly assembled, except that following an initial reading (t = 0), a 5000-fold molar excess of unlabeled RNA competitor was added to the binding mixture and rapidly mixed. Anisotropy measurements were taken in kinetic mode in intervals of 15 s, with five measurements taken at each time point. All non-linear regression fitting of anisotropic data and statistical evaluations were performed using PRISM software version 2.0 (GraphPad, San Diego, CA). Human TNFα mRNA contains an ARE in its 3′-UTR which contributes to its rapid turnover in vivo (32Raabe T. Bukrinsky M. Currie R.A. J. Biol. Chem. 1998; 273: 974-980Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar,33Lindsten T. June C.H. Ledbetter J.A. Stella G. Thompson C.B. Science. 1989; 244: 339-343Crossref PubMed Scopus (803) Google Scholar). It is also sufficient to destabilize a heterologous mRNA in transfected cell systems (12Xu N. Chen C.-Y.A. Shyu A.-B. Mol. Cell. Biol. 1997; 17: 4611-4621Crossref PubMed Scopus (308) Google Scholar), and cytoplasmic proteins have been identified binding this element in vitro (34Hel Z. Skamene E. Radzioch D. Mol. Cell. Biol. 1996; 16: 5579-5590Crossref PubMed Scopus (46) Google Scholar). The core sequence of the TNFα ARE is similar to others identified as high affinity AUF1-binding sites (23DeMaria C.T. Brewer G. J. Biol. Chem. 1996; 271: 12179-12184Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 25Zhang W. Wagner B.J. Ehrenman K. Schaefer A.W. DeMaria C.T. Crater D. DeHaven K. Long L. Brewer G. Mol. Cell. Biol. 1993; 13: 7652-7665Crossref PubMed Scopus (497) Google Scholar). Taken together, these features make the TNFα ARE a strong candidate for high affinity interaction with AUF1. In gel mobility shift assays, binding of His6-p37AUF1-(1–257) to the TNFα ARE and U32 sequences generated two distinct complexes (Fig.1, B and C). No detectable binding was observed to the rabbit β-globin substrate (Fig. 1 D), consistent with the selectivity of AUF1 for A + U-rich RNA sequences (23DeMaria C.T. Brewer G. J. Biol. Chem. 1996; 271: 12179-12184Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 25Zhang W. Wagner B.J. Ehrenman K. Schaefer A.W. DeMaria C.T. Crater D. DeHaven K. Long L. Brewer G. Mol. Cell. Biol. 1993; 13: 7652-7665Crossref PubMed Scopus (497) Google Scholar). For both the TNFα ARE and U32 probes, the distribution of complexes with RNA was dependent on protein concentration, with the faster migrating complexes (complex I) appearing at lower concentrations of His6-p37AUF1-(1–257) (0.5–5 nm). Levels of complex I diminished as the abundance of complex II increased, consistent with the possibility of a precursor-product relationship. The diffuse smearing observed below binding complexes is likely due to RNA-protein dissociation in the gel. No additional binding events were observed in these assays, with protein concentrations tested up to 500 nm (data not shown). Previous hydrodynamic studies demonstrated that AUF1 forms dimeric structures in solution involving an N-terminal alanine-rich region (24DeMaria C.T. Sun Y. Long L. Wagner B.J. Brewer G. J. Biol. Chem. 1997; 272: 27635-27643Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Monomeric AUF1 was not detected in these experiments, indicating that dimers are generated with high affinity. To determine whether AUF1 oligomerization might contribute to the formation of complexes with the TNFα ARE, RNA-protein binding reactions were treated with the chemical cross-linker DSP, permitting covalent linkage through primary amino groups. In the absence of cross-linker, His6-p37AUF1-(1–257) migrates at an apparentM r ≈ 43,000 by SDS-PAGE, larger than its predicted M r of 32,600 (data not shown). In cross-linking reactions lacking RNA, His6-p37AUF1-(1–257) was primarily detected as a dimer (Fig. 2, lane 1), consistent with the hydrodynamic studies of the full-length p37AUF1 (24DeMaria C.T. Sun Y. Long L. Wagner B.J. Brewer G. J. Biol. Chem. 1997; 272: 27635-27643Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Cross-links were generated specifically through the DSP linker, since they were cleaved following treatment with reducing agents (data not shown). The presence of a non-binding RNA substrate (Rβ) did not alter the distribution of cross-linked protein products (Fig. 2, lane 2). However, larger protein complexes up to and including tetramers were observed in the presence of high affinity RNA targets (U32 and TNFα ARE; Fig. 2,lanes 3 and 4). The generation of these larger AUF1 complexes in the presence of U32 indicated that these species were unlikely to be the result of RNA bridging, since there is a paucity of primary amino groups contained within U32 RNA. Furthermore, the lack of detectable AUF1 tetramers in the absence of a high affinity RNA target even at high concentrations of protein (5 μm) suggests that dimer-dimer association does not occur in the absence of U-rich RNA sequences. Two binding complexes of AUF1 and RNA were detected by gel mobility shift assay (Fig. 1, B and C). The slowest mobility complex (complex II) likely represents RNA associated with the AUF1 tetramer, since it represents the largest change in mobility relative to the free probe, and its abundance increases with protein concentration. Complex I is observed at lower concentrations of protein and could thus represent an AUF1 monomer, dimer, or trimer associated with RNA. However, because unbound AUF1 is dimeric (24DeMaria C.T. Sun Y. Long L. Wagner B.J. Brewer G. J. Biol. Chem. 1997; 272: 27635-27643Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar), interpretation of complex I as an AUF1 dimer rather than a monomer or trimer bound to RNA is the sole case in which monomeric AUF1 species are not required. Given both the absence of additional intermediate binding species observed by gel mobility shift analysis (Fig. 1) and the absence of detectable monomeric AUF1 by gel filtration and sedimentation velocity experiments (24DeMaria C.T. Sun Y. Long L. Wagner B.J. Brewer G. J. Biol. Chem. 1997; 272: 27635-27643Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar), the contribution of monomeric AUF1 to the binding equilibrium is likely to be negligible. Given the following findings, (i) His6-p37AUF1-(1–257) association with TNFα ARE and U32 RNAs generates two RNA-protein complexes, (ii) formation of these complexes is dependent on protein concentration, (iii) AUF1 complexes as large as tetramers are associated with these RNA targets, and (iv) AUF1 dimers do not interact in the absence of U-rich RNA targets, we propose that AUF1 associates with these RNA probes by sequential dimer binding. In this model, complex I (Fig. 1) represents the AUF1 dimer-bound RNA (P2R), and complex II represents an RNA-associated AUF1 tetramer (P4R). Whereas the re-iterative nature of AREs suggests that AUF1 oligomers may be the result of multiple binding sites on the RNA target, RNA-dependent tetramer cross-linking indicates that adjacent dimers are held in close proximity in the P4R complex, making interaction between these subunits likely. Furthermore, AUF1·ARE binding equilibria evaluated by remaining free probe concentration (Fig. 1, see also Refs.23DeMaria C.T. Brewer G. J. Biol. Chem. 1996; 271: 12179-12184Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 24DeMaria C.T. Sun Y. Long L. Wagner B.J. Brewer G. J. Biol. Chem. 1997; 272: 27635-27643Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 35Wagner B.J. DeMaria C.T. Sun Y. Wilson G.M. Brewer G. Genomics. 1998; 48: 195-202Crossref PubMed Scopus (239) Google Scholar) are not resolved by Scatchard analysis, suggesting that binding events involving multiple AUF1 dimers are not independent (data not shown). However, the model does not exclude the possibility that the initial dimer binding event may occur at one of several sites on a given RNA target. For this reason, the AUF1 dimer-RNA equilibrium may reflect an average of multiple simultaneous P2R variants. The generation of tetrameric His6-p37AUF1-(1–257) structures on U-rich RNA targets indicates that protein-protein interactions may be generated between AUF1 dimers in an RNA-dependent manner. Whereas previous studies demonstrated that formation of AUF1 dimers in the absence of RNA required sequences near the N terminus, sequences C-terminal to the RRMs were dispensable for dimer assembly (24DeMaria C.T. Sun Y. Long L. Wagner B.J. Brewer G. J. Biol. Chem. 1997; 272: 27635-27643Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). In order to evaluate the contribution of C-terminal sequences to tetramer formation in the presence of an RNA target, gel mobility shift assays were also performed using the truncation mutant His6-p37AUF1-(1–229), which lacks all sequences C-terminal of the RRMs. Association of His6-p37AUF1-(1–229) with the TNFα ARE generated primarily a single complex consistent with P2R (Fig. 3 A, complex I), whereas a complex consistent with P4R (complex II) was detected only weakly at higher concentrations of protein. Similar binding products were observed with the U32 probe, and no binding activity was observed to Rβ, indicating that RNA-binding specificity was not compromised by deletion of the C-terminal sequences of AUF1 (data not shown). Although association with TNFα ARE was observed at very low concentrations of His6-p37AUF1-(1–229) (0.5 nm), complete probe association was not detected (Fig. 3 A), even at protein concentrations up to 2 μm (data not shown). This suggests that maintenance of His6-p37AUF1-(1–229)-RNA complexes may be hindered by gel fractionation, possibly involving rapid dissociation in the sample wells or during electrophoresis. Covalent cross-linking of His6-p37AUF1-(1–229) with DSP confirmed that this protein forms dimers in the absence of RNA (Fig. 3 B, lane 1). A small amount of trimeric AUF1 was also detected but is likely the product of partial oxidation or aggregation in the protein preparation. Inclusion of the Rβ RNA oligonucleotide in the cross-linking reaction did not alter the distribution of covalently linked products (Fig. 3 B,lane 2). Similarly, only minimal changes in the recovery of trimeric AUF1 were observed by addition of the TNFα ARE (Fig.3 B, lane 4), and tetrameric species were not detected. However, binding reactions containing U32displayed both trimeric and tetrameric cross-linked species (Fig.3 B, lane 3). Taken together, these data indicate that the ability of AUF1 to form RNA-dependent tetramers is compromised but not completely abrogated by removal of sequences between amino acid residues 229 and 257. In particular, tetramer formation with His6-p37AUF1-(1–229) was observed only with the uridylate homopolymer, where the possibility of multiple identical binding sites exists on the RNA. This suggests that a bona fide ARE, like that in TNFα mRNA, presents a hierarchy of AUF1-binding sites consistent with a sequential binding model, in which AUF1 sequences between 229 and 257 contribute to secondary binding events. In order to evaluate the validity of the sequential dimer-binding model for AUF1 oligomerization on an ARE, it was necessary to first express the equilibrium relationships between each component mathematically. Subsequently, fluorescence-based solution binding experiments performed under equilibrium conditions were employed to test the accuracy of these equations in describing the sequential association of AUF1 dimers with a high affinity RNA target. The steady state concentrations of P2R and P4R may be described in terms of the concentrations of RNA [R] and dimeric protein [P2] by Equations 1 and 2. [P2R]=K1[R][P2]Equation 1 [P4R]=K1K2[R][P2]2Equation 2 By titrating excess protein [P2]totagainst a constant concentration of RNA substrate where [R]tot ≪ 1/K 1, [P2]free remains in vast excess over [P2R] and [P4R]. Accordingly, [P2]free is well approximated by [P2]tot and is henceforth referred to simply as [P2]. By using fluorescein-labeled RNAs, RNA-protein complexes are distinguishable in solution based on differences in the intrinsic fluorescence anisotropy exhibited by each species resulting from changes in molecular volume under conditions of constant temperature and viscosity (36Checovich W.J. Bolger R.E. Burke T. Nature. 1995; 373: 254-256Crossref Scopus (165) Google Scholar, 37Lundblad J.R. Laurance M. Goodman R.H. Mol. Endocrinol. 1996; 10: 607-612Crossref PubMed Scopus (217) Google Scholar). An initial experiment was performed to determine whether the quantum yield of the fluorescein-labeled RNA changed as a result of AUF1 binding. By using Fl-TNFα ARE and a titration of His6-p37AUF1-(1–229), no significant change in fluorescence intensity was observed with protein concentrations up to 250 nmHis6-p37AUF1-(1–229) dimer (Fig.4 A), demonstrating that AUF1 binding did not alter the quantum yield of the fluorescein-labeled RNA. Accordingly, the measured anisotropy A t of the fluorescent RNA probe may be interpreted using Equation 3. At=∑iAifiEquation 3 where A i represents the intrinsic anisotropy of each fluorescing species (in this case R, P2R, or P4R), and f i its fractional concentration (38Otto M.R. Lillo M.P. Beechem J. Biophys. J. 1994; 67: 2511-2521Abstract Full Text PDF PubMed Scopus (65) Google Scholar, 39Jameson D.M. Sawyer W.H. Meth