INTRODUCTION

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The generation of functional mRNA in eukaryotes requires the accurate removal of noncoding regions (introns) from pre-mRNA by a process termed pre-mRNA splicing (14, 25). Splicing takes place in the spliceosome, a dynamic RNA-protein complex that assembles in a stepwise manner on the pre-mRNA (10, 14). The spliceosome is composed of small nuclear ribonucleoprotein particles (snRNPs) and extrinsic (non-snRNP) factors. The recognition of exon/intron boundaries, the splice sites, by the splicing apparatus is a critical step in processing of both constitutively and alternatively spliced pre-mRNAs. U1 snRNP defines the 5' splice site, and U2 snRNP defines the branchpoint sequence (3, 10, 14, 17). Since in most cases the first AG dinucleotide downstream of the branchpoint is used as the 3' splice site, by defining the branchpoint, U2 snRNP defines the 3' splice site (18, 27). Targeting of U2 snRNP to the branch site requires the extrinsic splicing factor U2 snRNP auxiliary factor (U2AF). U2AF binds site specifically to the intron pyrimidine tract between the branchpoint sequence and 3' splice site at an early step in spliceosome assembly and recruits U2 snRNP to the branch site (22, 29, 42). Regulation of 3' splice site choice, both positive and negative, can be realized by influencing the pyrimidine tract binding of U2AF (33, 35, 45). Because U2AF is a major determinant in 3' splice site selection, it has been the subject of extensive biochemical and genetic investigation.

Human U2AF is a heterodimer composed of a 65-kDa large subunit (hU2AF⁶⁵) and a 35-kDa small subunit (hU2AF³⁵) (41). Both subunits are conserved in other organisms (40), and U2AF homologs have been identified in Drosophila melanogaster (9, 21), Schizosaccharomyces pombe (16, 36), and Caenorhabditis elegans (3a, 39). The Drosophila U2AF large (dU2AF⁵⁰)- and small (dU2AF³⁸)-subunit homologs are 50 and 38 kDa, respectively (9, 21). The U2AF large subunit contains three RNA recognition motifs (RRMs) and an amino-terminal arginine-serine-rich (RS) domain (42). The small subunit contains a highly degenerate RRM (pseudo-RRM) (2), two Zn²⁺ binding motifs (37), and a carboxyl-terminal RS domain and glycine-rich region (43).

Both U2AF subunits are involved in recognition of the intron pyrimidine tract. The large subunit (hU2AF⁶⁵ and dU2AF⁵⁰) is required for site-specific pyrimidine tract binding (9, 42). The small subunit acts as a cofactor to stabilize the large subunit on the pyrimidine tract, apparently through protein-protein interactions with constitutive and alternative splicing factors (38, 45). While it has been firmly established that all three RRMs on hU2AF⁶⁵ are necessary for high-affinity RNA binding, a role for the large-subunit RS domain in RNA binding remains unresolved (11, 42). In one study removal of the hU2AF⁶⁵ RS domain had a modest effect on RNA binding (42). In a second study, the RS domain was found to be absolutely required for RNA binding (11).

In vitro splicing assays using U2AF-depleted extracts prepared by two independent methods have identified independent and essential roles for the two U2AF RS domains: the large-subunit RS domain is required to target U2 snRNP to the branch site (34, 42), and in the immunodepleted extracts under certain conditions, the small-subunit RS domain is apparently necessary for protein-protein interactions with constitutive and alternative splicing factors to stabilize hU2AF⁶⁵ on the pyrimidine tract (38, 45). In contrast to the essential roles assigned to the two U2AF RS domains in vitro, molecular genetic analysis of the Drosophila U2AF RS domains indicates that either one of the RS domains is dispensable in vivo (19). Importantly, at least one RS domain on U2AF is essential for viability (19).

The observation that the dU2AF³⁸ RS domain is not essential in vivo (19) refocused our attention on domains present in the U2AF small subunit that are phylogenetically conserved. In an exhaustive database search for proteins containing RRMs, some of the signature sequences of this motif were identified in hU2AF³⁵ (2). These sequences are also present in the Drosophila and S. pombe small-subunit homologs (21, 36). Although some of the most conserved residues in the RNA recognition motif are present in the U2AF small subunits, the RNP-1 octamer is highly degenerate and the RNP-2 hexamer is absent. Since these defining submotifs and other conserved residues are not present in the U2AF small-subunit RRM, it was termed a degenerate RRM or pseudo-RRM (2). Degenerate RRMs have been identified in a collection of RNA binding proteins, including several of the SR proteins, the pyrimidine tract binding protein, and the large subunit of U2AF (10). The degenerate RRMs in SRp30a (ASF/SF2) (4, 46), pyrimidine tract-binding protein (15), and hU2AF⁶⁵ (42) were all found to be required for high-affinity RNA binding.

Two putative Zn²⁺ binding domains, one on either side of the pseudo-RRM, were recently identified in hU2AF³⁵ in a database search (37). These Cys₃His Zn²⁺ binding motifs are conserved in all three small subunit homologs. Though sequence-specific RNA binding has not been described for proteins that contain this type of Zn²⁺ binding motif, several proteins that have this domain are involved in RNA metabolism (37). The evolutionary conservation of the pseudo-RRM and the two Zn²⁺ binding motifs in all three U2AF small subunit homologs suggested to us that these domains are important for function.

The lack of requirement for the dU2AF³⁸ RS domain in vivo prompted us to search for novel biochemical activities associated with the small subunit. The phylogenetically conserved, degenerate RRM (2) and two Zn²⁺ binding motifs (37) in the small subunit suggested that it might participate in RNA binding. While we detected weak RNA binding activity for the Drosophila small subunit on its own, we found that when complexed with the large subunit, dU2AF pyrimidine tract binding affinity increased 20-fold. This increase in RNA binding activity was not specific to Drosophila U2AF; the human U2AF heterodimer bound RNA with 15-fold-higher affinity than hU2AF⁶⁵. Surprisingly, removal of the dU2AF³⁸ RS domain abolished the increase in binding activity of the dU2AF heterodimer, indicating that the RS domain is necessary for high-affinity binding. Deletion of the dU2AF⁵⁰ RS domain (dU2AF⁵⁰RS) dramatically reduced RNA binding activity of the large-subunit monomer. High-affinity binding was restored when the dU2AF³⁸ RS domain was supplied in trans to dU2AF⁵⁰RS. These data suggest that high-affinity RNA binding activity requires at least one RS domain on U2AF, which is consistent with the requirement for at least one RS domain in vivo.

MATERIALS AND METHODS

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Recombinant proteins. Expression plasmids used to purify His₆-dU2AF⁵⁰, His₆-dU2AF⁵⁰/dU2AF³⁸, His₆-dU2AF⁵⁰RS/dU2AF³⁸, hU2AF⁶⁵/His₆-hU2AF³⁵, and His₆-hU2AF⁶⁵ were as described previously (9, 20). His₆-dU2AF⁵⁰/dU2AF³⁸RS was made by insertion of an oligonucleotide linker (top strand, 5'CTCTACTAATAGCTGCA3'; bottom strand, 5'GCTATTAGTAGAGGTAC3') between KpnI and PstI sites in the dU2AF³⁸ coding sequence in pdr154 (20) to create pdr234.

Protein-RNA binding analysis. Apparent dissociation constants (K_ds) for interaction of U2AF large subunits and heterodimers with RNA were determined by use of native gel electrophoresis. Binding reactions were performed in a volume of 10 µl containing the indicated concentrations of proteins, 0.1 nM ³²P-labeled oligonucleotide, 20 mM HEPES-KOH (pH 7.6), 100 mM KCl, 0.2 mM EDTA, 10% glycerol, 0.5 mM DTT, 50 µg of bovine serum albumin per ml, and 10 mg of heparin per ml. Incubations were continued for 1 h at 4°C. One half of the reaction mixtures were subjected to electrophoresis through 4% polyacrylamide gels (60:1; 0.5× Tris-borate-EDTA [pH 8.3]) at 4°C for 100 min at 20 V/cm. All binding experiments were repeated a minimum of two times; most were performed four times. In some binding assays, a fraction of the U2AF heterodimer-RNA complex failed to enter the gel (see, for example, Fig. 3). However, in independent runs of the identical experiment, none of the complex was retained in the well. Importantly, the binding affinities were the same in both cases. RNA binding was quantitated with the use of the Fuji phosphorimager, and K_d values were obtained by fitting binding curves to the data obtained from the native gels. A simple two-state binding reaction was assumed; using DeltaGraph Pro, the data were fitted to the following equation: y = 1/[1 + (K_d/x)], in which y represents the fraction of RNA bound by protein and x equals the protein concentration. The variation in K_ds obtained from independent experiments was between 10 and 25%.

To examine whether the small subunit could bind RNA or contribute to the binding activity of the large subunit, we purified recombinant U2AF heterodimers (dU2AF and hU2AF), large-subunit monomers (dU2AF⁵⁰ and hU2AF⁶⁵), and the Drosophila small-subunit monomer (dU2AF³⁸) from E. coli. To purify recombinant U2AF heterodimers, both subunits were coexpressed in the same E. coli cells (20). One of the two subunits was His₆ tagged (dU2AF⁵⁰ in dU2AF and hU2AF³⁵ in hU2AF), and recombinant heterodimers were purified by affinity chromatography using Ni²⁺-NTA-agarose followed by ion-exchange chromatography. Recombinant large subunits His₆-dU2AF⁵⁰ and His₆-hU2AF⁶⁵) were expressed and purified similarly. However, His₆-dU2AF³⁸ was completely insoluble when expressed independently in E. coli. To avoid solubilization by denaturants, we coexpressed dU2AF³⁸ with the His₆-tagged interaction domain from dU2AF⁵⁰ (His₆-linker) (see Materials and Methods). The interaction domain contains 28 amino acids of dU2AF⁵⁰ followed by 15 unrelated residues that result from a frameshift mutation introduced after the interaction domain (20). Soluble dU2AF³⁸, complexed with the His₆-tagged dU2AF⁵⁰ interaction domain (linker/dU2AF³⁸), was purified under the same conditions as the recombinant heterodimers. Samples of the final preparations were analyzed by electrophoresis through a sodium dodecyl sulfate (SDS)-polyacrylamide gel (Fig. 1). The heterogeneity observed in recombinant dU2AF³⁸ was due to proteolysis at its carboxyl terminus. We detected a similar heterogeneity when the amino-terminal His₆-tagged dU2AF³⁸ was purified separately (data not shown).

View larger version (64K): [in this window] [in a new window]   FIG. 1.   SDS-polyacrylamide gel electrophoresis of purified recombinant U2AF proteins. Samples from the final preparations of U2AF monomers and heterodimers were run on an SDS-12% polyacrylamide gel and stained with Coomassie blue. All individually purified monomers are His6 tagged. dU2AF50 is His6 tagged in the dU2AF heterodimers, and hU2AF35 is His6 tagged in the hU2AF heterodimer. Marker sizes are indicated in kilodaltons.

To determine whether the soluble form of the small subunit could bind RNA on its own, we performed electrophoretic mobility shift analyses. The purified, recombinant small subunit (Linker/dU2AF³⁸) was incubated for 1 h with a ³²P-labeled pyrimidine tract RNA oligonucleotide derived from the adenovirus L1-L2 intron (MINX-WT) and a mutant derivative (MINX-MUT). The sequences of MINX-WT and MINX-MUT pyrimidine tracts are shown in Fig. 3. Complex formation was assessed by native gel electrophoresis. dU2AF³⁸ exhibited very weak but reproducible RNA binding activity (Fig. 2, lanes 2 and 4). Complex formation was detected in high salt (600 mM KCl) and with excess nonspecific competitor (heparin [10 mg/ml] or heparin [5 mg/ml] and tRNA [100 µg/ml]). There was some preference for U-rich tracts, as dU2AF³⁸ bound better to MINX-WT than to MINX-MUT (Fig. 2; compare lanes 2 and 4). A similar preference of dU2AF³⁸ RNA binding was observed in assays using the non-sex-specific, tra pyrimidine tract RNA and a tra mutant that disrupted the U-rich tract (reference 6 and data not shown).

View larger version (41K): [in this window] [in a new window]   FIG. 2.   dU2AF38 interacts weakly with pyrimidine tract RNA. For electrophoretic mobility shift analysis of dU2AF38 with MINX (WT and MUT) pyrimidine tract RNA, 200 pM 32P-labeled RNA oligonucleotide was incubated in the presence (+) or absence () of 5 µM dU2AF38 complexed with the His6-dU2AF50 interaction domain (L/d38). Protein-RNA complexes (C) and unbound RNA (F) were separated by electrophoresis through a native polyacrylamide gel and visualized by autoradiography.

Encouraged by the weak RNA binding activity of dU2AF³⁸, we examined whether association of dU2AF³⁸ with dU2AF⁵⁰ could influence the RNA binding activity of dU2AF⁵⁰. To rule out the possibility that any difference in activity between dU2AF⁵⁰ and the dU2AF heterodimer was due to differences occurring during the preparation of the recombinant proteins, we purified the dU2AF heterodimer and free dU2AF⁵⁰ monomer from the same E. coli cells. To do this, we took advantage of a second coexpression plasmid from which expression of the two subunits is not stoichiometric (20). Expression of His₆-dU2AF⁵⁰ was ~10-fold higher than that of dU2AF³⁸ in this expression plasmid. Heterodimer and His₆-dU2AF⁵⁰ monomer were first copurified on Ni²⁺-NTA-agarose and then resolved from each other by cation-exchange chromatography (see Materials and Methods). The binding activities of these proteins were determined by gel mobility shift analysis using the MINX-WT pyrimidine tract RNA (Fig. 3A). Consistent with the increased size of the dU2AF⁵⁰/dU2AF³⁸ complex, the dU2AF heterodimer retarded the mobility of the MINX-WT RNA oligonucleotide to a greater extent than the dU2AF⁵⁰ monomer (Fig. 3A; compare lanes 2 and 7). The apparent K_d of the dU2AF⁵⁰ monomer for MINX-WT was ~2.2 × 10⁶ M (Table 1), in good agreement with previous analysis (6). Significantly, the dU2AF heterodimer had a 20-fold-higher affinity for RNA than uncomplexed dU2AF⁵⁰ (apparent K_d of ~1.0 × 10⁷ M [Table 1]). The increased RNA binding was unaffected by high salt (350 mM KCl) or nonspecific competitor (10 mg of heparin or 450 µg of tRNA per ml). Thus, dU2AF⁵⁰ monomer can bind the pyrimidine tract RNA on its own, but the heterodimer binds with increased affinity.

View larger version (51K): [in this window] [in a new window]   FIG. 3.   The dU2AF heterodimer binds pyrimidine tract RNA with higher affinity than the dU2AF50 monomer. (A) Electrophoretic mobility shift analysis of dU2AF heterodimer and dU2AF50 monomer interaction with MINX-WT pyrimidine tract RNA. dU2AF50 monomer protein concentrations were 5,000, 1,000, 200, 40, and 8 nM (lanes 2 to 6). dU2AF heterodimer concentrations were 5,000, 1,000, 200, 40, and 8 nM (lanes 7 to 11). Proteins were incubated with 100 pM 32P-labeled RNA oligonucleotide. Protein-RNA complexes (C) and unbound RNA (F) were separated by electrophoresis through a native polyacrylamide gel and visualized by autoradiography. Occasionally, the dU2AF50/dU2AF38 heterodimer-RNA complex appeared to be retained in the sample well. However, this effect was variable. The sequence of the MINX-WT pyrimidine tract oligonucleotide is displayed below the autoradiogram. The apparent Kd for the dU2AF50 monomer was ~2.2 × 106 M. The apparent Kd for the dU2AF heterodimer was ~1.0 × 107 M. (B) Electrophoretic mobility shift analysis of dU2AF heterodimer and dU2AF50 monomer interaction with MINX-MUT pyrimidine tract RNA. Protein concentrations were identical to those in panel A. The sequence of the MINX-MUT pyrimidine tract oligonucleotide is displayed below the autoradiogram. The apparent Kd for the dU2AF50 monomer could not be determined. The apparent Kd for the U2AF heterodimer was ~1.1 × 106 M. Although it may appear that U2AF binding is cooperative, this is due to the broad titration of protein used in this experiment. When a finer titration is used, we detect no evidence of cooperative binding (Fig. 5B and data not shown) (9).

We analyzed human U2AF heterodimer and large-subunit monomer for RNA binding activity to determine whether this increase in RNA binding activity was specific to Drosophila U2AF (Fig. 4). The apparent K_d of the hU2AF⁶⁵ monomer for MINX-WT RNA was ~3.8 × 10⁸ M (Table 1). This value is ~8-fold lower than previously reported (9, 42). We have no explanation for the increased binding activity of our recombinant protein. By comparison, the human U2AF heterodimer bound RNA with 15-fold-higher affinity (apparent K_d of ~2.5 × 10⁹ M [Table 1]) than reported previously for the hU2AF⁶⁵ monomer. Taken together, these data indicate there is a similar increase in RNA binding affinity for both the human and Drosophila U2AF heterodimers over free large-subunit monomers and suggest that a role for the small subunit in RNA binding is a common feature of U2AF.

View larger version (34K): [in this window] [in a new window]   FIG. 4.   The hU2AF heterodimer binds pyrimidine tract RNA with higher affinity than the hU2AF65 monomer. (A) Electrophoretic mobility shift analysis of hU2AF heterodimer and hU2AF65 monomer interaction with MINX-WT pyrimidine tract RNA. hU2AF65 monomer protein concentrations were 2,000, 400, 80, 16, 3.2, 0.64, and 0.128 nM (lanes 2 to 8). hU2AF heterodimer concentrations were 400, 80, 16, 3.2, 0.64, and 0.128 nM (lanes 9 to 14). Proteins were incubated with 100 pM 32P-labeled RNA oligonucleotide. Protein-RNA complexes (C) and unbound RNA (F) were separated by electrophoresis through a native polyacrylamide gel and visualized by autoradiography. The apparent Kd for hU2AF65 was ~3.8 × 108 M. The apparent Kd for hU2AF heterodimer was ~2.5 × 109 M. (B) Electrophoretic mobility shift analysis of hU2AF heterodimer and hU2AF65 monomer interaction with MINX-MUT pyrimidine tract RNA. Protein concentrations were identical to those in panel A. The apparent Kd for hU2AF65 was ~2.6 × 107 M. The apparent Kd for the hU2AF heterodimer was ~6.1 × 109 M.

To determine whether the specificity of U2AF for pyrimidine tracts was maintained in the recombinant U2AF heterodimers, we analyzed RNA binding by using the MINX pyrimidine tract mutant (MINX-MUT). As previously reported, binding of both dU2AF⁵⁰ and hU2AF⁶⁵ to MINX-MUT was significantly reduced (compare lanes 2 in Fig. 3A and B and lanes 3 in Fig. 4A and B). dU2AF⁵⁰ binding to MINX-MUT was undetectable, and the affinity of hU2AF⁶⁵ was ~7-fold lower (apparent K_d of ~2.6 × 10⁷ M [Table 1]). Similarly, the U2AF heterodimers bound the MINX mutant with reduced affinity. The dU2AF heterodimer bound MINX-MUT with ~10-fold-lower affinity (apparent K_d of ~1.1 × 10⁶ M [Table 1]), and the hU2AF heterodimer binding affinity was reduced ~2.5-fold (apparent K_d of ~6.1 × 10⁹ M [Table 1]). Taken together, these data indicate that the U2AF heterodimer retains the specificity for pyrimidine tract RNA demonstrated by the large-subunit monomer.

The dU2AF⁵⁰ monomer and the dU2AF heterodimer lacking the small subunit RS domain (dU2AF⁵⁰/dU2AF³⁸RS) were analyzed for RNA binding activity to assess whether the RS domain on dU2AF³⁸ was responsible for the increased binding activity of the U2AF heterodimer. The dU2AF³⁸ RS domain deletion was identical to the deletion mutation used in the in vivo analysis of dU2AF³⁸ (20). This deletion removed the glycine-rich carboxyl terminus and the entire RS domain (see Materials and Methods). To minimize differences in the preparation of the proteins, we purified monomer and heterodimer from the same E. coli cells (see above and Materials and Methods). dU2AF⁵⁰ and dU2AF⁵⁰/dU2AF³⁸RS were analyzed for RNA binding activity by using MINX-WT (Fig. 5B). Surprisingly, removal of the RS domain on dU2AF³⁸ reduced the RNA binding activity of the heterodimer to within threefold of the activity of the dU2AF⁵⁰ monomer (Fig. 5B). Phosphorimager quantitation analysis of bound and free radiolabeled RNA indicates a 2.5-fold-higher binding affinity of the heterodimer (dU2AF⁵⁰/dU2AF³⁸RS) compared to the dU2AF⁵⁰ monomer (apparent K_d of dU2AF⁵⁰, ~8.0 × 10⁷ M; apparent K_d of dU2AF⁵⁰/dU2AF³⁸RS, ~3.0 × 10⁷ M [Table 1]). We note that this dU2AF⁵⁰ protein preparation binds RNA with higher affinity than our previous preparations (~3-fold) (Table 1). However, since the dU2AF⁵⁰ monomer and the dU2AF⁵⁰/dU2AF³⁸RS heterodimer were purified from the same E. coli cells, their binding activities can be compared directly. Because of the variation in the protein preparations, comparison of the RNA binding activity of the dU2AF⁵⁰/dU2AF³⁸RS heterodimer to the activity of the independent dU2AF⁵⁰ (or dU2AF⁵⁰/dU2AF³⁸ heterodimer) preparation described above would be misleading. We conclude that the RS domain on dU2AF³⁸ is necessary for the enhanced RNA binding activity of the dU2AF heterodimer and that the pseudo-RRM and two Zn²⁺ binding motifs make only a modest contribution to this binding activity.

View larger version (55K): [in this window] [in a new window]   FIG. 5.   At least one RS domain on U2AF is required for high-affinity RNA binding. (A) Electrophoretic mobility shift analysis of dU2AF50RS monomer and dU2AF50RS/dU2AF38 heterodimer interaction with MINX-WT pyrimidine tract RNA. dU2AF50RS monomer protein concentrations were 5,000, 1,000, 200, and 40 nM (lanes 1 to 4). dU2AF50RS/dU2AF38 heterodimer concentrations were 5,000, 1,000, 200, and 40 nM (lanes 5 to 8). Proteins were incubated with 100 pM 32P-labeled RNA oligonucleotide. Protein-RNA complexes (C) and unbound RNA (F) were separated by electrophoresis through a native polyacrylamide gel and visualized by autoradiography. The apparent Kd for dU2AF50RS could not be determined. The apparent Kd for dU2AF50RS/dU2AF38 was ~1.7 × 107 M. (B) Electrophoretic mobility shift analysis of dU2AF50 and dU2AF50/dU2AF38RS heterodimer interaction with MINX-WT pyrimidine tract RNA. dU2AF50 monomer protein concentrations were 4,000, 1,600, 640, 256, and 102 nM (lanes 2 to 6). dU2AF50/dU2AF38RS heterodimer protein concentrations were 4,000, 1,600, 640, 256, and 102 nM (lanes 7 to 11). The apparent Kd for dU2AF50 was ~8.0 × 107 M. The apparent Kd for the dU2AF50/dU2AF38RS heterodimer was ~3.0 × 107 M.

To determine whether the dU2AF⁵⁰ RS domain also plays a role in RNA binding in our assay, we analyzed dU2AF⁵⁰RS and dU2AF⁵⁰RS/dU2AF³⁸ for binding to the MINX-WT RNA pyrimidine tract. Interestingly, removal of the dU2AF⁵⁰ RS domain severely reduced RNA binding activity of dU2AF⁵⁰ (compare lane 1 in Fig. 5A and lane 3 in Fig. 3A). Significantly, when an RS domain was supplied to dU2AF⁵⁰RS in trans (from dU2AF³⁸), binding activity was restored (Fig. 5A). The RNA binding activity of dU2AF⁵⁰RS/dU2AF³⁸ was determined to be only ~1.7-fold lower than that of the wild-type dU2AF heterodimer (apparent K_d of ~1.7 × 10⁷ M [Table 1]). Taken together, these data indicate that the presence of at least one RS domain on U2AF, in addition to the three RRMs of the large subunit, is necessary for high-affinity RNA binding activity in vitro.

Although the known biochemical activities associated with the U2AF small subunit require the C-terminal RS domain and glycine-rich region, we have shown that these domains are dispensable in vivo (20). We therefore examined the amino-terminal 189 amino acids of dU2AF³⁸ for other biochemical activities. This region is the most conserved part of the U2AF small subunit and contains a degenerate RRM and two putative Zn²⁺ binding domains (21). Since pseudo-RRMs and related Zn²⁺ binding domains have been implicated in RNA binding of other proteins (10, 37), we analyzed the small subunit for RNA binding activity. We found that a soluble form of dU2AF³⁸ bound RNA weakly on its own and when complexed with the large subunit increased the pyrimidine tract binding affinity of dU2AF⁵⁰ 20-fold. The contribution of the U2AF small subunit to pyrimidine tract RNA binding was not specific to Drosophila; the human U2AF heterodimer also bound RNA with a 15-fold-higher affinity than the hU2AF⁶⁵ monomer. Like the large subunits, both human and Drosophila heterodimers bound with reduced affinity to mutant pyrimidine tract RNAs, indicating that binding specificity was retained. Surprisingly, removal of the RS domain and glycine-rich region from dU2AF³⁸ abolished virtually all of the increased binding activity of the dU2AF heterodimer. Although this result does not exclude a role for the degenerate RRM and Zn²⁺ binding motifs in RNA binding, it suggests that the RS domain on dU2AF³⁸ is responsible for the increased binding activity of the dU2AF heterodimer. Finally, we have found that in our binding assays, the RS domain on dU2AF⁵⁰ is required for RNA binding and that the dU2AF³⁸ RS domain supplied by association with the large subunit will restore high-affinity binding activity to dU2AF⁵⁰RS. These results suggest that at least one of the RS domains on dU2AF is required for high-affinity RNA binding.

What is the role of the U2AF RS domain in RNA binding? It has been shown previously that when hU2AF⁶⁵ is bound to an intron pyrimidine tract RNA, its RS domain can be UV cross-linked to the branchpoint adenosine. This result indicates that the large-subunit RS domain is in close proximity to the RNA and suggests that the positively charged residues in the hU2AF⁶⁵ RS domain are in a position to stabilize the base-pairing interaction between U2 snRNA and the branch site (34). The small-subunit U2AF RS domains could similarly stabilize the large-subunit RRMs on the pyrimidine tract through nonspecific interactions with the phosphodiester backbone of the pre-mRNA. However, the RNA binding activity of the heterodimer (and dU2AF³⁸) was resistant to high salt, suggesting that it is unlikely that these interactions are purely electrostatic in nature.

Requirement for the large-subunit RS domain for RNA binding. Our biochemical analysis of the U2AF heterodimer and the requirements for an RS domain for high-affinity RNA binding differ from previous studies of U2AF (11, 42). The first biochemical analysis of hU2AF⁶⁵ (42) detected a very modest decrease in RNA binding activity when the hU2AF⁶⁵ RS domain was deleted. In our analysis, dU2AF⁵⁰RS was severely impaired in RNA binding activity (Fig. 5A). There are several possible explanations for this difference. In the analysis of the hU2AF⁶⁵ RS domain, the RS deletion removed the entire RS domain but an adjacent region containing several positively charged residues was retained (42). If the large-subunit RS domain stabilizes U2AF on the pyrimidine tract RNA by interaction with the phosphodiester backbone (see above), this basic region retained in the original hU2AF⁶⁵RS deletion mutant might be sufficient for improved RNA binding activity. Significantly, this deletion derivative was originally determined to have no splicing activity; however, it was recently found that the positively charged region retained in the mutant could partially reactivate a U2AF-depleted extract (34). hU2AF⁶⁵ splicing activity was completely abolished only when this basic region was also deleted. The dU2AF⁵⁰ RS domain deletion used in the present study is similar to the previously characterized, more extensive hU2AF⁶⁵ RS domain deletion (dU2AF⁵⁰RS retains only three basic residues).