A Splicing Enhancer Complex Controls Alternative Splicing of doublesex Pre-mRNA

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1 Cell, Vol. 74, , July 16, 1993, Copyright by Cell Press A Splicing Enhancer Complex Controls Alternative Splicing of doublesex Pre-mRNA Ming Tian and Tom Maniatis Harvard University Department of Biochemistry and Molecular Biology Cambridge, Massachusetts Female-specific splicing of Drosophila doublesex (dsx) pre-mrna is regulated by the products of the transformer (fra) and transformer 2 (fra2) genes. In this paper we show that Tra and Tra2 act by recruiting general splicing factors to a regulatory element located downstream of a female-specific 3 splice site. Remarkably, Tra, TraP, and members of the serinelarginine-rich (SR) family of general splicing factors are sufficient to commit dsx pre-mrna to female-specific splicing, and individual SR proteins differ significantly in their ability to participate in commitment complex formation. Characterization of the proteins associated with affinity purified complex formed on dsx pre-mrna reveals the presence of Tra, Tra2, SR proteins, and additional unidentified components. We conclude that Tra, Tra2, and SR proteins are essential components of a splicing enhancer complex. Alternative splicing of individual nuclear pre-messenger RNAs (pre-mrnas) can lead to the production of multiple mrnas encoding functionally distinct proteins (for recent reviews see Maniatis, 1991; Green, 1991; Nadal-Ginard et al., 1991; Rio, 1992). A striking example of this phenomenon is the sex determination pathway of Drosophila melanogaster, which involves a cascade of regulated splicing events (for reviews see Baker, 1989; Steinmann-Zwicky et al., 1990). Alternative splicing of doublesex (dsx) premrna, the last step in this regulatory hierarchy, generates male or female-specific mrnas. In females, dsx premrna, which contains six exons, is spliced to produce a dsx mrna consisting of exons 1, 2, 3, and 4 (Figure 1; Burtis and Baker, 1989) and this mrna encodes a transcriptional repressor of male sexual differentiation (Baker and Ridge, 1980; Nothiger et al., 1987). In contrast, male dsx mrna is composed of exons 1,2,3,5, and 8 (Figure 1; Burtis and Baker, 1989), and it encodes a protein that represses the expression of genes required for female development (Baker and Ridge, 1980; Nothiger et al., 1987). Regulation of dsx pre-mrna splicing involves positive control by Tra and Tra2 of the female-specific 3 splice site immediately upstream of exon 4 (Hedley and Maniatis, 1991; Hoshijima et al., 1991; Ryner and Baker, 1991; Tian and Maniatis, 1992). The pyrimidine tract of the femalespecific 3 splice site, an important determinant of splicing efficiency (Green, 1991) is interrupted by purines (Burtis and Baker, 1989). The male-specific 3 splice site at exon 5 conforms to the consensus. As a result, the male-specific 3 splice site is used by default in males. In females, Tra and Tra2 activate the female-specific 3 splice site, leading to the female splicing pattern. Both Tra (Boggs et al., 1987) and Tra2 (Amrein et al., 1988; Goralski et al., 1989) contain motifs characteristic of proteins involved in RNA processing. Tra consists of an extended serinelarginine-rich domain (SR domain), while Tra2 contains two SR domains and a ribonucleoprotein consensus-type RNA recognition motif (RRM) (Bandziulis et al., 1989). Both the SR domain and RRM are also present in members of the SR family of splicing factors (Ge et al., 1991; Krainer et al., 1991; Fu and Maniatis, 1992a; Zahler et al., 1992) and in splicing factors U2AF (Zamore et al., 1992) Ul 70K protein (Theissen et al., 1988; Mancebo et al., 1990), and suppressor of white apricot (su(vp)) protein (Chou et al., 1987). Tra, Tra2, and several unidentified nuclear proteins bind specifically to a regulatory element (the repeat element) in the female-specific exon(hedleyand Maniatis, 1991; Tianandfvlaniatis, 1992; lnoue et al., 1992). This element is located about 300 nt downstream of the female-specific 3 splice site and contains six copies of a 13 nt repeat sequence (Nagoshi and Baker, 1990). The repeat element is both necessary and sufficient for mediating Tra- and Tra2-dependent regulation of splicing (Nagoshi and Baker, 1990; Hedley and Maniatis, 1991; Hoshijima et al., 1991; Ryner and Baker, 1991; Tian and Maniatis, 1992). In this paper, we study the mechanisms of Tra and Tra2 function using a previously established in vitro system in which the dsx female-specific splicing can be activated by recombinant Tra and Tra2 (Tian and Maniatis, 1992). We show that Tra and Tra2 cooperate with nuclear factors to commit dsx pre-mrna to the splicing pathway, and we identify the factors involved. Moreover, we use an affinity purification method to demonstrate that Tra and Tra2 act by recruiting general splicing factors to a regulatory sequence located downstream of the female-specific splice site. We propose that Tra and Tra2 activate dsx femalespecific splicing by promoting the formation of a splicing enhancer complex. Results Tra and Tra2 Commit dsx Pre-mRNA to Female-Specific Splicing Since Tra and Tra2 function as a binary switch in splice site choice, they most likely act at the early commitment stage of spliceosome assembly. To investigate this possibility, we designed a functional assay for dsx commitment complex (dsxcc) formation that involves competition and preincubation experiments. In the competition experiments, we added unlabeled RNAs with or without the repeat sequence to in vitro splicing reactions in which dsxfemale-specific splicing can be activated by recombinant Tra and Tra2 (Van and Maniatis, 1992; Figure 2, lanes 2 and 3). Since Tra and Tra2 bind

2 Cell 106 Figure 1. The Sex-Specific Splicing Pattern of dsx Pre-mRNA The open boxes, hatched box, and closed boxes represent common exons, the female-specific exon, and male-specific exons, respectively. The exon numbers are indicated, and the lines connecting the exons represent splicing. pa at the end of exon 4 and exon 6 represents the site for cleavage and polyadenylation. specifically to the repeat element, repeat-containing competitor RNAs should titrate away these factors and abolish Tra- and Tra2-dependent regulation. Consistent with this prediction, the addition of repeat-containing RNA competitors inhibits the female-specific splicing (Dl, D4, and D5; Figure 2, lanes 10-16) while RNAs without the repeat sequence have no effect (D3 and D6; Figure 2, lanes 4-6 and 17-l 6). The competitor RNA containing just one copy of the repeat sequence weakly inhibits the femalespecific splicing (D2; Figure 2, lanes 7-9). Based on these results, we designed a commitment complex assay in which labeled dsx Dl substrate was preincubated with Tra, Tra2, and micrococcal nuclease (MN)-treated nuclear extract (Krainer and Maniatis, 1965). MN-treated extract is splicing deficient because it lacks functional small nuclear RNAs (snrnas), but presumably contains all the protein components necessary for splicing. The reason for including MN-treated extract in the preincubation stems from our previous observation that Tra and Tra2 promote the specific binding of several nuclear proteins to the repeat element (Tian and Maniatis, 1992). The preincubation was followed by the addition of a splicing-competent nuclear extract plus an excess of unlabeled repeat-containing competitor RNA (D5), and the incubation was continued. If during the preincubation the binding of Tra, Tra2, and the nuclear proteins to repeat element results in the formation of a stable complex, the substrate will be spliced in the presence of the repeatcontaining competitor RNA during the second incubation period. After preincubation with Tra, Tra2, and MN-treated extract, female-specific splicing was indeed observed in the presence of repeat-containing competitors (Figure 3, lane 5). Thus, a stable complex that is resistant to competition was formed during preincubation. Since this stable complex commits the dsx pre-mrna to the female-specific splicing pathway, we refer to it as the dsx commitment complex (d.sxcc). Tra and Tra2 are required for dsxcc formation, since no splicing was observed when they are absent during the preincubation step (Figure 3, lane 3). However, Tra and Tra2 are not sufficient for dsxcc formation, since preincubation of the Dl substrate with Tra and Tra2 in the absence of MN-treated nuclear extract does not lead to &WCC formation (Figure 3, lane 4). dsxcc formation therefore requires both Tra and TraP and addi d.sx m I 4 I D4 05 D6 Figure 2. Repeat-Containing RNAs Competitively Inhibit In Vitro Activation of dsx Female-Specific Splicing by Tra and TraP AYP-IabeleddsxRNAsplicingsubstratecontaining therepeatelement (Dl) was incubated in a HeLa cell nuclear extract in the absence (lane 2) or presence (lane 3) of recombinant Tra and TraP. The reaction products were then fractionated on a denaturing polyacrylamide gel and visualized by autoradiography. To identify the dsx RNA sequences required for splicing, excess unlabeled competitor RNA containing various regions of dsx pm-mrna (Dl-D6) was added to the reaction at the same time as the labeled precursor. The competitor RNAs are diagrammed below the autoradiogram. The splicing substrate is Dl. The precursor and the splicing product are indicated at the right of the autoradiogram. Lane 1, unspliced Dl substrate; lane 2, incubation in the absence of Tra and TraP; lanes 3-18, splicing reactions carried out in the presence of Tra and Tra2 and different amounts of competitor RNAs as indicated above each lane. Minus indicates no competitor added. In the three titration points for D3, D2, and Di (lanes 4-12) the ratios of competitor to substrate are 1O:l,50:1, and 250:1, respectively. In the two titration points for D4, D5, and D6 (lanes 13-16) the ratios of competitor to substrate are 5O:l and 250:1, respectively. tional proteins present in the MN-treated nuclear extract. The presence of repeat-containing RNA competitors during preincubation abolished dsxcc formation, while the presence of nonspecific RNA had no effect (Figure 3, lanes 6-7). Thus, &WCC formation requires the factors binding to the repeat element. Significantly, the formation of the &WCC does not require ATP (Figure 3, lane 6). In mammalian systems, the formation of E complex, the earliest known prespliceosome, is also ATP independent (Michaud and Reed, 1991). However, E complex contains Ul small nuclear RNP (snrnp). The formation of the dsxcc in MN-treated extract, which lacks fully functional Ul snrnp, suggests that the formation of the dsxcc may precede E complex assembly. However, we can not eliminate the possibility that E complex and the dsxcc may be formed independently.

3 ;;;itive Regulation of dsx Pre-mRNA Splicing 4 5 TraiTraP s NEm Tra+Tra* s NE D5 1 tlr 2 h, NE Lx f h, 2 llr ing sequential precipitation with ammonium sulfate and MgCI? (Zahler et al., 1992). We carried out this purification procedure and followed the activities that complement Tra and Tra2 in dsxcc formation (Figure 4). After ammonium sulfate precipitation of HeLa cell whole-cell extracts, SR proteins are present in the 65%- 90% saturated fraction (Zahier et al., 1992). This fraction also complemented Tra and Tra2 in the commitment complex assay (Figure 4, lane 6). We then precipitated the SR proteins from the 65%~90% ammonium sulfate-saturated fraction with MgCl2. Significantly, the complementing activity is contained exclusively in the pellet fraction (Figure 4, lanes 7-6). When analyzed by SDSpolyacrylamide gel eiectrophoresis (SDS-PAGE), the pellet fraction contains the characteristic set of SR proteins and only low levels of contaminating non-sr proteins (Figure 4, lane SR; 6 NP Tra+TraP s D5 NE 1 hr 2 tlr NEm ~ra+tra* NE s D6 D5 ikdi M SR 94- ui, I* - sl3p75 ST-- a& 43 - u* -sip55 Figure 3. Tra and Tra2 Are Required for the Formation of the dsxcc This figure shows the results of a splicing assay used to identify factors required for the formation of a dsxcc on 32P-labeled Dl RNA. Analysis of the precursor and splicing products was as described in Figure 2. Lane 1, unspliced substrate; lane 2, incubation in nuclear extract in the presence of Tra and TraP; lanes 3-8, preincubation experiments, with the order of addition of various factors diagrammed below the autoradiogram. In these schemes, the horizontal lines represent the progress of the reactions, and the left ends are the beginning of the reactions. The addition order of reagents and incubation times are indicated above the lines. NE, nuclear extract; NE, MN-treated nuclear extract; S, splicing substrate; CP, creatine phosphate. - wp*o Members of the SR Family of General Splicing Factors Are Required for dsxcc Formation We used dsxcc formation as a functional assay to identify the nuclear proteins that complement Tra and TraP in the preincubation assay. The chromatographic behavior of the complementing activities in our initial purification efforts suggested that they may correspond to a group of known splicing factors, the SR proteins (Ge et al., 1991; Krainer et al., 1991; Fu and Maniatis, 1992a; Zahler et al., 1992). The SR proteins are characterized by the presence of an SR domain in the C-terminal half of the protein and an FIRM in the N-terminal half. These proteins can complement splicing-incompetent SlOO extracts, and they can infiuence splice site choice in pre-mrnas containing competing 5 or 3 spiice sites (Ge et al., 1991; Krainer et al., 1991; Mayeda et al., 1992; Fu et al., 1992; Zahier et al., 1992, 1993). A unique property of the SR proteins is that they can be precipitated as a group in the presence of 20 mm MgCi* (Zahier et al., 1992). Based on this property, a simpie two-step purification procedure was designed involv- Figure 4. hsr Proteins Are Required for the Tra- and T&-Dependent Formation of the dsxcc Thesplicingsubstrate is Dl. Thepositionsoftheprecursor and splicing product are indicated at the right of the autoradiogram. Lane 1, unspliced substrate; lane 2, incubation with nuclear extract in the presence of Tra and Tra2; lane 3, incubation in the nuclear extract in the presence of Tra, Tra2, and competitor RNA 05; lanes 4-8, preincubation experiments, with the experimental schemes used for each lane illustrated below as in Figure 3. AS, 85%-90% ammonium sulfatesaturated fraction of HeLa whole-cell extracts; Mg (P), the pellet of MgCk precipitation; Mg (S/N), the supernatant of MgCI, precipitation. SDS-PAGE analysis of the MgCI? precipitate is shown at the right of the autoradiogram. The proteins are visualized by silver staining. Lane M is the molecular weight standard. The sizes of the marker proteins are indicated at the left, Lane SR is the MgCl, precipitate. The SR proteins are labeled at the right.

4 Cell 108 AS rsrp20 rsf2/asf rsc35 cap55 s a +j* $ Figure 5. Complementation of Tra and TraP in ---a d $9 / r -3 $ Q dsxcc Formation by Individual SR Proteins The splicing substrate is Dl. Precursor and splicing product are indicated between the two autoradiograms. All lanes are preincubation experiments in the presence of Tra, Tra2, and varying levels of SR proteins indicated above each lane. Minus indicates no SR proteins added in the preincubation. In the left-hand autoradiogram, the three titration points for each rsr protein contain 1 pi, 2 ul, and 4 pl of protein, respectively. In the right-hand autoradio IO gram, 2 ul of rsc35 and 4 pl of each hsr protein are used. These amounts of SR proteins show comparable activities in complementing SlOO and in switching 5 splice sites, except for SRpPO (data not shown). Analogous to RBPI (Kim et al., 1992) SRpPO exhibits weak activities in these two assays. The amount of each protein given in microliters serves only as a relative number. The absolute amount of each protein is not used here for comparison because the percentage of active protein and the presence of inhibitory activities for each protein preparation are unknown. For this reason, these data are not meant to give detailed quantitation of the specific activities of each protein. Zahler et al., 1992). These results strongly suggest that the SR proteins can complement Tra and Tra2 in dsxcc formation. Individual SR Proteins Differ in Their Ability to Promote dsxcc Formation The MgCh precipitate contains at least six SR proteins (Figure 4, lane SR; Zahler et al., 1992). To determine which one or subset of the six SR proteins are responsible for the complementing activity, we generated recombinant baculoviruses that express individual full-length SR proteins: SRp20, SFPIASF, SC%, and SRp55. The recombinant SR (rsr) proteins were purified with procedures analogous to those used for HeLa SR (hsr) proteins (see Experimental Procedures). The other two SR proteins in the MgCl* precipitate, SRp40 and SRp75, have not been cloned (Zahler et al., 1992). Without recombinant versions of these proteins, we purified them from the MgC& precipitate by preparative SDS-PAGE (Zahler et al., 1992). We then tested the activities of these proteins in dsxcc formation. As shown in Figure 5, rsc35 and rsrp55 efficiently complement Tra and Tra2 in the formation of the dsxcc (Figure 5, lanes 9-14). Similarly, hsrp40, hsrp55, and hsrp75 also complement Tra and Tra2 in dsxcc formation (Figure 5, lanes 18-20). In contrast, rsrp20, hsrp20, and rsf2/ ASF showed little or no activity in thecommitment complex assay (Figure 5, lanes 3-8 and 17). We also tested various combinations of SR proteins in dsxcc formation and found no synergy among them (data not shown). Thus, individual SR proteins differ significantly in their ability to function in dsxcc formation. SR proteins have also been shown to function in two other assays: complementing SlOO for constitutive splicing activity and switching splice site usage (Ge et al., 1991; Krainer et al., 1991; Fu et al., 1992; Mayeda et al., 1992; Zahler et al., 1992, 1993; Kim et al., 1992). In these two assays, SR proteins also exhibit substratedependent differences in activity (Zahler et al., 1993). These observations and the distinct behavior of SR proteins shown in this study suggest that they may perform highly specific functions in regulated splicing and that distinct sets of SR proteins may function on different pre-mrnas. Tra and TraP Promote Complex Formation on the dsx Repeat Element The dsxcc may facilitate subsequent splicing by interacting with additional splicing components. For this reason, it is important to analyze complex formation on the repeat element in the context of complete nuclear extract. Gel filtration has been used to analyze the spliceosome and its precursors (Abmayr et al., 1988; Reed et al., 1988; Reed, 1990; Michaud and Reed, 1991; Bennett et al., 1992a, 1992b), and we took a similar approach to study complex formation on the repeat element. The RNA we used for complex formation, D7, corresponds to a region of the female-specific exon that contains five copies of the repeat sequence (see Experimental Procedures). As the negative control, we used an RNA that has no repeat sequence, D8 (see Figure 2). After incubating P-labeled RNAs in splicing reactions with or without Tra and Tra2, we loaded the reaction onto a Sephacryl S-500 gel filtration column. The elution profile is generated by measuring the radioactivity of the column fractions. Three peaks of radioactivity were observed with the repeat-containing RNA (Figure 8A). According to elution volume, the peak eluting at fractions represents degraded RNA. The peak eluting at fractions corresponds to H complex that results from the interaction of heterogeneous nuclear RNP (hnrnp) proteins with the input RNA (Bennett et al., 1992a, 1992b). Both peaks are present in elution profiles of a binding reaction with RNA that does not contain the repeat sequence (Figure 8A). The peak eluting at fractions is unique to the RNA containing the repeat sequence (Figure 8A). In addition, the formation of this complex is significantly decreased in the absence of Tra and Tra2 (Figure 8B). Thus, the formation of this complex is dependent on both the repeat element and Tra and Tra2. We call this complex the dsx repeat complex (dsxrc). The exact relationship between the dsxrc and the dsxcc remains to be established. The dsxcc is defined by its function in a commitment complex

5 ygitive Regulation of dsx Pre-mRNA Splicing B cpm C Figure 6. Gel Filtration Analysis of the Complex Formed on the dsx Repeat Sequence The graphs show the elution profiles of binding reactions with RNAs plus or minus repeat sequence (A and C) and plus or minus Tra and TraZ (B). These conditions plus the salt concentration used in binding reactions and in the column elution are indicated within each graph. R and H indicate the dsxrc and the H complex, respectively. assay, it can form with purified Tra, Tra2, and SR proteins, and it forms on a substrate containing both the repeat sequence and a functional intron. In contrast, the dsxrc has not been shown to be functional, it is formed in total nuclear extract in the presence of Tra and Tra2, and the RNA contains only the repeat sequence. As will be discussed later, the dsxrc may contain components in addition to Tra, Tra2, and SR proteins. Thus, the dsxcc may be the core or precursor of the dsxrc. In contrast with H complex, which is salt sensitive (Bennett et al., 1992b), the dsxrc can form and remains stable in the presence of 250 mm KCI (Figure 6C). Under high salt conditions, the amount of H complex is significantly decreased, while the amount of the dsxrc increases relative to the amount of complex formed under low salt conditions. This observation suggests that under low salt conditions H complex formation may compete with dsxrc formation on the limiting amount of input RNA. The dsxrc elutes primarily in the void volume of the Sephacryl S500column (Figure 6). Under the sameconditions, spliceosomes elute between the void volume and H complex (Abmayr et al., 1988; Reed et al., 1988; Reed, 1990; Michaud and Reed, 1991; Bennett et al., 1992b). The exclusion limit of Sephacryl S-500 resin is about 2 x 10 daltons, so the dsxrc may be an aggregate of monomeric complexes. The dsxrc Contains Tra, Tra2, and SR Proteins To analyze the composition of the dsxrc, we designed an affinity purification method to separate it from the components in the nuclear extract (Figure 7). The method combines a modification of a previously described R17 coat protein affinity method (Bardwell and Wickens, 1990) with a biotin-avidin affinity step (Grabowski and Sharp, 1986). As shown in Figure 7, two copies of the binding site for the bacterial phage R17 coat protein (Bardwell and Wickens, 1990) were inserted downstream of the dsx repeat element. Rather than covalently attaching purified R17 protein to agarose beads as previously described (Bardwell and Wickens, 1990) we constructed a glutathione Stransferase-R17 fusion protein (GST-R17) that could be attached to glutathione beads and then released with glutathione (Smith and Johnson, 1988). The RNA containing the dsx repeat sequences and the R17 coat protein-binding sites was synthesized in vitro in the presence of biotin- UTP. The dsxrc formed in soluble reactions fails to bind to glutathione-agarose through GST-R17, presumably owing to the aggregation of monomeric complexes. For this reason, we first immobilized the RNA on glutathioneagarose. The dsxrc is then formed on the immobilized RNA by mixing the resin with nuclear extract in the presence of Tra and Tra2 (Figure 7). The binding reaction was carried out in 250 mm KCI to favor the formation of the dsxrc relative to H complex. After incubation at 30% for 1 hr, the unbound components in the reaction were washed away, and the protein- RNA complex was eluted by free glutathione (Figure 7). The advantage of elution with glutathione is twofold. First, the proteins bound nonspecifically to the resin are not released, further improving purification. Second, the gentle elution condition does not disrupt the protein-rna complex, enabling further purification. When the complex was eluted from the glutathione-agarose, it was contaminated dsx m r 4 I R(i) RI-I Figure 7. Affinity Purification Scheme for the dsxrc The RNAs used in the purification are diagrammed at the top. B represents biotin. The other symbols are explained in the diagram.

6 Cell Figure 8. Analysis of the Complex with Antibodies Directed against Tra, TraP, and Total SR Proteins The antibody used in each case is indicated at the top of each blot. Lane M in each blot is the prestained protein standard. The sizes of the marker proteins are indicated at the left of each blot. The last lanes of each blot are purified proteins (Tra, Tra2, and SR proteins) as positive controls. The other lanes are complexes formed under conditions indicated above each lane. The identities of the stained proteins are indicated at the right of each blot. with several glutathione-binding proteins from the HeLa cell nuclear extract. To remove these proteins, the protein-rna complex was selected with avidin-agarose, and the proteins bound to the complex were then eluted with buffers containing SDS (Figure 7). After this second purification step, the protein composition of the eluted material is still complex when analyzed on SDS-PAGE. However, proteins unique to complex selected on repeat-containing RNA could be detected, suggesting substantial enrichment for the dsxrc (data not shown). As a first step in the analysis of the components in the complex, we carried out Western blotting experiments using antibodies against known splicing factors. When Western blots of the affinity-purified dsxrc were probed with antibodies against Tra or Tra2, both proteins were present in the complex assembled on RNA containing the repeat sequence, but were not detected on RNA lacking the repeat (Figure 8, lanes l-8). We also used the monoclonal antibody (MAb), MAb104, to detect SR proteins in the complex (Figure 8, lanes 9-l 3). MAb104 recognizes a phosphoepitope present in all SR proteins (Roth et al., 1991; Zahler et al., 1992). This antibody also cross-reacts with Traand Tra2, possibly owing to their phosphorylated SR domains (Figure 8, lane 12). Western analysis with this antibody detected SRpSO and SRp40 in the d.sxrc (Figure 8, lane 12). The staining just below SRp40 is present in all the complexes and is most likely due to the nonspecific staining of the GST- R17, because it is present in large amounts. SRp20 is barely detectable in the dsxrc (Figure 8, lane 12). The presence of SRp55 and SRp75 varies from very low (data not shown) to nondetectable (Figure 8, lane 12) depending on the batches of nuclear extracts used. The SR proteins are detected only in the dsxrc formed on RNAs containing the repeat sequence in the presence of Tra and TraP (compare lanes 10 and 11 with lane 12 in Figure 8). Occasionally, a small amount of SRp30 could be detected in the complex in the absence of Tra and Tra2 (data not shown). The presence of SR proteins in the dsxrc further substantiates their roles in the regulation of dsx femalespecific splicing. SRp30 and SRp40 are the most abundant SR proteins in the complex. SRp30 is a mixture of SC35 and SF2/ASF. Antibodies against SF2/ASF detected only a small amount of SF2IASF in the dsxrc (data not shown), suggesting that the majority of the SRp30 in the complex is SC35. However, we have not been able to prove this by staining with anti-sc35 antibody, since this antibody recognizes a phosphoepitope and cross-reacts with other SR proteins (Fu et al., 1992). If the SRp30 in the dsxrc is indeed accounted for by SC35, the presence of SC35 and SRp40 would be consistent with the fact that they efficiently complement Tra and Tr& in dsxcc formation. SRp55 and SRp75, although functioning in dsxcc formation, are not consistently detected in the dsxrc. It is possible that SRp55 and SRp75 interact with other factors in the nuclear extract. These interactions may modify their specificities in protein-protein or protein-rna interactions and prevent these two proteins from interacting with the repeat element, with Tra and Tra2, or with both. Small amounts of SF2/ASF and SRpPO are detected in the dsxrc. Although these two proteins show no activity in dsxcc formation, we cannot eliminate the possibility that they function at later stages. Analysis of the dsxrc by two-dimensional gel electrophoresis revealed the presence of additional non-sr proteins (data not shown). The identities of these proteins are currently being investigated. Discussion In this paper, we show that Tra, Tra2, and certain members of the SR family of general splicing factors play an essential role in the assembly of the dsxcc. Moreover, we show that these proteins stably associate with the dsxrc. Finally, we report that individual SR proteins differ significantly in their ability to cooperate with Tra and Tra2 in promoting dsxcc formation. Thus, individual SR proteins are not only able to function as essential splicing factors, they can also interact with splicing regulators to control alternative splicing. Cooperative Interactions among Tra, Tra2, and SR Proteins in dsxcc Formation The cooperativity among Tra, Tra2, and SR proteins in dsxcc formation is likely to involve both protein-rna and

7 Positive Regulation of dsx Pre-mRNA Splicing 111 protein-protein interactions. Only one of these proteins, Tra2, is capable of binding with high specificity to the dsx repeat sequence in the absence of other factors (Hedley and Maniatis, 1991) and this interaction requires the RRM (H. Amrein, M.-L. Hedley, and T. M., unpublished data). Tra can discriminate between oligonucleotides containing either the wild-type or mutant repeat sequence (Inoue et al., 1992). When longer RNA fragments with higher sequence complexity are used in the binding reaction, the binding specificity of Tra is variable, depending on the assay used. In ultraviolet cross-linking experiments, Tra binds RNA nonspecifically(tian and Maniatis, 1992) while in filter binding assays Tra binds to the repeat element with a low level of specificity (K. Wood and T. M., unpublished data). However, Tra binds to the repeat element with a high level of specificity in the presence of nuclear extracts (Tian and Maniatis, 1992; this paper) or in the presence of SR proteins (K. Wood and T. M., unpublished data). Similarly, specific recognition of the dsx repeat sequence by SR proteins requires Tra and Tra2 (this paper; K. Wood and T. M., unpublished data). Thus, specific binding of Tra and the SR proteins to the repeat element is likely to involve interactions among Tra, Tra2, and SR proteins. These observations and the fact that the dsx repeat element contains six copies of the repeat sequence suggest that the initiation of dsxrc formation involves binding of Tra2 to one or more of the repeats, followed by the recruitment of SR proteins and Tra to the complex, possibly through interactions with repeat sequences not occupied by Tra2 The RRMs of the SR proteins may be able to recognize specifically one or more of the six repeat sequences when held in place by protein-protein interactions with Tra, Tra2, or both. Once formed, this complex may facilitate the assembly of other splicing factors on the adjacent female-specific 3 splice site. Mechanisms of Splicing Activation The dsx female-specific 3 splice site deviates from the consensus sequence in that the pyrimidine tract immediately upstream of the AG dinucleotide is interrupted by purines (Burtis and Baker, 1989). Substitution of these purines by pyrimidine leads to the constitutive activation of this splice site in vivo (Hoshijima et al., 1991) and in vitro(m. T. andt. M., unpublisheddata). Thepurinesin the female-specific splice site most likely weaken the binding of the splicing factor U2AF to the pyrimidine tract, which is essential for the formation of the earliest ATP-dependent prespliceosome (A complex; Green, 1991). The Tra- and T&-dependent complex formed on the dsx repeat sequence could activate the female-specific splice site by stabilizing the weak interactions between U2AF with the defective pyrimidine tract. The splicing factor UPAF is first detected in E complex, the earliest known prespliceosome complex formed during mammalian spliceosome assembly in vitro (Bennett et al., 1992b). E complex formation occurs in the absence of ATP, and it commits pre-mrna to the splicing pathway (Michaud and Reed, 1991). E complex contains Ul snrnp. dsxcc formation may precede E complex assembly since neither ATP nor Ul snrnp is required in this process. The dsxcc may facilitate E complex formation by interacting with U2AF and stabilizing its interaction with the defective pyrimidine tract. However, the recognition of the 3 splice site by U2 snrnp involves components in addition to UPAF (Kramer, 1988; Fu and Maniatis, 1992b). These additional components could equally be the targets of Tra and Tra2 activation. Alternatively, Tra and Tra2 may interact with U2 snrnp directly. The characterization of the components in the dsxrc should help distinguish among these possibilities. A number of other examples of positive control of splice site selection have been reported recently. The splicing of the mouse immunoglobulin u (Igfvl) pre-mrna also involves a positive regulatory element (M2 element) (Watakabe et al., 1993). Analogous to the repeat element in dsx, the M2 element is situated in the exon downstream of the regulated 3 splice site. Furthermore, the M2 element can substitute for the repeat element in the dsxfemale-specific exon and activate the female-specific 3 splice site in a Tra- and T&-independent manner. Like the dsx repeat element, M2 serves as a binding site for splicing components that may include Ul snrnp. The M2 element consists of a purine-rich sequence, and similar sequence motifs have been found in the exons of other pre-mrnas. The dsx repeat sequence shares no homology with the purine-rich exon elements, consistent with its nature as a specialized regulatory element. The role of exon sequences in splicing has been recognized in early studies (Reed and Maniatis, 1988); however, their exact function remains unclear. The present observations with the dsx repeat element and the IgM M2 element suggest that exon sequences may contain binding sites for regulatory complexes. Another case of positive regulation is the neuronalspecific splicing of c-sfc (Black, 1992). In contrast with the dsx repeat element and IgM M2 elements that are in the exon, the c-src regulatory element is situated in the intron downstream of the regulated splice sites. Although different in location, the C-SC element also serves as a binding site for splicing activators, the identities of which are unknown at present. Finally, in exon definition, factors binding to 5 and 3 splice sites communicate across the exon (Robberson et al., 1990) and the 3 splice sitesof terminal exons can interact with polyadenylation factors (Niwa and Berget, 1991; Niwa et al., 1992). In the most recent example involving the alternative splicing of exon 4 in preprotachykinin pre-mrna, the binding of Ul snrnp to the 5 splice site at the 3 end of exon 4 facilitates the binding of U2AF to the 3 splice site at the 5 end of this exon through exon-bridged interactions (Hoffmann and Grabowski, 1992). All these cases of splicing activation involve positive regulatory complexes. The elements differ in their position: the dsx repeat element and IgM M2 element are located in exons downstream of the affected 3 splice site, the c-srcelement maps to an intron, and the preprotachykinin element is positioned at the intron-exon boundary. However, these elementsshare one thing in common: they are all located adjacent to and downstream of the regulated splice sites. This arrangement is reminiscent of tran-

8 Cell 112 scription activation by enhancers. Thus, these elements The SR proteins were purified from the dialyzed fractions by MgClz may function as splicing enhancers. In contrast with the positive regulation discussed above, regulatory proteins can also antagonize the function of general splicing factors. Sex lethal (Sxl) regulates the alternative splicing of tra pre-mrna by binding to the pyrimidine tract of the regulated splice site and precluding the interaction of U2AF with this splicing signal (Valcarcel et al., 1993). The germline-specific splicing of P element premrna is controlled by inhibitory complexes assembled on pseudod splice sites that interfere with the recognition of the adjacent authentic 5 splice site (Siebel and Rio, 1990; Siebel et al., 1992). Su(w ) protein represses the splicing of both its own primary transcript and the whiteapricot pre-mrna (Singham et al., 1988). Given the various interactions between splicing regulators and general splicing components, studies in this and other alternative splicing systems should contribute to the understanding of both regulated and constitutive splicing. Expsrlmental Procedures In Vitro Splicing Reactions In vitro splicing reactions were carried out as previously described (Tian and Maniatis, 1992). In commitmentcomplexassays, thesplicing substrate was preincubated with the reagents indicated plus the basic components in the splicing reaction (ATP, creatine phosphate, MgCI,, polyvinyl alcohol) unless otherwise specified. After preincubation at 30 C for 1 hr, the reagents indicated at the second addition point were added, and the incubation was continued for another 2 hr. Proteins and Antibodles The antibody for Tra is a mouse polyclonal antibody generated against purified recombinant Tra. The antibody for Tra2 is a mouse MAb generated against purified recombinant Tra2. The antibody for SF2/ASF is a gift from Drs. A. Krainer and A. Mayeda. MAb104 is obtained from the hybridoma provided by Dr. M. Roth. The cdnas for SRpPO and SRp55 were amplified from HeLa cell and Drosophila embryo cdnas. To facilitate detection, the 5 ends of the cdnas of SRpPO and SRp55 were fused to a 10 amino acid myc tag. The SF2/ASF cdna was not modified. The cdnas containing the complete coding regions of SRpPO, SRp55, and SFP/ASF were cloned into the baculovirus expression vector pvl941 (PharMingen), and the recombinant baculoviruses were generated according to the protocols of the manufacturer (PharMingen). The recombinant virus for SC35 was described previously (Fu and Maniatis, 1992a). For protein production, Sf9 cells were infected with individual virus. All the procedures were carried out at 4 C. After 72 hr of infection, the cells were spun down at 2000 rpm (TJ-6 rotor) for IO min and washed once with phosphate-buffered saline. The cells from a 100 ml culture were lysed in 20 ml of buffer A (20 mm Tris-HCI [ph 7.51, 100 mm KCI, 0.2 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 1 mm dithiothreitol [OTT]) by sonication. The cell debris was spun down at 8000 rpm (HB-4 rotor) for 20 min and the supernatant saved. The debris was reextracted with 20 ml of buffer B (20 mm Tris-HCI [ph mm ammonium sulfate, 0.2 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 0.5% p-mercaptol ethanol) for 30 min. The mixture was spun at 6900 rpm (HE-4 rotor) for 20 min. The supernatant was pooled with that from the first spinning. Ammonium sulfate was added to the supernatant to 40% saturation. After stirring for 2 hr, the mixture was spun at 10,000 rpm (HB-4 rotor) for 30 min. The supernatant was loaded onto a 2 ml Phenyl-Sepharose (Pharmacia) column equilibrated in buffer C (20 mm Tris-HCI ]ph 7.51, 0.2 mm EDTA, 1 mm DTT) containing 1.8 M ammonium sulfate. The elutions were carried out in steps using buffer C containing 1.3 M. 1.O M. and 0.5 M ammonium sulfate, respectively. rsr2/asf and rsc35 were eluted in the 1.3 M and 1.O M fractions, and rsrp20 and rsrp55 were eluted in the 1.O M and 0.5 M fractions. The fractions containing SR proteins were dialyzed against BClOO (20 mm Tris-HCI [ph 751,100 mm KCI, 0.2 mm EDTA, 0.5 mm Dll). precipitation (Zahler et al., 1992). As positive controls for the activities of the rsr proteins, they all function in complementing SlOO and switching 5 splice site assays, although rsrp20 displays weak activities in both assays, analogous to the RNA-binding protein RBPl (Kim et al., 1992). GST-R17 was produced by Escherichia coli transformed with pgst-r17. pgst-ri 7 was constructed by cloning the RI 7 coding sequence (Gott et al., 1991) into GST expression vector (pgem-a) (Smith and Johnson, 1988). The transformed E. coli (HBlOl) (800 ml) was grown to OD, = , and isopropyl 6-D-thiogalactopyranoside was added to a final concentration of mm. The cells were induced for 3 hr. The cells were spun down at 4000 rpm (J-6 rotor) and washed with 20 ml of buffer I (50 mm Tris-HCI [ph 6.01, 25% sucrose, 10 mm EDTA). The pellet was resuspended in 20 ml of buffer I. Four milliliters of 20 mglml lysozyme in buffer I were added to the cells, and the digest was incubated on ice for 1 hr. The cells were spun down at 8000 rpm (HE-4 rotor) for 10 min and then lysed by sonication in 20 ml of buffer II (10 mm Tris-HCI [ph 7.51, 1 mm EDTA, 1 mm DlT, 1 mm phenylmethylsulfonyl fluoride, 1 mm benzimidine). The cells were lysed by sonication. KCI and Triton X-100 were added to 100 mm and 0.1% final concentrations, respectively. The debris was pelleted by spinning at 8000 rpm (HB-4 rotor) for 30 min. The supernatant was loaded onto a 2 ml glutathione-agarose column (Sigma) equilibrated with buffer Ill (20 mm Tris-HCI [ph 7.51, 100 mm KCI, 0.2 mm EDTA, 1 mm DTT). GST-R17 was eluted with buffer Ill containing 5 mm glutathione. The eluted GST-R17 was dialyzed against buffer III for 24 hr. Gel Flltratlon Analysis Splicing reactions were scaled up to 200 pl for gel filtration analysis. Polyvinyl alcohol was left out of the reaction, and KCI concentration was as specified. The reactions were incubated at 30 C for 1 hr and then loaded onto a Sephacryl S-500 (Pharmacia) column (1.5 cm x 50 cm). The column was equilibrated and run in 20 mm Tris-HCI (ph 7.5) 60 or 250 mm KCI (as specified), 0.2 mm EDTA, 0.1% Triton X-i 00 at a speed of 7 mllhr. Fractions (1.8 ml) were collected, and 0.2 ml samples of each fraction were counted by Cherenkov counting. RNA8 Dl, D2, D3, D4, D5, and D6 were as described previously (Ran and Maniatis, 1992). D7 was transcribed from T3F260 linearized with Hindlll. T3F260 was constructed by cloning into Bluescript SK(+) vector a 260 bp Fspl fragment of the dsx female-specific exon. The R(+) RNA used in the purification of the dsxrc is transcribed from R(S)- R17 linearized with Bglll. R(S)-R17 was constructed by ligating a 390 bp Mlul-Aflll fragment from the female-specific exon to two copies of the R17-binding site (Bardwell and Wickens, 1990). The fusion construct is contained in the SP73 vector (Promega). The R(-) RNA is transcribed from Afl-RI7 linearized with Bglll. AR-RI7 was constructed by ligating a 380 bp Aflll fragment from the female-specific exon to two copies of the R17-binding site (Bardwell and Wickens, 1990). Afflnlty Purlflcatlon of the dsxrc RNA (4 ug) was incubated with 24 ug of GST-R17 in TMK buffer (0.1 M Tris-HCl [PH 8.01, 10 mm MgC12, 0.1 M KCI) in 25OC for 50 min. A 1:l slurry (1 ml) of glutathione-agarose (equilibrated in TMK buffer) was added to the reaction. Triton X-100 was added to 0.01% final concentration. The mixture was rocked at 4OC for 1 hr. The resin was washed four times with 4 ml each of S buffer (20 mm Tris-HCI [ph 7.51, 250 mm KCI, 3 mm MgCI,, 0.01% Triton X-100). The resin was mixed with a 2 ml splicing reaction. The composition of the splicing reaction is the same as that in normal splicing except that the KCI concentration is 250 mm and the polyvinyl alcohol is omitted. The reaction was rocked at 30 C for 1 hr. The resin was washed eight times with 4 ml each of ST buffer (S buffer plus 0.1% Triton X-100). After washing, 2.5 ml of ST buffer containing 50 mm glutathione was added to the resin, and the mixture was rocked at 4OC for 30 min. The resin was spun down, and the supernatant was saved. The elution was repeated once more. The two elutions were pooled. A I:1 slurry (200 ul) of avidin-agarose (equilibrated in S250: 20 mm Tris-HCI [ph 7.51, 250 mm KCI, 0.2 mm EDTA, 0.1% Triton X-100) was added to

9 y$ive Regulation of dsx Pre-mRNA Splicing the eluant. and the mixture was rocked at 4OC overnight. The resin nuclear ribonucleoprotein particles at the 3 splice site. Proc. Natl. was washed four times with 10 ml each of S250. SDS buffer (0.8 ml) Acad. Sci. USA 89, (20 mm Tris-HCI [ph 7.51, 2% SDS, 5% p-mercaptol ethanol) was Fu, X.-D., Mayeda, A., Maniatis, T., and Krainer, A. (1992). General added to the resin. The mixture was incubated at 85OC for 10 min. splicing factors SF2 and SC35 have equivalent activities in vitro, and The resin was spun down and the supernatant saved. The elution was repeated once more. The supernatants were pooled, and the proteins were precipitated with 4 vol of acetone. The pellet was resuspended in SDS sample buffer and run on SDS-PAGE. Western analysis was done according to standard procedures (Promega). Acknowledgments We thank Adrian Krainer and Akila Mayeda for the SlOO extract and anti-sf2/asf antibody; Robin Reed for the 5 D-16X plasmid; Mark Roth for the hybridoma producing MAb104; Marvin Wickens and Olke Uhlenbeck for plasmids used in the R17 procedure; and Hubert Amrein, James Bruzik, Xiang-Dong Fu, Mary-Lynne Hedley, Edward C. Hsiao, Robin Reed, Tom Schaal, Kristen Wood, and Jane Wu for discussions and comments on the manuscript.this work is supported by a grant from the National Institutes of Health to T. M Received April 6, 1993; revised May 10, References Abmayr, S. M., Reed, R., and Maniatis, T. (1988). Identification of a functional mammalian spliceosome containing unspliced pre-mrna. Proc. Natl. Acad. Sci. USA 85, Amrein, H., Gorman, M., and NISthiger, R. (1988). Thesexdetermining gene tra-2 of Drosophila encodes a putative RNA binding domain. Cell 55, 1025-I 035. Baker, 8. S. (1989). Sex in flies: the splice of life. Nature Baker, B. S., and Ridge, K. A. (1980). 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