The SRm160/300 splicing coactivator is required for exon-enhancer function

Abstract

Exonic splicing enhancer (ESE) sequences are important for the recognition of splice sites in pre-mRNA. These sequences are bound by specific serine-arginine (SR) repeat proteins that promote the assembly of splicing complexes at adjacent splice sites. We have recently identified a splicing “coactivator,” SRm160/300, which contains SRm160 (the SR nuclear matrix protein of 160 kDa) and a 300-kDa nuclear matrix antigen. In the present study, we show that SRm160/300 is required for a purine-rich ESE to promote the splicing of a pre-mRNA derived from the Drosophila doublesex gene. The association of SRm160/300 and U2 small nuclear ribonucleoprotein particle (snRNP) with this pre-mRNA requires both U1 snRNP and factors bound to the ESE. Independently of pre-mRNA, SRm160/300 specifically interacts with U2 snRNP and with a human homolog of the Drosophila alternative splicing regulator Transformer 2, which binds to purine-rich ESEs. The results suggest a model for ESE function in which the SRm160/300 splicing coactivator promotes critical interactions between ESE-bound “activators” and the snRNP machinery of the spliceosome.

Pre-mRNA splicing occurs within the spliceosome, a ≈60S complex composed of four small nuclear ribonucleoprotein particles (U1, U2, U4/U6. and U5 snRNPs) and many non-snRNP splicing factors (1, 2). A large number of non-snRNP splicing factors have been identified that contain domains rich in serine-arginine repeats (SR proteins) (3–5). A subgroup of these proteins, the “SR family,” contain one or two N-terminal RNA recognition motifs (RRMs) and a C-terminal domain rich in serine and arginine residues (RS domain) in which many of the serines are phosphorylated. SR family proteins are required for both general and regulated pre-mRNA splicing and function at multiple stages of spliceosome assembly. It is thought that SR family proteins function by promoting interactions with each other and with snRNP-associated proteins containing RS domains (6–8). For example, it has been proposed that splice site recognition and pairing across introns is promoted by a network of interactions involving the association of the SR family proteins SC35 and ASF/SF2 with the U1 snRNP 70-kDa protein at the 5′ splice site and with the U2 snRNP auxiliary factor 35-kDa subunit (U2AF-35kDa) bound at the polypyrimidine tract adjacent to the 3′ splice site (6); both of the latter proteins contain short RS domains. The phosphorylated RS domains of these proteins are most likely required for the protein–protein interactions proposed to be involved in this network (6, 7, 9, 10).

Elevated concentrations of SR family proteins promote the selection of alternative splice sites in vitro (11–14), and in vivo (15–17). SR family proteins, and other RS domain-containing proteins, also function in splice site recognition by interacting with specific intron or exon sequences called “enhancers.” In a prototypic example, regulation of alternative splicing of the Drosophila doublesex (dsx) pre-mRNA, which is part of a cascade of regulatory splicing events that determines the sex of Drosophila, involves the assembly of a multi-SR protein complex on an exonic splicing enhancer (ESE) within exon 4 of the dsx pre-mRNA (18–20). The assembly of this complex, which contains SR family proteins and the RS domain proteins Transformer (Tra) and Transformer 2 (Tra2), promotes the recognition of a weak, upstream, female-specific 3′ splice site, thereby promoting exon 4 inclusion. The dsx ESE can function in heterologous pre-mRNAs and, similarly, it can be replaced functionally by purine-rich ESEs from alternatively spliced mammalian pre-mRNAs (21, 22). Recently, it was demonstrated that hTra2α and hTra2β (23, 24), the human homologs of Drosophila Tra2, preferentially bind to purine-rich ESEs containing GAA repeats and, in conjunction with specific SR family proteins, promote ESE-dependent splicing (25). However, the mechanism by which ESEs promote splice site recognition and splicing through communication with the general splicing machinery is not well understood.

We have previously identified a complex of SR-related nuclear matrix proteins of 160 and 300 kDa (SRm160/300) that is required for the splicing of specific pre-mRNAs (26). SRm160 is an SR repeat protein that lacks an RNA recognition motif and, together with the 300-kDa subunit, associates with pre-mRNA through multiple interactions with factors bound directly to pre-mRNA (26). Here we demonstrate that SRm160/300 is also required for a purine-rich ESE to promote the splicing of a pre-mRNA derived from exons 3 and 4 of the dsx pre-mRNA. This function of SRm160/300 depends on the formation of an early splicing complex containing U1 snRNP and involves interactions between SRm160/300, U2 snRNP, and hTra2β. The results suggest a model for the mechanism by which ESEs function, in which multiple cooperative interactions involving the SRm160/300 splicing coactivator play a critical role.

MATERIALS AND METHODS

Antibodies.

Antibodies used in this study are the murine monoclonals mAb-B1C8 (27), mAb-B3 (28), mAb-104 (29), and the rabbit polyclonals rAb-SRm160 (26) and rAb-SRm300 (unpublished results).

Nuclear Extracts.

HeLa nuclear extracts were prepared essentially as described in ref. 30. Nuclear extracts depleted of SRm160/300, U1 snRNP, or U2 snRNPs were identical to those described in ref. 26. The depletion of snRNPs from nuclear extracts has been described in detail in ref. 31.

Immunoprecipitation Assays.

Immunoprecipitation of splicing complexes was performed as described in ref. 32. The coimmunoprecipitation assays in Figs. 3B and 4 were also performed as described in ref. 32 except for the following changes. Nuclear extract was incubated under standard splicing conditions for 10 min (without the addition of exogenous RNA) before mixing with staphylococcal protein A-Sepharose coupled to antibody. The nuclear extract used in Fig. 4 was simultaneously preincubated with or without DNase-free RNase (Boehringer Mannheim). Preincubation of the extract with the RNase (at a final concentration of 6 units/μl of extract) resulted in the efficient degradation of small nuclear RNAs (snRNAs), as assayed by the analysis of remaining RNA recovered from the treated extract on a denaturing 10% polyacrylamide gel stained with ethidium bromide (data not shown). The immunoprecipitation wash buffers were as described in ref. 32, but contained either 300 mM KCl (Fig. 3B) or 150 mM KCl (Fig. 4). For the coimmunoprecipitation assay in Fig. 4, bound proteins were eluted from protein A-Sepharose by incubating with 100 μl of a 2 M KCl “SR dialysis buffer” [ref. 41; 2 M KCl SRDB: 2.0 M KCl/10 mM Hepes, pH 7.6/1 mM Na₂EDTA/2 mM DTT/5 mM KF/5 mM β-glycerophosphate/0.2 mM Pefabloc (Boehringer)]. The beads were then washed in 100 μl of regular SRDB. The eluate and wash fractions were pooled and incubated with fresh protein A-Sepharose to remove IgG, and remaining protein in the “Ig-cleared” supernatant was recovered by precipitation with trichloroacetic acid.

Figure 3.

Interactions between SRm160/300 and snRNPs in the assembly of dsx splicing complexes. (A) U1 snRNP binds to the dsx pre-mRNA independently of the ESE, whereas the ESE and U1 snRNP are required for the assembly of U2, U4/U6, and U5 snRNPs on the dsx pre-mRNA. Biotinylated dsx pre-mRNAs were incubated in mock-depleted or snRNP-depleted splicing reaction mixtures for 40 min before affinity selection on streptavidin-agarose. RNA recovered from the beads was separated on a denaturing 10% polyacrylamide gel and analyzed by Northern hybridization using riboprobes specific for the five spliceosomal snRNAs. Lanes 3–11 contain RNA recovered after affinity selection with biotinylated dsx pre-mRNAs, and lane 2 contains RNA recovered after a control selection performed in the presence of a nonbiotinylated dsx(GAA)₆ pre-mRNA. Selections were performed using the dsx pre-mRNA with no enhancer sequence (lanes 3, 6, 9), with the (GAA)₃ enhancer (lanes 4, 7, 10), or with the (GAA)₆ enhancer (lanes 5, 8, 11, 12) from splicing reaction mixtures containing “mock-depleted” extract (CtrlΔ, lanes 2–5), U1 snRNP-depleted extract (U1Δ, lanes 6–8), or U2 snRNP-depleted extract (U2Δ, lanes 9–11). The selection in lane 12 was performed from a splicing reaction mixture containing an equal mixture of the U1 and U2 snRNP-depleted extracts. Lane 1 contains RNA recovered directly from nuclear extract, representing approximately 3% of the amount of extract used in each selection. (B) A subpopulation of U2 snRNP associates with SRm160/300 in the absence of exogenous pre-mRNA. RNA recovered after immunoprecipitation with rAb-SRm160 (lane 3) or a corresponding preimmune serum (lane 2) from HeLa nuclear extract (lane 1) was analyzed as in A. The amount of nuclear extract represented in lane 1 corresponds to approximately 5% of the amount used for each immunoprecipitation.

Figure 4.

SRm160/300 interacts with the ESE-binding protein hTra2β. Immunoprecipitates were collected from HeLa nuclear extract by using mAb-B1C8 (lanes 4 and 5) and a control mAb (B3; specific for the hyperphosphorylated large subunit of RNA polymerase II; lane 3), transferred to nitrocellulose, and immunoblotted with an affinity-purified anti-peptide antibody specific for hTra2β. Total nuclear extract separated in lanes 1 and 2 represents approximately 10% of the amount of extract used in each immunoprecipitation. Nuclear extract was preincubated in the presence (lanes 1, 3, and 4) or absence (lanes 2 and 5) of ribonuclease before immunoprecipitation. Size markers (in kDa) and the rabbit IgG (Ig) heavy chain, derived from rabbit anti-mouse antibody used to couple mAbs B1C8 and B3 to protein A-Sepharose, are indicated.

Splicing Complex Selection Assays.

Affinity selection of splicing complexes assembled on biotinylated RNAs was performed under conditions described in ref. 33. Selections were carried out from 25-μl splicing reaction mixtures containing near-saturation levels of (unlabeled) dsx pre-mRNA transcribed in vitro in the presence of biotin-11-UTP (Sigma), using T7 RNA polymerase (Pharmacia) according to the manufacturer’s instructions. The final concentrations of ribonucleotides in the transcription reaction mixtures were 0.075 mM bio-11-UTP, 0.425 mM UTP, and 0.5 mM each of ATP, CTP, and GTP.

Northern Blotting.

Northern blotting was performed with a mixture of [α-³²P]UTP-labeled riboprobes specific for the five spliceosomal snRNAs, as described in ref. 34.

Splicing Assays.

Splicing assays with the dsx pre-mRNAs were carried out essentially as described in ref. 26, using the following conditions: reactions were performed in 15-μl mixtures containing 5 μl of nuclear extract, 0.5 mM ATP, 20 mM creatine phosphate, 3.2 mM MgCl₂, 2 mM DTT, 0.4 units of RNasin (Promega), 60 mM KCl, and 5.6 mM Hepes at pH 7.6 (all final concentrations). The dsx templates were as described previously (22). The splicing reaction products were analyzed on denaturing 7% polyacrylamide gels.

SDS/PAGE and Immunoblot Analysis.

SDS/PAGE and immunoblotting were performed essentially as described in ref. 35. The immunoblot in Fig. 4 was developed by using a secondary antibody conjugated to horseradish peroxidase and chemiluminescence detection (NEN), according to the manufacturer’s instructions.

RESULTS

SRm160/300 Is Required for ESE Function.

To determine whether SRm160/300 is required for ESE-dependent splicing of a pre-mRNA containing a weak 3′ splice site, pre-mRNAs containing sequences from exons 3 and 4 of the dsx gene, with or without an ESE consisting of three or six GAA repeats in exon 4 [dsxΔE, dsx(GAA)₃, and dsx(GAA)₆ pre-mRNAs; kind gift of J. Yeakley and X.-D. Fu (22)], were tested for activity in HeLa nuclear extracts in the presence or absence of SRm160/300 (Fig. 1). Nuclear extracts were specifically immunodepleted of SRm160/300 with a polyclonal antibody specific for SRm160 [rAb-SRm160 (26)] or mock-depleted with the corresponding preimmune serum. These extracts contained normal levels of other SR proteins detected by mAb104 (36) and are identical to the preparations characterized in ref. 26 (see figure 3 in this previous study).

Figure 1.

SRm160/300 is required for ESE-dependent splicing. The splicing activity of a Drosophila doublesex (dsx) pre-mRNA that is dependent on a GAA-repeat ESE in the 3′ exon was assayed in the presence or absence of SRm160/300. Splicing reaction mixtures containing SRm160/300-immunodepleted nuclear extract (lanes 1, 3, and 5), or nuclear extract mock-depleted with preimmune serum (lanes 2, 4, and 6), were incubated for 1 hr with a dsx pre-mRNA with no enhancer sequence (dsxΔE; lanes 1 and 2), with a dsx pre-mRNA containing three GAA repeats [dsx(GAA)₃; lanes 3 and 4], or with a dsx pre-mRNA containing six GAA repeats [dsx(GAA)₆; lanes 5 and 6]. The splicing reaction intermediates and products are indicated on the right of the gel.

Consistent with previous reports, increasing the number of GAA repeats from zero to six resulted in a significant stimulation of dsx pre-mRNA splicing in the mock-depleted extract (Fig. 1; compare lanes 2, 4, and 6). However, specific immunodepletion of SRm160/300 markedly reduced this level of ESE-dependent splicing (compare lanes 5 and 6). This loss of splicing activity was not due to a nonspecific effect, since splicing activity in the SRm160/300-depleted extract can be restored by addition of highly purified SRm160/300 proteins (26). Moreover, a longer exposure of the gel in Fig. 1 revealed that depletion of SRm160/300 resulted in little to no inhibition of the low levels of splicing of the dsxΔE and dsx(GAA)₃ pre-mRNAs (compare lanes 3 and 4, data not shown). These results indicate that the ESE-dependent splicing of the dsx pre-mRNA requires SRm160/300, whereas the low level of constitutive splicing of this substrate in the absence of an efficient ESE is not dependent on SRm160/300.

The ESE Promotes the Recruitment of SRm160/300 to the dsx pre-mRNA.

To investigate the mechanism by which SRm160/300 promotes the ESE-dependent splicing of the dsx pre-mRNA, it was next determined if the ESE mediates the association of SRm160/300 with the pre-mRNA (Fig. 2). To determine whether SRm160/300 specifically associates with factors bound directly to the ESE, immunoprecipitations were performed with antibodies that are highly specific for either SRm160 [mAb-B1C8 (27)] or SRm300 (rAb-SRm300; unpublished work) from splicing reactions incubated with short RNAs consisting of (GAA)₁₂ or (GUU)₁₂ repeats (Fig. 2A; kind gift of J. Yeakley and X.-D. Fu). An equal amount of the dsxΔE pre-mRNA was included in these reaction mixtures as an internal control for recovery. Both antibodies immunoprecipitated the (GAA)₁₂ RNA, but not the (GUU)₁₂ RNA (compare lanes 8 and 9, 10 and 11). Although the (GUU)₁₂ RNA is less stable in nuclear extract than the (GAA)₁₂ RNA (compare lanes 4 and 5), quantitation of the amounts of RNA recovered in the pellets vs. totals demonstrated that both antibodies immunoprecipitated approximately 6-fold more of the (GAA)₁₂ RNA than the (GUU)₁₂ RNA (data not shown). Moreover, this difference was not due to nonspecific losses, because an equivalent level of the dsxΔE pre-mRNA was immunoprecipitated by both antibodies from the different reaction mixtures. The level of immunoprecipitation of the (GAA)₁₂ RNA was significantly lower (by 4- to 5-fold) than that observed for the dsx(GAA)₆ pre-mRNA by the same antibodies (e.g., compare lanes 3 and 8 in Fig. 2B; data not shown). These results indicate that SRm160/300 can specifically associate with GAA-repeat sequences. However, other sequence elements are also required for its stable recruitment to the pre-mRNA.

Figure 2.

ESE-dependent association of SRm160/300 with the dsx pre-mRNA. (A) Association of SRm160/300 with GAA repeats. Equal amounts of the dsxΔE pre-mRNA and a (GAA)₁₂ or (GUU)₁₂ RNA (lanes 2 and 5) were incubated together in nuclear extract under splicing conditions for 40 min. Complexes assembled on these RNAs were immunoprecipitated with mAb-B1C8 or rAb-SRm300. The input RNAs are shown in lanes 1–3. RNA recovered directly after incubation in nuclear extract is shown in lanes 4–6, and RNA recovered after immunoprecipitation is shown in lanes 7–11. Immunoprecipitations were performed with a nonspecific control Ab (rabbit anti-mouse, lane 7; the corresponding input and total are in lanes 3 and 6, respectively), mAb-B1C8 (lanes 8 and 9), or rAb-SRm300 (lanes 10 and 11). The corresponding inputs for the latter two sets are in lanes 1 and 2, and the corresponding totals are in lanes 4 and 5. The RNA samples were separated on a denaturing 15% polyacrylamide gel. Loading was 12.5% of the total amount of RNA from the inputs and totals and 50% of the total amount of RNA recovered from the pellets (Pels). (B) The association of SRm160/300 with the dsx pre-mRNA requires U1 snRNP in addition to ESE-bound factors. Immunoprecipitations were performed with mAb-B1C8 (lanes 6–8, 15–17, 24–26) or rAb-SRm300 (lanes 9–11, 18–20, 27–29) from sets of splicing reaction mixtures incubated for 40 min containing either a control (mock-depleted) nuclear extract (lanes 1–11), a U1 snRNP-depleted nuclear extract (lanes 12–20), or a U2 snRNP-depleted nuclear extract (lanes 21–29). Each set of reactions was incubated with the three dsx pre-mRNAs described in Fig. 1, as indicated above the gel. RNA recovered directly from splicing reactions (lanes 1–4, 12–14, 21–23) and RNA recovered after immunoprecipitation (lanes 5–11, 15–20, 24–29) was separated on a denaturing 7% polyacrylamide gel. The amounts of RNA loaded are as described for A. A control immunoprecipitation was performed with preimmune serum (lane 5) from a reaction mixture containing mock-depleted nuclear extract and the dsx(GAA)₆ pre-mRNA. The corresponding total is shown in lane 4.

U1 snRNP and the ESE Function Together to Recruit SRm160/300 and U2 snRNP to the dsx pre-mRNA.

In addition to interactions mediated directly through the ESE, the stable association of SRm160/300 with the dsx pre-mRNA could also involve more indirect interactions mediated through the formation of one or more snRNP-containing splicing complexes. To determine whether this is the case, the association of SRm160/300 with the dsx pre-mRNAs was assayed in splicing reaction mixtures depleted of U1 or U2 snRNPs (Fig. 2B).

Consistent with the results in Fig. 2A, in a mock-depleted reaction mixture, antibodies to SRm160 (mAb-B1C8) and SRm300 (rAb-SRm300) immunoprecipitated increasing levels of dsx pre-mRNA as the numbers of GAA repeats in the ESE increased (Fig. 2B; e.g., compare lanes 6–8; data not shown). However, neither antibody immunoprecipitated the dsx pre-mRNA, with or without an ESE, in the absence of U1 snRNP (Fig. 2B, lanes 17 and 20). In the absence of U2 snRNP, both antibodies also immunoprecipitated increasing levels of pre-mRNA as the number of GAA repeats increased (compare lanes 24–26 and 27–29); however, these levels of pre-mRNA immunoprecipitation in the U2 snRNP-depleted reaction mixture were not as high as the levels observed in a mock-depleted splicing reaction mixture containing both U1 and U2 snRNPs (compare lanes 26 and 29 with lanes 8 and 11). Immunoprecipitation could be fully restored by mixing of the U1- and U2-depleted extracts, indicating that the reduced levels observed in the snRNP-depleted extracts were not due to a nonspecific effect (see Fig. 3A, lane 12; data not shown). These results indicate that both U1 snRNP and factors bound to the ESE are required to promote the stable association of SRm160/300 with the pre-mRNA, whereas U2 snRNP is not absolutely required but further stabilizes the SRm160/300 association.

To determine whether binding of U1 snRNP to the dsx pre-mRNA and factors to the ESE are independent or interdependent events required for the recruitment of SRm160/300, the ability of U1 snRNP to bind to the three dsx pre-mRNAs was analyzed by using a splicing complex affinity-selection assay (Fig. 3A; ref. 33). Complexes assembled on biotinylated dsx pre-mRNAs in the different snRNP-depleted nuclear extracts used in Fig. 2B were selected on streptavidin agarose beads, eluted, and analyzed for their snRNP composition by Northern hybridization using snRNA-specific riboprobes (refer to Materials and Methods).

U1 snRNP binds efficiently and at an approximately equivalent level to all three dsx pre-mRNAs in the mock-depleted and U2 snRNP-depleted reaction mixtures (Fig. 3A, compare lanes 3–5 and 9–11). This observation shows that binding of U1 snRNP to the dsx pre-mRNA occurs independently of the presence of an ESE. Therefore, binding of U1 snRNP, although required, is not sufficient for the recruitment of SRm160/300 to the dsx pre-mRNA; binding of factors to the ESE is also important, consistent with the specific association of SRm160/300 with GAA repeats observed in Fig. 2A. Moreover, consistent with the increased levels of splicing promoted by the ESE in the mock-depleted extract, binding of U2, U4/U6, and U5 snRNPs to the pre-mRNA is promoted by increasing numbers of GAA repeats only in this extract (compare lanes 3–5 with 6–11).

An Association Between SRm160/300 and U2 snRNP.

Interestingly, depletion of U1 snRNP prevented not only the association of SRm160/300 but also the binding of U2 snRNP to the dsx pre-mRNA, even in the presence of six GAA repeats (Fig. 3A, lanes 6–8). This observation indicates that U1 snRNP and ESE-bound components cooperate to recruit both SRm160/300 and U2 snRNP to the dsx pre-mRNA. By contrast, as described above, depletion of U2 snRNP resulted in a partial loss of the SRm160/300 association with the GAA repeat-containing pre-mRNAs (Fig. 2B, lanes 24–29). Similarly, depletion of SRm160/300 resulted in a partial loss of binding of U2 snRNP to the (GAA)₆ repeat dsx pre-mRNA (data not shown).

Given the dependence on U1 snRNP and the partial dependence on U2 snRNP for the association of SRm160/300 with pre-mRNA (Fig. 2B), and the parallel entry of these latter components into splicing complexes (Fig. 3A), it was next determined whether SRm160/300 interacts with U1 and U2 snRNPs. rAb-SRm160 immunoprecipitates prepared from HeLa nuclear extract were probed for the five spliceosomal snRNAs (Fig. 3B). rAb-SRm160 did not immunoprecipitate U1 snRNP, again indicating that one or more factors that associate with U1 snRNP after its entry into splicing complexes promotes the association of SRm160/300. However, rAb-SRm160, but not a corresponding preimmune serum, specifically immunoprecipitated a low level of U2 snRNP from nuclear extract (compare lanes 2 and 3). This immunoprecipitation was not prevented by masking U2 snRNA with an antisense oligonucleotide complementary to the branch site-pairing region (data not shown), indicating that it is not mediated through the binding of U2 snRNP to RNA in the extract. These results suggest that U2 snRNP and SRm160/300 can associate, consistent with the proposal that they form mutually stabilizing interactions within splicing complexes.

SRm160/300 Interacts with the ESE-Binding Protein hTra2β.

In a recent study it was shown that two human homologs of the Drosophila alternative splicing regulator Transformer-2, hTra2α and hTra2β, bind to ESEs containing GAA repeats (25). Unlike SR family proteins, the hTra2 proteins contain a single RNA recognition motif located between two RS domains (23, 24). The hTra2 proteins are present in HeLa nuclear extracts and are both detected as an ≈40-kDa species in SDS/polyacrylamide gels by mAb104 (25). It has been observed that rAb-SRm160 and mAb-B1C8 specifically coimmunoprecipitate a subset of SR proteins detected by mAb104, including 75- and 40-kDa species (ref. 26; Y.L. and B.J.B., unpublished observations). To determine whether this 40-kDa species corresponds to one of the hTra2 proteins, immunoprecipitates from HeLa nuclear extract were probed with an affinity-purified anti-peptide antibody specific for hTra2β (kind gift of R. Tacke and J. Manley; Fig. 4). mAb-B1C8, but not a control mAb, specifically immunoprecipitated hTra2β (compare lanes 3 and 4). This coimmunoprecipitation of hTra2β by mAb-B1C8 was resistant to extensive preincubation of the nuclear extract with RNase (compare lanes 4 and 5; refer to Materials and Methods), indicating that the association between hTra2β and SRm160/300 is mediated by protein–protein interactions and is not “tethered” by endogenous RNA in the nuclear extract. Probing of the same immunoblot with mAb104 revealed a comparable level of enrichment for the mAb104-reactive 40-kDa antigen(s), indicating that a significant fraction of the 40-kDa protein(s) immunoprecipitated by mAb-B1C8 corresponds to hTra2β (data not shown). Similar coimmunoprecipitation results were obtained with rAb-SRm160 (data not shown). We conclude that SRm160/300 interacts with the ESE-binding protein hTra2β and that this interaction, in conjunction with additional interactions involving U1 and U2 snRNPs and SR proteins, is critical for the promotion of splicing by an ESE containing GAA repeats.

DISCUSSION

SRm160/300 is required for a GAA-repeat ESE to promote the splicing of a pre-mRNA derived from the Drosophila doublesex (dsx) gene, which contains a weak 3′ splice site. The association of SRm160/300 with this pre-mRNA requires the formation of a splicing complex containing both U1 snRNP and factors bound to the ESE. The detection of specific interactions between SRm160/300, U2 snRNP, and the ESE-binding SR domain protein, hTra2β, indicates that SRm160/300 promotes ESE-dependent splicing by mediating critical interactions between U1 snRNP bound to the 5′ splice site, hTra2β bound to the ESE, and U2 snRNP bound to the pre-mRNA branch site. These results support a model in which SRm160/300 functions as a coactivator of ESE-dependent splicing by bridging between “basal” snRNP components of the spliceosome and SR protein “activators” bound to an ESE (Fig. 5).

Figure 5.

Model for the role of the SRm160/300 splicing coactivator in ESE function. Interactions involving the binding of hTra2β to a GAA-repeat ESE and U1 snRNP to the 5′ splice site are required to recruit SRm160/300 to the pre-mRNA. Since neither of these interactions alone is sufficient for SRm160/300 recruitment, it is proposed that interactions mediated by one or more SR proteins between SRm160/300 and U1 snRNP, and between SRm160/300 and the ESE, promote spliceosome formation and splicing. These interactions simultaneously promote the binding of U2 snRNP to the pre-mRNA, which also interacts with SRm160/300. It is proposed that these interactions promote the pairing of specific pairs of exons during the regulation of splice site selection.

These results demonstrate the critical importance of multiple cooperative interactions involving U1 snRNP and SR proteins in the recruitment of SRm160/300 to an ESE-dependent pre-mRNA. The association of SRm160/300 with GAA repeats was specific, but weak, in the absence of other pre-mRNA sequences. Moreover, the association of SRm160/300 with the dsx pre-mRNA containing an ESE with six GAA repeats in the present study was prevented by the depletion of U1 snRNP and was weak in the absence of the ESE. U2 snRNP has similar factor requirements for recruitment to the pre-mRNA. Thus U1 snRNP, in addition to promoting the stable association of SRm160/300, is also required for the stable binding of U2 snRNP to an ESE-dependent substrate. SRm160/300 does not detectably interact with U1 snRNP in the absence of pre-mRNA. Therefore, it is likely that one or more factors that associate with pre-mRNA after the binding of U1 snRNP to the 5′ splice site and the binding of factors to the ESE recruit both SRm160/300 and U2 snRNP.

The specific interaction detected between SRm160/300 and U2 snRNP is consistent with the parallel entry of these components into splicing complexes. The observation that depletion of either SRm160/300 or U2 snRNP weakens but does not prevent the association of the other component with the pre-mRNA provides evidence that these components can also interact with splicing complexes independently (this study; A.G.E., P.A.S., and B.J.B., unpublished observations). The interactions between SRm160/300, U2 snRNP, and ESE-bound components detected in this study may occur in conjunction with interactions previously proposed to be important for ESE function. These include interactions involving the RS domains of specific SR family proteins, such as ASF/SF2, which binds to purine-rich ESEs, and of U2AF-35kDa, which binds to the polypyrimidine tract through U2AF-65kDa (37). However, very recent evidence suggests that this latter interaction is not required for ESE function, since binding of U2AF-65kDa to ESE-dependent pre-mRNAs occurs with equal efficiency in the presence or absence of an ESE (ref. 38; Y.L. and B.J.B., unpublished observations), and ESE-dependent splicing can function in the absence of U2AF-35kDa in vitro (38). Moreover, it has also been observed that the RS domain of the Drosophila homolog of U2AF-35kDa is dispensable for the regulation of dsx pre-mRNA splicing and viability (39). Taking these results together with the results in the present study, we conclude that SRm160/300 has a more direct role than U2AF in mediating cross-intron interactions required for ESE-dependent splicing.

The requirement of SRm160/300 for the ESE-dependent recognition of a weak 3′ splice site is consistent with its having a critical role in the regulation of splice site selection. SRm160/300 may promote the joining of specific pairs of exons dependent on GAA-repeat enhancer sequences. However, other ESE sequences that promote splicing through interactions involving specific SR family proteins (40) could also require SRm160/300. We have recently observed that SRm160/300 is required for splicing in vitro of a subset of pre-mRNAs that contain relatively strong splice sites, but are not dependent on a GAA-repeat ESE (26). It is possible that these substrates contain distinct ESE sequences that promote splicing through SRm160/300.

In summary, the SRm160/300 complex functions in the ESE-dependent splicing of a dsx pre-mRNA by forming multiple interactions with factors bound directly to the ESE and snRNPs bound at splice sites. Since the RS domains of different SR proteins are known to interact (6, 7, 9, 10), the large size and SR-rich nature of the SRm160 and SRm300 proteins are consistent with their proposed role in the formation of multiple interactions with other RS domain proteins bound to pre-mRNA (Fig. 5). The ratio of SRm160/300 to other specific RS domain proteins could regulate the selection of splice sites in alternatively spliced pre-mRNAs, as well as influence the activity of constitutively spliced pre-mRNAs (26). Moreover, the abundant consensus phosphorylation sites and observed phosphorylation of SRm160 and SRm300 (41) indicate that the regulation of splicing by differential phosphorylation mechanisms could be influenced by kinases and phosphatases that target SRm160/300. Finally, the stable association of SRm160/300 with the nonchromatin “matrix” of the nucleus indicates that it may function in association with this substructure in vivo (32). In particular, it is possible that SRm160/300 functions as a substructure on which specific pairs of splice sites are juxtaposed after their initial recognition by U1 snRNP and SR proteins.

ABBREVIATIONS

ESE

exonic splicing enhancer

snRNP

small nuclear ribonucleoprotein particle

snRNA

small nuclear RNA

SR

serine-arginine

SRm160/300

complex containing the SR matrix proteins of 160 and 300 kDa

RS domain

C-terminal domain rich in serine and arginine residues

U2AF

U2 snRNP auxiliary factor

U2AF-35kDa

U2AF 35-kDa subunit