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.

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.

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.

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.

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.