Cell, Vol. 62, 25-34,

July 13, 1990, Copyright

0 1990 by Cell Press

A Protein Factor, ASF, Controls Cell-Specific Alternative Splicing of SV40 Early Pre-mRNA In Vitro Hui Ge and James L. Manley Department of Biological Sciences Columbia University New York, New York 10027

Summary SV40 early pre-mRNA is alternatively spliced by utilization of two different 5’ splice sites and a shared 3’ splice site to produce large T and small t mRNAs. The ratio of small t to large T mRNAs produced in human embryonic kidney 293 cells is lo- to 20-fold greater than in other mammalian cells, suggesting the existence of a 293 cell-specific factor that modulates alternative splicing. Here we show that nuclear extracts from 293 cells give rise to significantly more small t splicing than do extracts from HeLa cells. Using an in vitro complementation assay, we have characterized and extensively purified a factor from 293 extracts that brings about striking increases in small t splicing with concomitant decreases in large T splicing. The factor is heat sensitive and micrococcal nuclease resistant, suggesting that it is a protein lacking an accessible RNA component. Purification of the alternative splicing factor indicates that the activity is contained in one of several possibly related polypeptides of 3035 kd. Introduction Alternative splicing of pre-mRNA is a widely used mechanism to increase the coding potential of genes in animal cells and their viruses. Selection of alternative splicing pathways has also been found to be an important regulatory step in the expression of many genes (for reviews see Leff et al., 1986; Breitbart et al., 1987). There are a number of ways that an RNA can be alternatively spliced, which have all been documented. Well-studied examples of each include the following: multiple 5’ splice sites and a single 3’ splice site in the SV40 early (Berk and Sharp, 1978a) and adenovirus Ela (Berk and Sharp, 1978b) premRNAs; a common 5’ splice site and multiple 3’ splice sites, as in the Drosophila rransformer(Boggs et al., 1987) and double-sex (Burtis and Baker, 1989) genes; exon “jumping” in the rat calcitonin/calcitonin gene-related peptide gene (Amara et al., 1982); mutually exclusive exon “skipping” as occurs in the rat a- and j3-tropomyosin genes (Wieczorek et al., 1988; Helfman et al., 1986); and intron “skipping,” as in the Drosophila P element transposase (Laski et al., 1986) and suppressor-of-white-apricot (Chou et al., 1987) genes. While regulation of alternative splicing is in most cases essentially a qualitative type of control, i.e., the amount of mRNA (and protein) ultimately produced is not significantly affected, there are now examples where regulated splicing can function as an on/off switch in gene expression. The best examples of this to

date are in Drosophila, where several genes are known in which either a functional or a nonfunctional mRNA is produced, depending on the alternative splicing pathway utilized (for review see Bingham et al., 1988). The cis-acting sequences required for splicing of simple (nonalternative) introns have been largely determined, and many of the transacting factors that constitute the spliceosome have been identified (for review see Padgett et al., 1986; Green, 1986; Krainer and Maniatis, 1988). Conserved consensus sequences define both the 5’ and 3’ splice sites (Mount, 1982) and mutations in these elements frequently disrupt splicing. The site of lariat branch formation, usually found between 18 and 40 nucleotides upstream of the 3’ splice site, is characterized rn metazoans by a weaker consensus sequence. However, mutations here frequently do not block splicing, but result instead in the use of “cryptic” branch points situated nearby (Padgett et al., 1985; Ruskin et al., 1985). Four snRNPs, Ul, U2, U4/6, and U5, are currently the best characterized of the factors required for splicing (for review see Steitz et al., 1988). Ul and U2 snRNPs interact via mechanisms involving base pairing with the 5’ splice site and lariat branch site, respectively, in yeast (Parker et al., 1987; Seraphin et al., 1988; Siliciano and Guthrie, 1988) as well as in mammals (Zhuang and Weiner, 1986; Wu and Manley, 1989; Zhuang and Weiner, 1989). The U5 snRNP appears to associate in some manner with the 3’splice site (Chabot et al., 1985). This interaction does not involve base pairing and is perhaps mediated instead by a 70-100 kd intron binding protein that may be associated with U5 snRNP (Tazi et al., 1988; Gerke and Steitz, 1986). The polypyrimidine stretch-3’ splice site is also bound by another protein, U2AF, which may function earlier in the splicing reaction, as it appears to be required to stabilize the initial interaction of U2 snRNP with the branch site during spliceosome assembly (Ruskin et al., 1988). In addition, several hnRNP proteins have also been shown to recognize the 3’ splice site (Swanson and Dreyfuss, 1988). Some progress has been made in elucidating &-acting sequences that can influence alternative splicing, and candidate transacting regulators have also been identified. Perhaps not surprisingly, a considerable number of different sequences in pre-mRNAs have been shown to be capable of influencing alternative splicing. These include: largely uncharacterized sequences in exons (Somasekhar and Mertz, 1985; Reed and Maniatis, 1986; Mardon et al., 1987; Helfman et al., 1988; Cooper and Ordahl, 1989; Laski and Rubin, 1989; Hampson et al., 1989; Streuli and Saito, 1989; Nagoshi and Baker, 1990); the nature of the 3’ splice site-pyrimidine stretch (Fu et al., 1988b; Sosnowski et al., 1989; Emeson et al., 1989); sequences between the 3’ splice site and branch point region (Helfman et al., 1990); the branch site region itself (Noble et al., 1988; Reed and Maniatis, 1988; Zhuang et al., 1989); the distance separating the 5’ splice site and branch site (Fu et al., 1987; Smith and NadaCGinard, 1989); and finally, relative strengths of competing 5’splice

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Figure 1. Enhanced clear Extracts

254

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15’S,

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(A) Analysis of RNA products of in vitro splicing. [o-32P]GTP-labeled SV40 early pre-RNA (pSPHd) was synthesized and processed in vitro with either HeLa (HeLa) or 293 cell (293) nuclear extract. RNA was extracted and analyzed on a 5% polyacrylamide-6 M urea gel. M: 32P-labeled Hpall digest of pBR322. Sizes (in nucleotides) are indicated on the left. The structures of products and intermediates are schematically indicated on the right. (The lower half of the gel was exposed four times longer than the top to facilitate detection of the small t intron). (B) Sl nuclease mapping of in vitro splicing products. RNA samples from (A) were hybridized to a 3’ end-labeled DNA probe at 39oC for 4 hr and digested with Sl nuclease at 39X for 60 min. Sl nuclease-resistant DNA fragments were fractionated on a 5% denaturing polyacrylamide gel. M, Hpall-digested pBR322 marker; Cos, Sl analysis of RNA isolated from SV40-transformed Cos monkey cells; HeLa, RNAs processed in HeLa nuclear extract; 293, RNAs processed in 293 nuclear extract; Pre, unprocessed pm-RNA. T and t indicate the protected DNA fragments representing large T and small t mRNA products. (C) Schematic diagrams of the SV40 early region pm-RNA, DNA probe, and the protected fragments corresponding to small t and large T mRNAs. The sizes (in nucleotides) of each species and the positions of the large T and small t 5’ splice sites and the shared 3’ splice site are indicated.

sites (Fu and Manley, 1987; Zhuang et al., 1987). Many of these sequences have been implicated in cell- or tissuespecific splicing control, although how they function is unknown. Several genes that encode frans-acting factors involved in the control of splicing in Drosophila have been isolated. These include the regulatory gene suppressor-of-whifeapricot (Chou et al., 1987) and the sex determination genes transformer (Boggs et al., 1987) sex-lethal (Bell et al., 1988) and transformer-2 (Amrein et al., 1988; Goralski

et al., 1989). Although all of the proteins encoded by these genes share homology with known RNA binding proteins (for reviews see Mattaj, 1989; Bandziulis et al., 1989) the mechanisms by which they function are unknown. The existence of developmental stage-specific snRNAs has also been documented (for review see Mattaj and Hamm, 1989) but no evidence exists that any of these are involved in splicing control. We have used the early pre-mRNA of the DNA tumor virus SV40 as a model to study the control of alternative splicing. The mRNAs for the large T and small t tumor antigens are produced from this precursor by alternative splicing using two 5’ splice sites and a shared 3’ splice site. Our previous studies (Fu and Manley, 1987) have shown that the ratio of small t to large T mRNAs produced in the adenovirus-transformed human embryonic kidney cell line 293 (Graham et al., 1977) is lo- to 20-fold higher than in many other mammalian cells, suggesting the existence of a cell-specific factor(s) that can influence alternative splicing of this pm-mRNA. Consistent with this, the pattern of branch site selection during in vitro splicing of large T pre-mRNA is different in extracts of HeLa and 293 cells (Noble et al., 1987) and mutations in the branch site region show cell-specific effects on alternative splicing in vivo (Fu et al., 1988a; Noble et al., 1989). However, our ability to understand the molecular basis for this cellspecific difference in alternative splicing has been hampered by our inability to detect significant levels of small t splicing in vitro. Indeed, the lack of suitable in vitro assays in any system has largely blocked progress in understanding how alternative splicing is controlled. Here we describe conditions that allow the detection of both small t and large T splicing in vitro. 293 nuclear extracts reproducibly give rise to significantly more small t splicing products than do HeLa extracts. Using an in vitro complementation assay, we have extensively purified a protein from 293 cells that enhances small t splicing in HeLa extracts while simultaneously reducing large T splicing. Results Elevated Small t Splicing Occurs in 293 Cell Nuclear Extracts Previous studies from our laboratory failed to detect significant levels of small t splicing in vitro from precursors containing both the large T and small t Ysplice sites (e.g., Noble et al., 1986). We (Noble et al., 1987) and others (van Santen and Spritz, 1986) observed, however, that deletion of the large T 5’ splice site “activated” small t splicing, which allowed us to characterize in some detail the products and intermediates of small t splicing (Noble et al., 1987, 1988). To identify conditions that would allow more efficient small t splicing from a pre-RNA also competent for large T splicing, we first analyzed the splicing of a mutant pre-RNA, pSVLi, under a variety of conditions. This precursor, which contains an insertion in the pyrimidine stretch at the 3’ splice site, had been shown previously to result in a higher ratio of small t to large T mRNA in vivo (Fu et al., 1988b). Using the conditions described in the Experimental Procedures, significant levels of small t

Alternative

Splicing

Factor

27

from this pre-RNA were detected in vitro (data not shown). We next asked whether a 770 nucleotide wild-type preRNA (pSPHd; see Figure 1C) could also be spliced to both small t and large T RNAs under the conditions established above, and furthermore, whether a difference in the splicing pattern could be detected between HeLa and 293 nuclear extracts. To this end, 32P-labeled pSPHd pre-RNA was incubated in reaction mixtures containing either HeLa or 293 nuclear extract, and the RNA products were extracted and analyzed by denaturing polyacrylamide gel electrophoresis (Figure 1A). Strikingly, while small t products and intermediates were detected in both reactions, the efficiency of small t splicing was considerably higher in the 293 extract. In addition, accumulation of large T products was lower in the 293 extracts than in the HeLa extract. While increased small t splicing in 293 extracts was found to be completely reproducible (see below), the concomitant decrease in large T splicing seen in Figure 1 was somewhat variable. Several lines of evidence demonstrate that the products indicated as small t related were in fact derived from small t splicing. First, both branched and debranched forms of the intron comigrate precisely with previously characterized authentic small t introns (Noble et al., 1987; data not shown). Second, the sizes of the 5’ exon intermediate and spliced RNA product as well as the linearized intron-exon intermediate, which can be detected on longer exposures following debranching, all agree with those calculated for authentic small t products. Finally, nuclease Sl analysis using a 3’ end-labeled DNA probe revealed protected fragments that comigrate with those produced by in vivo SV40 early mRNA (Figure 16). Note again in Figure 16 that significantly more small t products (the Sl analysis detects both 5’ exons and spliced RNA) were formed in 293 extracts than in HeLa extracts. Although cell type-specific differences in alternative splicing in vitro have not been reported previously, such factors as extract (Reed and Maniatis, 1986) and salt (Schmitt et al., 1987) concentrations have been reported to influence splice site selection in vitro. Therefore, despite the fact that the protein and nucleic acid concentrations in the HeLa and 293 extracts used were nearly identical, as were the salt concentrations during both extract preparation and in vitro processing, we wished to rule out the possibility that the observed differences in splicing were due to perhaps trivial factors. We note first that more than 20 separate preparations of HeLa and 293 extracts have been made during the course of these experiments, and results similar to those shown in Figure 1 have been reproducibly obtained. As an example, Figure 2A shows the splicing products obtained using three separate prepThe 293 extracts show arations of HeLa and 293 extracts. roughly 5-fold higher levels of small t splicing than do the HeLa extracts, a difference that corresponds well to that observed in vivo. The results of an experiment in which the concentration of nuclear extract in reaction mixtures was varied is shown in Figure 28. At most, only very low levels of spliced small t mRNA were detected at any concentrasplicing

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Figure 2. Enhanced Small t Splicing in 293 Extracts and Independent of Extract Concentration

Is Reproducible

(A) Three separate preparations of HeLa (lanes l-3) and 293 (lanes 4-6) nuclear extracts were used for in vitro splicing reactions, and products were analyzed as in Figure 1A. (B) In vitro splicing reactions were performed in 25 ul reactions as in Figure lA, using the indicated amounts (in microliters) of HeLa or 293 nuclear extract, Products were analyzed on a 6% denaturing polyacrylamide gel. Cl and C2 on the left designate the intron-exon intermediate and spliced RNA, respectively, produced using a cryptic 5’ splice site upstream of small t (see text).

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Figure 4. Fractionated Small t and Represses

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The in in lane plus lane right

Extract

Enhances

The ASF-containing DEAE-Sepharose fraction isolated from 293 cell nuclear extract was added to either HeLa or 293 extracts for in vitro splicing, and the processed RNAs were analyzed as in Figure 1A. Lane 1, HeLa extract alone; lane 2, 293 extract alone; lane 3, 7 nl of HeLa extract plus 3 11 of DEAE-Sepharose fraction; lane 4, 7 t.tI of 293 extract plus 3 nl of DEAE-Sepharose fraction; lane 5, 3 frl of DEAESepharose fraction.

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Figure 3. 293 Cell Nuclear Facilitate Small t Splicing

ASF from 293 Cells Simultaneously Large T Splicing

Can Complement

HeLa Extracts

to

indicated amounts of HeLa and 293 extract were mixed and used vitro splicing reactions as in Figure 1A. Lane 1, HeLa extract alone; 2, 7.5 ul of HeLa plus 2.5 ul of 293 extract; lane 3, 5 pl of HeLa 5 ul of 293 extract; lane 4, 2.5 ul of H&a plus 7.5 pl of 293 extract; 5, 293 extract alone. Products are indicated schematically on the of the Figure.

found at all but the lowest concentration of 293 extract. In addition to small t and large T-related products, two species indicated as Cl and C2 are present in Figure 28. These represent the intron-exon intermediate and spliced RNA resulting from a splice using a cryptic 5’ splice site 47 nucleotides upstream of the small t 5’ splice site together with the authentic 3’ splice site. This splice has been consistently observed with precursors lacking the large T 5’ splice site (Noble et al., 1988) but for reasons not completely clear, was detected only irreproducibly with wild-type pre-RNA. Unlike small t splicing, the efficiency of this cryptic splice was the same in HeLa and 293 extracts, and accumulation of cryptic products was reduced at higher extract concentrations. We also examined

the effects of variations in MgC12 and KCI concentrations on cell-specific splicing. The results (not shown) indicated that small t splicing in 293 extracts was considerably more efficient than in HeLa extracts under all conditions tested. An Activity in 293 Extracts Simultaneously Enhances Small t Splicing and Represses Large 1 Splicing The above results indicate that a factor(s) present in 293 nuclear extracts can bring about enhanced small t premRNA splicing, reflecting the in vivo situation. Characterization of the factor(s) responsible would be facilitated if the 293 activity were dominant; i.e., if addition of increasing amounts of 293 extract to HeLa extract resulted in a proportional increase in small t splicing. The experiment shown in Figure 3 indicates that this is so. The amount of small t spliced RNA, as well as 5’ exon, increased proportionally to the amount of 293 extract present in the reaction mixtures.

Alternative 29

Splicing

Factor

This complementation assay allowed further characterization of the activity that enhances small t splicing. As a first step, 293 nuclear extract was passed through a DEAE-Sepharose column, as described in Experimental Procedures. All of the activity bound to the column and was eluted in one peak at a moderate salt concentration. Figure 4 shows the effects of this fraction on pSPHd preRNA splicing when added to either HeLa or 293 nuclear extracts. A striking increase in small t splicing was observed. Interestingly, this increase in small t splicing was accompanied by a corresponding decrease in large T splicing. Specifically, accumulation of both the lariat intron and spliced RNA products of the large T splice was substantially reduced. This finding suggests that the 293 activity, which we have tentatively called alternative splicing factor (ASF), does not only function by enhancing small t splicing from otherwise unreacted precursor, but rather somehow influences a competition between small t and large T splicing pathways.

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ASF Is Heat Sensitive and Micrococcal Nuclease Resistant Using the DEAE fraction containing ASF activity, we investigated two properties of the factor: its sensitivities to heat and to micrococcal nuclease (MN). Figure 5A shows the effects of preincubating the ASF-containing activity at various temperatures prior to addition to splicing reactions containing HeLa extract and pSPHd pre-RNA. ASF was resistant to moderate heat treatment (up to 55’%), but was largely inactivated by heating to 70% or higher. A small fraction of activity remained, however, even after heating to 95%. Note that the small t-enhancing and large T-suppressing activities displayed the same heat inactivation profile, supporting the view that they are both due to the same protein-containing factor. To determine whether ASF contains a required RNA component (e.g., an snRNP), ASF was preincubated with increasing concentrations of MN to degrade endogenous RNAs. The nuclease was inactivated by addition of EGTA, and samples were then added to HeLa splicing reactions. The results, shown in Figure 5B, indicate that both the small t-enhancing and large T-repressing activities were resistant to all concentrations of MN tested. The two highest concentrations of MN tested completely degraded all detectable snRNAs present in the ASF fraction, as monitored by ethidium bromide staining of RNAs extracted from aliquots of treated extract (results not shown). We conclude that ASF does not require an accessible RNA component for activity. In-

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5. ASF Is Heat Sensitive

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(A) Heat inactivation. ASF-containing heated at the indicated temperatures

DEAE-Sepharose fraction was for 10 min. In vitro splicing reac-

tions were carried out by mixing 3 ~1 of heated ASF fraction with 7 ul of HeLa extract. The resulting products were analyzed as in Figure 1A. Lane 1, HeLa extract alone: lane 2, HeLa extract plus 3 ul of unheated ASF fraction; and lanes 3-7, HaLa extract plus ASF fraction heated to the indicated temperatures. C’ : spliced product from cryptic 5’splice site as in Figure 28. (8) MN Digestion. Lane 1, pre-RNA was processed with 10 ul of HeLa extract alone; lane 2, 7 ul of HeLa extract plus 3 ul of ASF-containing DEAE-Sepharose fraction; lanes 3-7, 7 ul of HeLa extract plus 4 ul of ASF fraction pretreated with the indicated amounts (units/microliter) of MN; lane 6, HeLa extract plus MN (1 U/uI)-pretreated ASF in the presence of EGTA.

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Figure

7. Analysis

of ASF by Glycerol

Gradient

Centrifugation

ASF fractions from a Mono Q column (see Experimental Procedures) were pooled and further fractionated by glycerol gradient centrifugation. Thirteen fractions were collected, concentrated, and dialyzed against buffer D. ASF activity was assayed by mixing 5 ul of each fraction with 7 pl of HeLa nuclear extract for in vitro processing, and RNA products were analyzed as in Figure IA. HeLa, HeLa nuclear extract alone; H+Mono Q, 7 ul of HeLa extract plus 5 ul of Mono Q fraction; lanes 1-13, pre-RNA was processed in the mixture of 7 pl of HeLa extract and 5 ul of the indicated fractions from the glycerol gradient.

242-

below suggest that ASF is a

t intron (Fu and Manley, 1987), was processed in HeLa extracts with or without added ASF (purified through an additional step, Superose 6 gel filtration; see below). The results, shown in Figure 8, reveal that ASF greatly increased the splicing efficiency of the expanded small t introt-r. Indeed, splicing of the 132 nucleotide small t intron was increased to an even greater level than was the wildtype 66 nucleotide intron. The enhanced splicing of the small t intron brought about by ASF in vitro is therefore not due to the limiting size of the intron, which is completely analogous to the situation in 293 cells in vivo.

ASF Enhances Splicing of an Expanded Small t lntron The small t intron is unusual in that its size (66 nucleotides) approximates the apparent minimum for mammalian introns. Our previous studies showed that mutations that reduced the length of the intron by as little as 2 nucleotides eliminated small t splicing in vivo (Fu et al., 1988a), while mutations that increased its size enhanced small t splicing (Fu and Manley, 1987). However, the limiting size of the small t intron is not related to the increase in small t splicing detected in 293 cells in vivo: mutants with expanded introns displayed the same 293 cell-specific increase in small t splicing as did the wild-type, while deleted introns were not spliced to small t in either cell type. To test whether the factor we isolated from 293 cell nuclear extracts behaves in the same fashion, we asked if ASF could enhance splicing of a pre-RNA containing an expanded small t intron. Specifically, pre-RNA synthesized from pSVi68, which contains a 132 nucleotide small

ASF Is a 30-35 kd Polypeptide To characterize ASF further, the DEAE fraction was subjected to additional purification steps, using the complementation assay described above. The fractionation scheme employed after the initial DEAE step, sequential chromatography on FPLC Superose 6, poly(U)-Sepharose, and FPLC Mono Q, followed by sedimentation through a glycerol gradient is described in Experimental Procedures. Each step provided a significant purification, and in all cases ASF activity was recovered as a single peak. The activities of fractions obtained from glycerol gradient centrifugation are shown in Figure 7. Activity was found predominantly in fractions 8 and 9, with lesser amounts in adjacent fractions. Comparison with protein standards centrifuged in a parallel gradient suggests that the molecular size of ASF is approximately 30-35 kd. A similar size estimate was obtained from gel filtration of the Mono Q fraction on Superose 12 (data not shown). Note that large T splicing was not specifically inhibited in the fractions that enhanced small t splicing. Although we cannot rule

Figure

6. ASF Enhances

Splicing

of an Expanded

Small t lntron

ASF-containing Superose 6 fraction was added to HeLa processing reactions with either wild-type (pSPHd) or mutant (pSVi66) pre-RNAs, and RNA products were analyzed as in Figure IA. M, DNA marker; Pre, pre-RNA; HeLa, the indicated pre-RNA processed in HeLa extract alone; H+ASF, pre-RNA processed in HeLa extract complemented with ASF fraction. The products and intermediates are schematically indicated on the right.

deed, the results described single polypeptide.

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6. 30-35

kd Polypeptides

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Concentrated fractions from a glycerol gradient were subjected to electrophoresis through a 12.5% SDS-polyacrylamide gel and visualized by Coomassie blue staining. Mono 0, Mono Q fraction applied to the glycerol gradient; lanes l-13, corresponding fractions from the glycerol gradient; M, protein size marker. The sizes of marker proteins (in kilodaltons) are shown on the right. Fractions containing peak ASF activity (6 and 9) are indicated by the bracket. The arrow indicates the deduced position of ASF.

out the possibility that these two activities are in fact distinct and were separated in the glycerol gradient, we favor the explanation that large T suppression was masked by a nonspecific inhibition of large T splicing observed with all the gradient fractions. To determine whether any specific polypeptides could be correlated with ASF activity, aliquots of the glycerol gradient fractions were concentrated and analyzed by SDS-polyacrylamide gel electrophoresis. The results, shown in Figure 8, reveal that two to three polypeptides of similar size cofractionated exactly with ASF activity. Given that their size (30-35 kd) agrees precisely with that obtained from sedimentation and gel filtration analyses, and because these are the only species detected in the fraction containing maximal ASF activity (fraction 9) we conclude that at least one of these polypeptides corresponds to ASF Discussion The experiments described above have identified a protein factor that is able to modulate, in a cell type-specific fashion, the alternative splicing of SV40 early pre-mRNA in vitro. This protein, which we have tentatively called ASF, was purified from 293 cell nuclear extracts using an in vitro complementation assay. ASF influences the selection of alternative 5’splice sites in SV40 early pre-mRNA, resulting in an increased utilization of the small t 5’ splice site and decreased use of the large T 5’ splice site. Both native and denaturing size estimates suggest that ASF is an approximately 32 kd polypeptide. Two to three proteins of very similar gel mobilities were detected in our purest

preparations, raising the possibility that ASF may exist in multiple forms. How does ASF function to modulate splice site selection? Although it is clear that further studies are required to understand the underlying mechanism, several points merit discussion. An important question is whether ASF is solely an alternative splicing factor, or whether it might be a general splicing factor that is required for all splices, but can in some way also influence alternative splice site selection. This is at the moment a difficult question to address and will most likely require the production of antiASF antibodies to perform immunodepletion experiments. We do note, though, that ASF activity can be recovered from HeLa cell nuclear extracts by binding to and elution from DEAE-Sepharose (unpublished data), suggesting that ASF is present in HeLa cells, although apparently at a lower level than in 293 cells. If indeed ASF is a general splicing factor, this would indicate that certain types of alternative splicing can be modulated by alterations in the concentration (or activity) of ageneral splicing factor. However, because simply increasing the amount of 293 (or HeLa) nuclear extract in in vitro splicing reactions does not enhance small t splicing, it appears that it is not the absolute amount of ASF that determines the pattern of alternative splicing, but rather the ratio of this factor to other splicing components. This could conceivably provide a subtle mechanism for altering the splicing pattern of a class of pre-mRNAs. On the other hand, ASF may not be required for general splicing, but perhaps functions only to influence alternative splice site selection. It is not yet known whether ASF can affect any types of alternative splicing other than selection of alternative 5’ splice sites. Our previous studies have shown that large T (but not small t) splicing involves the utilization of multiple lariat branch sites, and that the predominant branch site utilized varies in a cell-specific fashion (Noble et al., 1987). Thus, 293 nuclear extracts use predominantly upstream branch sites for large T splicing, while in HeLa extracts the downstream-most branch site, which coincides with the unique small t branch site, is used most frequently. Furthermore, mutations in the upstream branch site region have cell type-specific effects on splicing in vivo (Fu et al., 1988a; Noble et al., 1989). Taken together, these results suggest that ASF may in some manner influence branch site selection, which in turn helps define the 5’ splice site. How ASF might participate in this reaction is not known. Preliminary studies indicate that the purified protein can be cross-linked by UV light to RNA substrates, although whether this cross-linking reflects a sequence-specific interaction is not clear. Does ASF correspond to a previously identified splicing factor? Several snRNP proteins are roughly the same size as ASF (Hinterberger et al., 1983; Bringmann and LOhrmann, 1986), but we consider it unlikely that ASF is one of these, for several reasons. First, ASF activity is found exclusively in the nuclear fraction of cell lysates (unpublished data), while a considerable fraction of the snRNPs are recovered in the cytoplasm during fractionation. Second, during both the Superose 6 gel filtration step and the final glycerol gradient centrifugation, ASF activity is de-

tected only in fractions corresponding to 30-40 kd, inconsistent with snRNP association. Finally, purified ASF fails to react with an anti-Sm monoclonal antibody (unpublished data), which indicates that ASF does not contain the Sm epitope found in several snRNP proteins. We also note that purified ASF is not recognized by anti-hnRNP Al and C protein monoclonal antibodies (unpublished data). Several recent studies have identified non-snRNP-associated proteins that appear to play important roles in mammalian pre-mRNA splicing, and some of these are similar in size to ASP UPAF is a factor initially shown to be required to facilitate stable U2 snRNP binding to premRNA (Ruskin et al., 1988). U2AF has recently been purified to apparent homogeneity and shown to consist of two subunits, a 64 kd protein that contains RNA binding activity and an associated 35 kd protein of unknown function (Zamore and Green, 1989). While it is clear that ASF is distinct from LEAF, it is conceivable that the small subunit of UPAF and ASF are related. A 35 kd protein required for spliceosome assembly has also been identified recently by use of a monoclonal antibody obtained from a panel of antibodies prepared against partially purified spliceosomes (Fu and Maniatis, 1990). Again, the relationship, if any, between this protein and ASF (or UPAF) is not known. Finally, an MN-resistant factor, SF2, was initially identified as an activity recovered in nuclear extracts that could complement a cytoplasmic SlOO fraction to restore efficient splicing (Krainer and Maniatis, 1985). This protein has now been extensively purified, and its size is similar to that reported here for ASF (Krainer et al., 1990). Although we have not found ASF to be able to complement HeLa SlOO extracts (unpublished data), several of the chromatographic properties of ASF and SF2 are similar, and SF2 can also influence selection of 5’ splice sites in a synthetic P-globin pre-mRNA (Krainer et al., 1990). It is thus possible that these two proteins are related or identical. If ASF is indeed equivalent to one (or more) of the above factors, this would provide strong support for the idea, discussed above, that changes in the concentration or activity of a general splicing factor can influence alternative splice site selection in vitro and in vivo. Our immediate goals are to determine the identity and to investigate the mechanism of action of ASF. Experimental

Piasmid

Procedums

Constructions

and Syntheeis

of RNA Pmcurscn

Plasmid pSPHd was described previously (Fu et al., 1966b). pSVi66 was created by inserting a Hindlii fragment (SV40 nucleotides 51714002) from pSTERi66 (Fu and Manley, 1987) into an Escherichia coli RNA polymerase promoter-containing vector pViac (described in Noble et al., 1967). Precursor RNAs were made by in vitro transcription of templates linearized at the unique Styi site at SV40 nucleotide 4410. Capped precursor RNAs were synthesized using SP6 RNA polymerase with m7G(5’)pppG(5’) and [a-32P]GTP (pSPHd) or E. coli RNA polymerase with m7G(5’)pppA(5’) and [a-32P]ATP (pSVi66), and purified on 5% denaturing polyacrylamide gels.

In Vitro Splicing

Aasaya

small t splicing: the final concentrations of HEPES (pH 7.9) and glycerol were reduced to 6 mM and S%, respectively, and the amount of nuclear extract was increased to 40% (v/v). The inclusion of 20 mM KCI, in addition to the (NH&S04 contributed by the dialyzed nuclear extract, was also found to be important for efficient small t splicing. Reaction mixtures routinely contained 10 pl of nuclear extract in a volume of 25 ul. For analysis of 293 fractions by compiementation, 3-5 ul of the appropriate fractions was added to reaction mixtures containing 7 ul of HeLa extract. For MN pretreatment, 30 pi samples of 293 DEAE fraction were treated with increasing amounts of MN, as indicated in the legend to Figure 5, for 30 min at 3O@C in the presence of 1 mM CaCi2. Reactions were stopped by addition of EGTA to 2 mM. Four microliters of each sample was added to 7 ul of HeLa extract for in vitro splicing, 25 ui was deproteinized, and the extent of RNA degradation was analyzed by gel electrophoresis. Heat inactivation of the 293 DEAE fraction was done by heating aiiquots to the temperatures indicated in Figure 5 for 10 min. Products of in vitro splicing reactions were analyzed directly by electrophoresis through 5% polyacrylamide-6 M urea gels. In one case (Figure lB), Sl nuclease analysis of processed RNAs was performed using a 3’end-labeled Hindiil to Bsmi (nucieotides 5171-4526) fragment of SV40 DNA, exactly as described (Fu and Manley, 1967).

Fractionation

of 293 Ceil Nuclear

Extracts

Fractionations routinely began with nuclear extract from 90 liters of 293 ceils. Dialyzed extract (120 ml) (~1.0 g of protein) was first chromatographed on two SO ml DEAE-Sepharose CL-GB columns (Pharmacia), which were preequilibrated with buffer A-50 (20 mM HEPES [pH 7.91, 10% glycerol, 3 mM MgCi2, 0.1 mM EDNA, 0.5 mM dithiothreitol, 05 mM phenytmethyisulfonyi fluoride, and 60 mM (NH&SO.,. After washing with 240 ml of buffer A-50. the columns were eluted with buffer A-250 (containing 250 mM (NH4)2S04). Approximately 200 ml total was collected from both columns and concentrated to 10 ml (20 mg/ml protein) with a CentrifloCF25 (Amicon). Insoluble material was removed by centrifugation at 10,000 rpm for 3 min in a microfuge. An aliquot of the supernatant was dialyzed against buffer D (20 mM HEPES, 20% glycerol, 0.2 mM EDTA, 0.5 mM dithiothreitol, 42 mM (NH&S04) for use in splicing reactions, and the remainder was applied to a 2.6 x 65 cm FPLC Superose 6 column equilibrated with buffer A-200 at a flow rate of 0.6 milmin. Proteins were collected in 7 ml fractions. We note that ASF activity at this stage of purification elutes more rapidly from gel filtration columns than at later stages, perhaps reflecting specific or nonspecific association with RNA or other proteins. ASF-containing fractions (35-36) were concentrated, dialyzed against buffer D, and loaded onto a 10 ml poiy(U)-Sepharose column (Pharmacia) equilibrated with buffer &SO. After washing with 50 ml of buffer A-50, bound material was eluted with buffer A-250 (approximately 10 ml), concentrated to 0.5 ml, and dialyzed against buffer D as above. The resulting ASF-containing dialysate from the poiy(U)Sepharose column was centrifuged for 3 min to remove insoluble material, and the supernatant was applied to an FPLC Mono Q column (HR5/5) equilibrated with buffer A-50 at a flow rate of 0.5 ml/min. After washing with 5 column volumes of the same buffer, proteins were eluted with a 20 ml linear gradient of (NH&SO, from 50-500 mM in buffer A at a flow rate of 0.5 mllmin. One milliliter fractions were collected, concentrated to 0.1 ml, and dialyzed against buffer D. Glycerol gradient sedimentation was carried out by diluting 100 ul of dialyzed Mono Q fractions (6 and 7) with an equal volume of buffer D lacking glycerol, and loading the sample onto the top of a 4.6 ml glycerol gradient (10%-30% v/v) made in buffer A-50. A mixture of protein markers (apoferritin, 440 kd; 5-amyiase, 200 kd; ovaibumin, 44 kd; and myoglobin, 17 kd) was loaded onto a parallel tube for size estimation After centrifugation at 47,ooO rpm at 4°C for 22 hr in an SW50.1 rotor, 13 fractions (0.36 ml each) were collected, concentrated to 0.1 ml, and dialyzed against buffer D. Five microliters from each fraction was used for in vitro splicing reactions and 20 ul was subjected to electrophoresis through a 12.5% polyacrylamide-SDS slab gel. Proteins were detected by staining with Coomassie brilliant blue.

and RNA Anaiyals

HeLa and 293 cell nuclear extracts were prepared, and in vitro splicing reactions were performed, essentially as described previously (Noble et al., 1967). However, the following changes in the composition of the reaction mixture were found to give rise to an increased efficiency of

Acknowledgments We are grateful to X.-Y. Fu, L. Ryner, and J. Noble for helpful discussions during the early stages of this work, to J. Harper and Y. Takagaki

$pive

Splicing

Factor

for many useful suggestions, and to A. Krainer for communicating results prior to publication. We thank S. Pifiol Roma and G. Dreyfuss for anti-hnRNP Al and C protein monoclonal antibodies. We also thank M. Wang for excellent technical assistance, and M. A. Scott for preparing the manuscript, This work was supported by National Institutes of Health grant CA33620. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received

March

20, 1990; revised

April 24, 1990.

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