separation of splicing factor sf3 into two components and

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 268, No. 23, Issue of August 15, pp. 17640-17646,1993 Printed in U. S.A.

Separation of Splicing Factor SF3 into Two Components and Purification of SF3a Activity*

(Received for publication, March 15, 1993)

Reto BrosiS, Hans-Peter Hauris, and Angela KramerSV From t h SDipartement de Biologie Cellulaire, Sciences III, Uniuersite de Geneue, CH-1211 Geneve 4, and the SAbteilung Pharmakologie, Biozentrum der Uniuersitat, CH-4056 Basel, Switzerland

Components required for the splicing of nuclear messenger RNA precursors in vitro have been isolated from HeLa cells. Here we describe the separation of splicing factor SF3 into two components, SF3a and SF3b. Both activities are required together with several other protein factors and U1 and U2 small nuclear ribonucleoproteins for the assembly of a presplicing complex which represents the first ATP-dependent step in the assembly of the active spliceosome. SF3a has been purified to homogeneity by a combination of ion-exchange chromatography, gel filtration, and glycerol gradient sedimentation. It consists of a complex of three polypeptides of 60, 66, and 120 kDa. The association of SF3a activity with these polypeptides has been confirmed by immunoprecipitation and depletion experiments using a monoclonal antibody directed against the 66-kDa subunit.

The splicing of nuclear messenger RNA precursors occurs within large multicomponent complexes, termed spliceosomes, that are assembled in a stepwise fashion by interactions between pre-mRNA,’ snRNPs, and a number of non- snRNP protein factors (for reviews see Refs. 1-3). In an ATP- independent reaction U1 snRNP associates with the pre- mRNA generating complex E that is committed to the splicing pathway. After binding of U2 snRNP, which requires energy in the form of ATP, presplicing complex A is formed. U4/U6 and U5 snRNPs join this complex as a preformed triple snRNP to give rise to complex B and, after a conformational change, the active spliceosome (complex C) is generated in which the pre-mRNA is processed in two transesterification reactions. The conservation of these reactions between nuclear pre-mRNA splicing and the autocatalytic removal of group I1 introns have led to the proposal that the catalysis of nuclear pre-mRNA splicing is RNA-based (4-6). The exist- ence of extensive base pairing interactions between pre- mRNA and snRNAs as well as intermolecular base pairing

*This work was supported by grants from the Schweizerischer Nationalfonds and the Kantons of Basel-Stadt, Basel-Landschaft, and Geneva (to A. K.). 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.

7 To whom correspondence should be addressed DBpartement de Biologie Cellulaire, Sciences 111, Universitk de Genive, 30 quai Er- nest-Ansermet, CH-1211 Genive 4, Switzerland. Tel.: 41-22-702- 6750; Fax: 41-22-781-1747.

The abbreviations used are: pre-mRNA, nuclear messenger RNA precursor; snRNA, small nuclear RNA snRNP, small nuclear ribo- nucleoprotein particle; AdML, adenovirus 2 major late; mAb, monoclonal antibody.

between different snRNAs during spliceosome assembly have lent further support to this hypothesis (see Refs. 7 and 8 for references and discussion). Thus, the spliceosomal snRNPs appear to play a major role in the alignment of the splice sites and most likely also in the catalysis of the two transesterification reactions that generate spliced mRNA (9).

In addition to snRNPs, however, a number of proteins are essential for splicing to proceed. The complexity of proteins participating in the overall reaction is reflected in the large number of complementation groups in Saccharomyces cerevis- i u e that encode proteins essential for splicing (see Ref. 3 for review). Moreover, between 30 and 50 polypeptides are associated with splicing complexes in mammalian cells (10, 11). Several of these proteins have been identified as hnRNP proteins (that bind to the pre-mRNA independently of the presence of splice sites (12)) or snRNP-associated polypeptides (11). Whereas the role of hnRNP proteins during split- ing remains to be established, some of the polypeptides that are characteristic for particular snRNPs appear to have essential functions during spliceosome assembly (13, 14).2*3

Several proteins involved in splicing have been characterized and purified from mammalian cells. Most is known about proteins that participate in the early events of the splicing reaction, i.e. in the formation of the ATP-dependent presplicing complex. U2AF consists of two subunits of 65 and 35 kDa, the larger one of which binds to the polypyrimidine tract upstream of the 3’ splice site and is required for the U2 snRNP/branch site interaction (15,16). Moreover, U2AF has been detected in the ATP-independent complex E (11). SC35 (35 kDa) was first identified with a monoclonal antibody raised against purified spliceosomes (17) and is required for the initial binding of U1 snRNP to the pre-mRNA (18). ASF/ SF2 (33 kDa) plays a role in the selection of alternative splice sites in addition to its function in presplicing complex formation (19-21). Both SC35 and ASFISF2 are members of the SR (serine-arginine) family of splicing factors (22, 23). Other less well characterized proteins include SF1 (241, SF3 (251, HRF (26), and an 88-kDa protein (27). Furthermore, SF4 has been shown to function in the conversion of complex B into the active spliceosome C (28) and two activities appear to be required for the second cleavage/ligation reaction, but are dispensable at earlier steps (29).

In an attempt to identify and characterize essential splicing components we have extensively fractionated HeLa cell nuclear extracts. We have previously shown that a minimum of five fractions containing protein factors SF1, SF2, SF3, SF4, and U2AF as well as two fractions that are enriched in the

R. Brosi, K. Groning, S. Behrens, R. Luhrmann, and A. Kramer,

S. Behrens, P. Legrain, F. Galisson, and R. Luhrmann, submitted submitted for publication.

for publication.

17640

Purification of Splicing Factor SF3a 17641

spliceosomal snRNPs are required for in uitro splicing (25, 30). Of these, SF1, SF3, UBAF, and snRNPs suffice for the assembly of presplicing complex A (25). SF1 has recently been purified to homogeneity and is represented by a 75-kDa heat- stable polypeptide (24). Here we report the separation of SF3 into two distinct activities, SF3a and SF3b, that are both required for complex A formation. SF3a, which has been purified to homogeneity, consists of three polypeptides of 60, 66, and 120 kDa.

EXPERIMENTAL PROCEDURES

Purification of SF3a-Chromatographic materials were purchased from Pharmacia LKB Biotechnology Inc. except for spermine-agarose which was from Sigma. All steps were performed at 4-10 "C. After each purification step fractions were dialyzed against buffer BH (20 mM Hepes-KOH, pH 7.9; 20% (v/v) glycerol; 3 mM MgC1,; 0.2 mM EDTA; 0.5 mM dithiothreitol) and tested for activity in presplicing complex formation.

SF3 was separated from splicing factors SF1, SF2, SF4, UZAF, and snRNPs by chromatography on DEAE-Sepharose Fast Flow, heparin- Sepharose and Mono Q as previously described (25, 30). Mono Q fractions containing SF3 activity were applied to a spermine-agarose column (175 x 16 mm, 0.5 mg of protein/ml column volume, flow rate 50 ml/h) equilibrated in buffer AT (50 mM Tris-HC1, pH 7.9; 10% (v/v) glycerol; 3 mM MgCl,; 0.2 mM EDTA; 0.5 mM dithiothreitol; 0.5 mM phenylmethylsulfonyl fluoride; 1 pg of leupeptin/ml) + 0.1 M KC1. After washing with two volumes of the same buffer, bound proteins were eluted with 0.3, 0.6, and 1 M KC1 in buffer AT. The 0.3 M KC1 step fraction that contained SF3 was loaded onto a 1-ml Mono S column equilibrated with buffer AH (as buffer AT but containing 20 mM Hepes-KOH, pH 7.9, instead of Tris-HC1) + 0.1 M KC1 (17.5 mg of protein/ml column volume, flow rate 48 ml/h). After removal of unbound material, bound proteins were eluted with a 30-ml linear salt gradient from 0.1 to 0.5 M KC1 in buffer AH. This step separated SF3 into two activities. SF3a does not bind to Mono S under the conditions used, whereas SF3b binds and elutes between 0.2 and 0.35 M KC1.

SF3a was further purified by chromatography of the Mono S flow- through on heparin-Sepharose (38 X 10 mm, 0.15 mg of protein/ml column volume, flow rate 9 ml/h). After washing with buffer AH + 0.1 M KC1, the column was developed with a 30-ml linear gradient from 0.1 to 0.5 M KC1 in buffer AH. SF3a activity eluted between 0.22 and 0.33 M KCl. Peak fractions were pooled, and 2 ml were loaded onto a Superose 12 column (505 X 16 mm, flow rate 50 ml/h) in buffer AH + 0.1 M KCl. SF3a activity eluted with an apparent molecular mass of about 400 kDa and a Stokes radius of 61 A, as determined by comparison with molecular mass standards (Pharma- cia). Finally, 200 pl of a Superose 12 fraction were sedimented through a 15-30% (v/v) glycerol gradient (4.2 ml in buffer AH + 0.1 M KCl) for 15 h at 50,000 rpm in a TST.60 rotor (Kontron; 255,000 X g). Fractions of 200 pl were collected from top to bottom. By comparison to protein standards separated in parallel gradients the sedimentation constant of SF3a was determined to be 5.5 S.

Purification of Other Proteins"U2AF was purified as described previously (16, 25).

PTB/hnRNP I (31, 32) was purified as a side product during the isolation of splicing factor SF1. In brief, HeLa cell nuclear extracts were fractionated through the Mono S column as described (24). Mono S fractions containing PTB/hnRNP I were applied to poly(U)- Sepharose in buffer AH + 0.05 M KCl. Proteins were eluted from the column with a linear salt gradient of 0.05-0.4 M KC1 followed by step elution with 1 M KC1 in buffer AH. PTB eluted as a characteristic triplet of polypeptides of 56-57 kDa between 0.3 and 0.7 M KCl.

HnRNP U (33) was obtained as a side product during the isolation of U2AF (see above). The protein was detected by Western blotting with anti-hnRNP U antibodies (provided by S. Piiiol-Roma and G. Dreyfuss) in poly(U)-Sepharose fractions eluted between 0.5 and 1 M KC1. It was further purified by Mono S chromatography where it eluted around 0.5 M KCl.'

Presplicing Complex Assembly-Preparation of the AdML pre- mRNA substrate and analysis of presplicing complex formation were performed as described (24, 34). A standard reaction contained 0.1 pl

' A. K r h e r , unpublished results.

of SF1 (ammonium sulfate-concentrated DSlOO), 1 pl of U2AF (HSlOOO), 0.5 pl each of Mono Q fractions enriched in U1 and U2 snRNPs, and 3 p1 of SF3 or the amounts of SF3a and SF3b (Mono S-bound fraction) indicated in the figure legends. Incubation was for 30 min at 30 "C.

Production of Monoclonal Antibodies-A female BALB/c mouse was immunized intracutaneously with 50 pg of SF3a (Superose 12 fraction) emulsified with complete Freund's adjuvant. A second injec- tion of 50 pg of SF3a in incomplete Freund's adjuvant was given 30 days later. After another 30 days 25 pg of SF3a (in buffer BT) was applied intraperitoneally. Three days later spleen cells were fused to PA1 (35) cells and plated into ten 96-well culture plates containing peritoneal macrophages (36). Hybridomas were screened in a dot-blot assay with 20 ng of SF3a spotted onto nitrocellulose filters. Positive clones were detected with horse radish peroxidase-coupled anti-mouse antibodies (Cappel) and staining with chloronaphthol. Two hybridoma lines tested positive against the 66-kDa subunit of SF3a and one (mAb 66) was successfully cloned.

IgG was purified from tissue culture supernatants by ammonium sulfate precipitation (50% saturation) and protein A-Sepharose chromatography. Although the monoclonal antibody was of the IgGl subtype (as determined by Ouchterlony double immunodiffusion using isotype-specific anti-mouse antibodies), it was eluted from protein A-Sepharose between two steps with 0.1 M sodium citrate buffer, pH 4.5 and 3.0.

Western Blot Analysis-Proteins were transferred from 7.5% SDS- polyacrylamide gels (37) onto nitrocellulose (38). The filter was incubated with mAb 66 cell culture supernatant, horseradish peroxidase-coupled rabbit anti-mouse I& (1:2000 dilution, Dako-Immu- noglobulins), and enhanced chemiluminescence Western blotting re- agents (Amersham) following the manufacturer's instructions. Pro- tein markers (Sigma) were as follows: myosin, 205,000 Da; p- galactosidase, 116,000 Da; phosphorylase b, 97,400 Da; bovine serum albumin, 66,000 Da; ovalbumin, 45,000 Da; carbonic anhydrase, 29,000 Da.

Zmrnunodepletion-mAb 66 or control-IgG (anti-TFIIIA (39)) were bound to 10 p1 of packed protein A-Sepharose beads equilibrated in NET-2 buffer (50 mM Tris-HC1, pH 7.9; 150 mM NaCI; 0.05% (v/v) Nonidet-P 40; 0.5 mM dithiothreitol). The resin was washed three times with NET-2 to remove unbound material, followed by three washes with buffer AH + 0.1 M KCl. SF3a (15 pl) was added and incubated for 60 min at 4 "C. Protein A-Sepharose was removed by centrifugation, and the supernatants were assayed for activity in splicing complex assembly. For the analysis of the material bound to mAb 66-protein A-Sepharose, the pelleted resin was washed three times in NET-2. Proteins were eluted into SDS sample solution (37) and analyzed in SDS-polyacrylamide gels. (The concentration of antibodies and the SF3a fractions used are given in the figure legends.)

RESULTS

Chromatography of HeLa cell nuclear extracts on DEAE- Sepharose, heparin-Sepharose, and Mono Q results in the separation of several splicing activities, five of which suffice for the assembly of a presplicing complex on an AdML pre- mRNA (25,30). These are SF1, SF3, UBAF, and two fractions that are enriched in U1 and U2 snRNPs (Fig. 1). SF3 was further fractionated on spermine-agarose, where it eluted in the 0.3 M KC1 step fraction (not shown). Chromatography of this fraction on a Mono S column resulted in the separation of SF3 activity into two components. One activity (SF3a) resided in the flow-through (0.1 M KC1); the other activity (SF3b) bound to the resin and eluted between 0.2 and 0.35 M KCI. Fig. 2 shows that neither the flow-through fraction nor individual fractions eluted from the column displayed a significant level of complex formation when tested in the presence of SF1, UZAF, and snRNPs. However, when flow- through and bound fractions were combined, presplicing complexes formed as efficiently as with the input fraction. Further titration experiments confirmed that at no concentration of the individual fractions tested could presplicing complex formation be observed, but complexes were assembled in the

17642 Purification of Splicing Factor SF3a

presence of both fractions (not shown). Thus, SF3 activity consists of at least two components that are both required at an early stage of spliceosome assembly.

SF3a was further purified from the Mono S flow-through by chromatography on heparin-Sepharose and elution with a salt gradient (not shown) which was followed by gel filtration on a Superose 12 column. Most of the activity is detected in fractions 37-40 (Fig. 3A). Inspection of the proteins present in these fractions reveals three major polypeptides of 60, 66, and 120 kDa and several less abundant proteins (Fig. 3B). Upon sedimentation of SF3a (Superose fraction 38) in a glycerol gradient the major polypeptides are again found in fractions that display activity in presplicing complex formation (Fig. 4; the SDS-polyacrylamide gel shown was inten- tionally overloaded to detect minor proteins that could cofractionate with SF3a activity), whereas several high molecular mass polypeptides as well as a polypeptide of 45 kDa that cofractionated with SF3a on the Superose 12 column (cf. Fig. 3) could be separated from the activity. In the preparation shown, a minor protein of -30 kDa can be detected in the

H.L.N”

DEAE-S.ph.mu 7

l o o

SF1 sm I

H.puln-s.p(u- 1 I 1

T o o sm l o m

SF2 - MAQ U2AF - 350 JYHQ) 450

spum~k~pwou U1 SF4 U2 u5 U W 6

l o o ?a 0 lom

d o S A

l o o - b~~~d*$chsrwa SAb

2203p

SUP+ 12 cuvtrol Q d h t

SF3a

FIG. 1. Schematic representation of the fractionation steps employed in the purification of splicing factor SF3a. The chromatographic resins are indicated. Numbers designate the KC1 concentrations at which splicing activities were eluted. When splicing factors were desorbed with a salt gradient, the approximate KC1 concentration required for elution is indicated. For further details, see “Experimental Procedures.”

active fractions, however, it was not consistently observed in other SF3a preparations and its association with SF3a activity is unclear. This protein does not corresponds to the hnRNP A1 protein as determined by Western blotting (not shown). From these results we conclude that SF3a comprises three polypeptides of 60, 66, and 120 kDa. Densitometer scanning of SDS gels indicated that these polypeptides are present in a roughly equimolar ratio in all column fractions analyzed (not shown).

The apparent molecular masses of the SF3a polypeptides are similar to other splicing factors or RNA binding proteins. A direct comparison of SF3a with U2AF (16), PTB/hnRNP I (31, 32), and hnRNP protein U (33) by SDS-gel electrophoresis clearly shows that the SF3a subunits differ from these proteins (Fig. 5). Furthermore, Western blotting of purified SF3a with anti-hnRNP U antibodies (kindly provided by S. Piiiol-Roma and G. Dreyfuss) did not reveal any cross-reaction with the 120-kDa polypeptide of SF3a (not shown). SF3a is also clearly different in size and subunit composition from a number of other known splicing factors (18-20, 24, 26, 27). An example of a fraction containing SF3b is shown in the last lane of Fig. 5. Thus far, we have not been able to identify any polypeptide(s) that cofractionates with the activity.

To aid in the further characterization of SF3a, monoclonal antibodies were generated. Two hybridoma cell lines were established that secrete antibodies directed against the 66- kDa polypeptide as shown by Western blotting of SF3a with one of these antibodies (mAb 66; Fig. 6A). A single protein of the same size is also detected in nuclear extracts. The fact that only the 66-kDa and not the 120-kDa polypeptide reacts with the antibody makes it highly unlikely that the two smaller subunits are derived from the larger one by proteolytic breakdown, as might be suggested by a comparison of the apparent molecular masses of the polypeptides.

We next performed a depletion experiment to verify that SF3a activity is associated with the purified polypeptides. mAb 66 was bound to protein A-Sepharose and incubated with a heparin-Sepharose fraction containing partially purified SF3a. Bound antigen was removed by centrifugation, and the supernatant was tested for residual activity. Depletion of the SF3a fraction with increasing concentrations of mAb 66 resulted in a gradual decrease of activity in presplicing complex formation, whereas depletion with a control antibody

I SF1 + U2AF + snRNPs 1 1- Mono SFlowthrough -1

”*””. .“” - - c ””””.””“ IP F T 1 FT2FT3 50 52 54 56 58 60 62 64 66 68 70 50 52 54 56 58 60 62 64 66 68 70

FIG. 2. Separation of SF3a and SF3b by Mono S chromatography. Left, input (IF‘), flow-through (FT), and bound fractions (3 pl) were individually added to a standard reaction containing SF1, UZAF, and snRNPs and tested for presplicing complex assembly. Right, fractions containing Mono S-bound material (1.5 p l ) were tested in a standard reaction in the presence of the Mono S flow-through fraction (1.5 pl). The posi- tion of presplicing complex A is indicated. For a comparison with complexes formed in nuclear extracts, see Fig. 3.

A

FIG. 3. Gel filtration of SF3a on Superose 12. A, input (IP; 2 pl) and Superose 12 fractions (2 pl) were tested in a standard assay in the presence of 1 pl of SF3b as indicated above the figure. The second lane shows a reaction performed in the absence of SF3a. In the first lone complex formation in 1 p1 of nuclear extract (NXT) is shown. The migration of complexes A and B is indicated. B, protein analysis. Proteins from 7.5 pl of the input (ZP) and Superose 12 fractions were separated in a 7.5% SDS- polyacrylamide gel and stained with silver. In lone M the sizes of protein markers are indicated on the left.


A I-' SF1 + SF3b + U2AF + snRNPs '-1

1- Superose 12 fractions -1 5 IP 34 35 36 37 38 39 40 41 -42 43 44 45 47 49 51 53 55 57 """e " I b S a # 8 . 8 .

B -

A-

B kDa IP M 34 35 36 37 30 39 40 41 42 43 44 45 46 47 49

205 - 116- 1

97.4 - ,

45-

29 -

had no effect on SF3a activity (Fig. 7). Quantitation of the radioactive RNA present in the presplicing complex revealed that the depletion was complete. Thus, the monoclonal antibody directed against the 66-kDa polypeptide can efficiently remove SF3a activity from a partially purified fraction.

To examine whether the three major polypeptides that cofractionate with SF3a activity are present in one complex, the proteins that were bound by mAb 66-protein A-Sepharose were analyzed. For this experiment a Mono Q fraction was used. As shown in Fig. 6B, the three polypeptides of 60, 66, and 120 kDa are the major proteins that are bound by the antibody, and they appear to be present at a similar ratio as in purified SF3a. In this experiment, the protein A-Sepharose- mAb 66-antigen complex was washed with a buffer containing 0.15 M NaCl before elution of the antigen. Similar results were obtained when the protein A-Sepharose-bound antibody- antigen complex was washed with increasing NaCl concentrations. The three polypeptides remained bound to the antibody up to 2 M NaCl and were again present in the eluate at roughly equimolar ratios (not shown). Thus, these polypeptides appear to be tightly associated. Two minor proteins were detected in the eluate as well. A polypeptide of 70 kDa is not found in fractions containing purified SF3a, nor is a similar protein seen when the experiment is performed, for example, with Mono S fractions containing SF3a. It is therefore likely that the 70-kDa polypeptide represents a contaminant. The protein of 100 kDa probably results from an aggregation of the antibody, since it was also observed in immunoprecipitates of

purified SF3a fractions that did not contain a polypeptide of similar size (not shown). Taken together, these results indicate that SF3a activity is associated with a complex of three polypeptides of 60,66, and 120 kDa.

To analyze the function of SF3a during presplicing complex formation in further detail, the purified factor was tested for several activities that have been shown (or implied) to be associated with splicing activities (see Refs. 2 and 3 for reviews). The purified protein did not exhibit any binding to the AdML pre-mRNA or RNA substrates that do not contain splice sites (as tested by native polyacrylamide gel electrophoresis or by cross-linking with ultraviolet light). We have also failed to observe binding of any of the subunits of SF3a to the pre-mRNA substrate under conditions where presplicing complexes are formed. Furthermore, no ATPase (40), RNA helicase (41), or RNA annealing activities (20) have been detected in fractions containing purified SF3a (results not shown).

Titration of nuclear extracts with mAb 66 by Western blotting and comparison of the signal with the signal obtained for known amounts of SF3a allowed us to determine that the 66-kDa subunit of SF3a is present at a concentration of about 50 ng/pl nuclear extract (results not shown). From this value we calculated that approximately lo6 molecules of SF3a are present per HeLa cell nucleus. Compared to 0.5-1.0 X lo6 molecules of each of the spliceosomal snRNPs per cell (421, SF3a appears to be a rather abundant protein.

17644 Purification of Splicing Factor SF3a

A 1- SF1 + SF3b + U2AF + snRNPs -1

2 5 I P 3 > ~ ~ ~ 9 & 1 1 12 1 4 l 6 i 8 - 2 0 1- Glycerol gradient fractions -1

B -

A-

w -

B I-' Glycerol gradient fractions -, I P M 1 3 5 6 7 8 9 1011 1 2 1 4 1 6 1 8 2 0 M 18a

205

97.4 116

66

45

29

FIG. 4. Analysis of SF3a in glycerol gradient fractions. A , presplicing complex assembly. Input (ZP; 2 pl) and glycerol gradient fractions (2 pl) were tested in a standard assay in the presence of 1 pl of SF3b as indicated above the figure. The migration of pre-mRNA incubated in the absence of splicing components is shown in the first l a n e ; a reaction with 1 pl of nuclear extract (NXT) is shown in the second lane. B, protein analysis. Input (7.5 pl) and glycerol gradient fractions (150 pl) were precipitated with trichloroacetic acid, precip- itates were washed with acetone, and proteins were separated in a 7.5% SDS-polyacrylamide gel followed by silver staining. The sizes of marker proteins (lanes M ) are indicated on the right.

DISCUSSION

Splicing factor SF3 functions in the formation of a presplicing complex (25, 34) and has been separated into two activities. Both, SF3a and SF3b are required at the first ATP- dependent step in the splicing reaction. We have purified SF3a to homogeneity and shown that it consists of a complex of polypeptides of molecular masses of 60,66, and 120 kDa.

The native molecular weight of SF3a is 141,000, as determined from the data obtained by gel filtration and glycerol gradient sedimentation (43). This value is unexpectedly low when compared to the migration of the individual polypeptide components of SF3a in SDS-polyacrylamide gels. From the data obtained by Western blotting and immunoprecipitation with mAb 66, it can be ruled out that the two smaller polypeptides are derived from the larger one by proteolytic breakdown. The low native molecular weight might reflect an unusual shape of the protein complex or modifications within one or more of the subunits that could lead to an abnormal behavior during SDS-polyacrylamide gel electrophoresis.

kDa

200 -

116 - 97.4 -

66-

45 -

29-

"- "

FIG. 5. Comparison of SF3a to other splicing factors and RNA binding proteins. Purified SF3a, UZAF, PTB/hnRNP I, and hnRNP U were separated in a 7.5% SDS-polyacrylamide gel (37) and stained with silver. The last lane shows an example of the state of purification of SF3b. The molecular mass of protein markers is indicated on the left.

By the criteria of apparent polypeptide size and subunit composition SF3a is distinct from other purified splicing activities and RNA-binding proteins. The amino acid sequences obtained for several peptides of the 60- and 120-kDa subunits of SF3a has furthermore not revealed any significant homologies to mammalian protein sequences in current data bases? In addition, neither SF3a nor proteins present in the SF3b-containing fraction appear to belong to the SR class of splicing factors (22, 23). These proteins contain extensive repeats of Ser-Arg residues and cross-react with a monoclonal antibody (mAb 104) that is directed against a phosphoepitope (44). SR proteins were detected by Western blotting with mAb 104 (kindly provided by M. Roth) in Mono Q fractions eluting from 0.2-0.5 M KC1 including those that display SF3 activity (cf. Fig. l).5 However, the SR proteins are separated from SF3 by spermine-agarose chromatography where they are detected in the 0.6 M KC1 step fraction. This fraction is not essential for presplicing complex formation nor for the cleavage-ligation reactions.5 For both assays, the SR proteins are most likely supplied in sufficient concentrations by the snRNP-containing Mono Q fractions.

Purified SF3a does not exhibit any RNA binding, ATPase, or RNA annealing activities. These activities have been found associated with several mammalian and/or yeast splicing factors (see Refs. 2 and 3 for review). We have, however, obtained evidence that SF3a comigrates, after two-dimensional gel electrophoresis, with three proteins (SAP 61, 62, and 114) that are specifically associated with purified presplicing and splicing complexes (11).6 Consistent with the observation that SF3a is a rather abundant nuclear protein, SAP 61, 62, and 114 are among the most prominent proteins in the isolated complexes (cf. Ref. 11). These findings might hint at a stoi-

' R. Brosi and A. Kramer, unpublished results. M. Bennett, R. Reed, R. Brosi, and A. Kramer, unpublished

results.


P

29- - 1 2 3 4 5

E

116 97.4

29- 0 -. - L

1 2 3 4 5

FIG. 6. Immunological analysis of SF3a with a monoclonal antibody (mAb 66) directed against the 66-kDa subunit. A, Western blot analysis. Nuclear extract (2 pl) and SF3a (heparin- Sepharose) were separated in a 7.5% SDS-polyacrylamide gel, blotted to nitrocellulose, and processed as described under “Experimental Procedures.” Lanes 2 and 3, silver-stained proteins in nuclear extract and in the SF3a fraction; lanes 4 and 5, Western blot after reaction with mAb 66. The sizes of marker proteins (lane 1 ) are indicated on the left. B, immunoprecipitation analysis. A SF3a-containing Mono Q fraction (15 pl; lane 2) was incubated with protein A-Sepharose (PAS) in the absence (lane 3 ) or in the presence (lane 4 ) of mAb 66 and processed as outlined under “Experimental Procedures.” Lane 5 shows SF3a (heparin-Sepharose fraction) for comparison. Lane I , marker proteins, the sizes are indicated on the left. The arrows on the right indicate SF3a polypeptides. The heavy and light chains of mAb 66 are indicated by Hand L, respectively.

chiometric, rather than an enzymatic, function for SF3a during spliceosome assembly.

Additional experiments have shown that an antibody directed against the yeast splicing factor PRP9, which is required for U2 snRNP addition to the spliceosome (45), cross- reacts with the 60-kDa subunit of SF3a.2 This finding is supported by a significant homology between the amino acid sequence of PRP9 and the sequence derived from a cDNA encoding the 60-kDa polypeptide of SF3a? Interestingly, the anti-PRP9 antibody also cross-reacts with a polypeptide that is specifically associated with the 17s U2 snRNP which appears to be active in pre-mRNA splicing in contrast to a 12s U2 snRNP that is comprised of the core-snRNP proteins and the U2-specific proteins A’ and B” (46): These obser-

’ A. Krtimer and G. Bilbe, unpublished results.

w e

1 2 3 4 5 6 7 a 9 1 0

FIG. 7. Depletion of SF3a activity with mAb 66. A heparin- Sepharose fraction (15 pl) containing SF3a activity was incubated in the absence of antibody ( l a n e 5), with increasing amounts of mAb 66-IgG bound to protein A-Sepharose (PAS; lanes 6,0.5 pg; lane 7,5 pg; lane 8.50 pg; lane 9,200 pg) or with 200 pg of protein A-Sepharose- bound control-IgG (lane 10) and processed as described under “Ex- perimental Procedures.” Unbound material (0.5 pl) was tested in a standard reaction in the presence of 0.5 p1 of SF3b for presplicing complex formation. Lanes 1 and 2 show pre-mRNA incubated in the absence or presence of 1 pl of nuclear extract, respectively. Lanes 3 and 4: standard reactions performed in the absence or presence of 0.5 pl of the untreated heparin-Sepharose fraction.

vations provide further evidence that SF3a plays an essential role in the assembly of the ATP-dependent presplicing complex.

Acknowledgments-We are grateful to Kaethi Bucher for her in- valuable help with the preparation of the monoclonal antibodies. We thank Diana Blank for excellent technical assistance; Andrea Buhler for cell culture; Seraphin Piiiol-Roma and Gideon Dreyfuss for the gift of anti-hnRNP A1 and anti-hnRNP U antibodies; Mark Roth for the gift of mAb 104; and Graeme Bilbe and Karsten Groning for comments on the manuscript.

REFERENCES

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