The EMBO Journal vol. 1 0 no. 1 pp. 207 - 214, 1 991

Biochemical characterization of U2 snRNP auxiliary factor: an essential pre-mRNA splicing factor with a novel intranuclear distribution Phillip D.Zamore1 and Michael R.Green' Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, MA 02138, USA 'Present address: Program in Molecular Medicine, University of Massachusetts Medical Center, Worcester, MA 01605, USA

Communicated by J.Tooze

U2 auxiliary factor (U2AF) is a non-snRNP protein required for the binding of U2 snRNP to the pre-mRNA branch site. Purified U2AF comprises two polypeptides of 65 and 35 kd. We have performed biochemical complementation and immunological assays to characterize U2AF in greater detail. First, we use an extract lacking only U2AF activity to show that U2AF is an essential splicing factor. Second, we show that all U2AF activity in vitro resides in the 65 kd U2AF polypeptide. Third, based upon both immunological and functional criteria, we show that U2AF is evolutionarily conserved. Most significantly, a Drosophila melanogaster nuclear extract contains proteins that are antigenicaily related to both human U2AF polypeptides and can substitute for human U2AF in vitro. Finally, we show that U2AF has an unexpected intranuclear distribution. Although diffusely present throughout the nucleoplasm, U2AF is also concentrated in a small number (between one and five) of nuclear 'centers.' This localization differs strikingly from that reported for snRNP antigens and splicing factors. Our data, in conjunction with those in the accompanying paper [Carmo-Fonseca et al. (1991) EMBO J., 10, 195-206.], suggest that these centers represent novel aspects of nuclear organization. Key words: Drosophila/human/pre-mRNA splicing/U2AF

Introduction In higher eukaryotes, most mRNAs are transcribed as premRNAs which must be spliced before they can be translated into proteins. Pre-mRNA splicing takes place in a protein -RNA complex called the spliceosome (reviewed in Maniatis and Reed, 1987; Sharp, 1987; Guthrie and Patterson, 1988). Spliceosome formation follows an ordered pathway. At each step in the pathway snRNPs or protein factors bind either to sites on the pre-mRNA or to factors already assembled into the complex. The binding of U2 snRNP to the pre-mRNA produces the first stable complex in the mammalian spliceosome assembly pathway (Maniatis and Reed, 1987; Sharp, 1987; Guthrie and Patterson, 1988). Binding of U2 snRNP to a specific sequence, the branch site, is thought to delineate the adenosine residue at which the RNA branch will form. In turn, the selection of a branch site helps define the 3' splice site (Smith and Nadal-Ginard, 1989). Stable binding of U2 snRNP to produce a pre-spliceosome Oxford University Press

is an ATP-dependent reaction that requires several auxiliary protein factors (Ruskin et al., 1988; Kramer and Keller, 1985; Kramer, 1988) as well as Ul snRNP (Barabino et al., 1990). Ul snRNP has been shown to bind the 5' splice site and may interact directly with U2 snRNP (Mattaj et al., 1986; Bindereif and Green, 1987). Two auxiliary factors have been defined as partially purified chromatographic fractions from a HeLa cell nuclear extract (Kramer, 1988). A third factor, U2AF, has been purified to homogeneity (Zamore and Green, 1989). U2AF binds the polypyrimidine tract/3' splice site, a sequence required for the efficient binding of U2 snRNP. It comprises two polypeptides, -35 and -65 kd, in appproximately 1: 1 stoichiometry. The two polypeptides copurify on a variety of chromatographic matrices and cosediment on a glycerol gradient (Zamore and Green, 1989). Previously, U2AF was shown to be required for prespliceosome assembly (Ruskin et al., 1988; Zamore and Green, 1989). In this report, we demonstrate that U2AF is essential for pre-mRNA splicing. We also present evidence that the two U2AF polypeptides are antigenically distinct proteins and that, in vitro, U2AF activity resides in the 65 kd polypeptide. We examine the distribution of U2AF activity in various organisms and find that U2AF from evolutionarily distant species can substitute for human U2AF in a HeLa cell nuclear extract. Finally, we show that the 65 kd U2AF polypeptide has a striking intranuclear localization that differs from that reported for other protein factors implicated in premRNA splicing.

Results U2AF is an essential pre-mRNA splicing factor U2AF was identified as an activity required for stable binding of U2 snRNP to the pre-mRNA branch site (Ruskin et al., 1988; Zamore and Green, 1989). In those studies, we showed that a typical HeLa cell cytoplasmic extract (S100), which contains functional U2 snRNP, cannot support formation of a pre-spliceosome because it lacks U2AF. The S100 provided a convenient complementation system for assaying U2AF. However, we were unable to reconstitute pre-mRNA splicing by adding U2AF to the S100, presumably because the S100 lacks other splicing factors. To establish the role of U2AF in pre-mRNA splicing, we developed a nuclear extract deficient only in U2AF. U2AF binds extraordinarily tightly to poly(U) -Sepharose (Zamore and Green, 1989) enabling the selective removal of U2AF from nuclear extract (NE). When NE was chromatographed on poly(U) - Sepharose in the presence of 1 M KCl, U2AF bound to the column but most proteins flowed through. To demonstrate that this depleted NE did not contain U2AF, we developed antibodies directed against both U2AF polypeptides. First, antibodies were raised against synthetic 207

P.D.Zamore and M.R.Green

peptides that corresponded to partial amino acid sequences from the two U2AF polypeptides (see Materials and methods). The antisera were purified on peptide-Sepharose columns to produce mono-specific antibodies directed against the 65 kd U2AF polypeptide (U2AF 65 kd; anti-pep A and anti-pep D) and the 35 kd U2AF polypeptide (U2AF 35 kd; anti-pep C). Anti-pep A and anti-pep D antibodies recognized the 65 kd protein in crude NE and purified U2AF (Figure 1). The anti-pep C antibody recognized only U2AF 35 kd in NE and purified U2AF. Next we used these antibodies to analyze the NE that had been passed through the poly(U) -Sepharose column in 1 M KC1. Figure 1 shows that neither U2AF 65 kd nor U2AF 35 kd was detected in this high salt flow-through. Therefore, we designate this fraction AU2AF-NE. By itself, the AU2AF-NE could not form pre-spliceosomes (Figure 2A) or splice pre-mRNAs (Figure 2B and C). However, adding purified U2AF to the AU2AF-NE fully reconstituted its ability to form splicing complexes and to splice both an Adenovirus Major Late (MINX; Figure 2B) and a human ,B-globin pre-mRNA (A9; Figure 2C). These results demonstrate that U2AF is a splicing factor in vitro. The amount of purified U2AF required to restore maximal splicing differed for the two pre-mRNAs examined. Reconstituting maximal levels of splicing in the AU2AFNE required 10- to 20-fold more U2AF for A9 than for MINX (compare Figure 2b, lane 7 with 2c, lane 6). Previously, we found that U2AF binds to the MINX polypyrimidine tract appproximately 20-fold more strongly than it does to human f-globin IVS 1 (Zamore and Green, 1989). We conclude that when U2AF is the limiting component, the affinity of a specific polypyrimidine tract for U2AF 65 kd determines splicing efficiency. The 65 and 35 kd U2AF polypeptides are antigenically distinct U2AF 65 kd and U2AF 35 kd could be distinct proteins, or they might be related, for example, through posttranslational modifications or proteolysis. To distinguish among these possibilities, we analyzed purified U2AF using our anti-peptide antibodies (see above). Two antibodies against peptides from U2AF 65 kd recognized the 65 kd

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Fig. 1. Nuclear extract (NE) depleted by chromatography on poly(U)Sepharose in 1 M KCI (AU2AF-NE) does not contain U2AF. NE, AU2AF-NE and purified U2AF were analyzed by immunoblotting with anti-U2AF 65 kd (anti-pep D and anti-pep A) and anti-U2AF 35 kd (anti-pep C) anti-peptide antibodies. The positions of molecular weight markers (in kd) are indicated on the left. 208

polypeptide (Figure 1, anti-pep A and anti-pep D). However, the anti-65 kd antibodies did not recognize U2AF 35 kd, indicating that the epitopes identified by these antibodies are not present in U2AF 35 kd. In the reciprocal experiment, the anti-peptide antibody against U2AF 35 kd recognized the 35 kd protein in purified U2AF but did not bind to U2AF 65 kd (Figure 1, anti-pep C). Thus, we conclude that the two U2AF polypeptides are antigenically distinct. Only the 65 kd U2AF polypeptide is needed for premRNA splicing in vitro We could not separate the two U2AF polypeptides using conventional chromatography matrices. Therefore, to determine whether one or both of the two U2AF polypeptides participated in pre-mRNA splicing, we employed C4 reverse-phase chromatography to separate them. Purified U2AF was injected onto a C4 column, washed to remove buffer components, and then eluted with a gradient of acetonitrile. Two protein peaks eluted from the column (Figure 3A). The A210 ratio of peak 1 to peak 2 was 1:2, consistent with their corresponding to equimolar amounts of U2AF 35 kd and U2AF 65 kd. Immunoblotting the protein present in each peak (Figure 3B), confirmed that peaks 1 and 2 corresponded to U2AF 35 kd and U2AF 65 kd, respectively. After separating the U2AF polypeptides, we tested the ability of each to restore splicing to the AU2AF-NE. When added to the AU2AF-NE, U2AF 35 kd reconstituted neither splicing (Figure 3C, lane 3) nor pre-spliceosome assembly (data not shown). In contrast, U2AF 65 kd restored the ability of the AU2AF-NE to splice both MINX (lane 2) and human 13-globin-derived pre-mRNAs (data not shown). Adding both polypeptides to the AU2AF-NE reconstituted splicing to the same level achieved by adding only the isolated U2AF 65 kd (compare lanes 2 and 1). These results imply that only U2AF 65 kd and not U2AF 35 kd functions in pre-mRNA splicing in vitro. -

U2AF is evolutionarily conserved To determine whether U2AF is conserved in evolutionarily divergent organisms, we performed immunoblot analysis using our anti-peptide antibodies on nuclear extracts from F9 (mouse), CHO (hamster), and QT6 (quail) cell lines (Figure 4A). In each of these cells, the anti-pep A or antipep D antibodies detect a 65 kd protein. Furthermore, addition of micrococcal-nuclease digested nuclear extracts from these cells to HeLa cell AU2AF-NE restores spliceosome assembly and splicing (data not shown), indicating that these cells contain functional U2AF activity. These results with vertebrate cell lines encouraged us to test whether the lower eukaryote, Drosophila melanogaster, also contains U2AF activity. When a nuclear extract from the D.melanogaster cell line Kc (Kc NE) was analyzed by immunoblotting, both antibodies against U2AF 65 kd (antipep A and anti-pep D) detected a - 55 kd protein (Figure 4B). The antibody against U2AF 35 kd (anti-pep C) detected a protein slightly larger (- 37 kd) than human U2AF 35 kd. To determine whether KC NE contains functional U2AF activity, we added it to AU2AF-NE. Figures 4C and 4D show that Kc NE restored the ability to splice to AU2AFNE. (This amount of Kc NE is insufficient to support splicing in the absence of the HeLa AU2AF-NE.) As expected, the Drosophila U2AF activity is resistant to treatment with

U2 snRNP auxiliary factor

To provide evidence that the cross-reactive 55 and 37 kd polypeptides are responsible for U2AF activity in Drosophila, we analyzed chromatographic fractions of KC NE (Figure 4D). Fractions from both DEAE- and heparin-Sepharose chromatography of Kc NE showed a

micrococcal nuclease (Figure 4C), consistent with its being a protein. Thus D.melanogaster, although evolutionarily distant from vertebrates, contains a protein activity which can replace U2AF and interact with components of the human pre-mnRNA splicing machinery.

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1 Fig. 2. A: U2AF reconstitutes pre-spliceosome formation in the U2AF depleted nuclear extract (AU2AF-NE). Increasing amounts of purified U2AF and 1 u1 of the were added to AU2AF-NE in a splicing reaction with MINX pre-mRNA. Twenty pl splicing reactions containing 9 s4l AU2AF-NE and concentration) final (1 with mg/ml treated heparin was of the reaction $l Five 15 min. for at component indicated below were incubated 30°C analyzed on a 0.5% agarose/3.5% acrylamide (80:1, acrylamide:piperizine diacrylamide) native gel buffered with 50 mM Tris-glycine (Konarska and Sharp, 1987). Added component: HeLa NE (lane 1), buffer (lane 2), increasing amounts of U2AF (lanes 3-10, 0.015 to 2 jl). Purified U2AF (-50 jg/ml) was diluted in buffer (see Materials and methods) to yield dilutions equivalent to 0.015 1l U2AF in lane 3, 0.03 in lane 4, 0.06 inIL lane 5, etc. B and C: U2AF reconstitutes splicing in the AU2AF-NE. Increasing amounts of purified U2AF were added to the AU2AF-NE in a 20 splicing reaction with either MINX (B) or A9 (C) pre-mRNAs and incubated at 30°C for 2 h. (B) Splicing products were analyzed on a 13% left. polyacrylamide gel. Lanes 1-9 correspond to lanes 1-9 in (A). Various intermediates in the splicing pathway are indicated symbolically on the They represent, from top: lariat intermediate, lariat product, pre-mRNA, spliced mRNA, exon 1. (C) A comparable analysis of the (3-globin derivative A9 using 8 ILI AU2AF and 2 Al of NE, buffer, or diluted U2AF. Lane 1, NE; lane 2, buffer; lanes 3-6, 0.25, 0.5, 1 and 2 11 U2AF. Splicing products were analyzed on a 10% polyacrylamide gel. The small A9 first exon was not retained on the gel. 209

P.D.Zamore and M.R.Green

perfect correlation between the presence of the 55 and 37 kd polypeptides and the presence of U2AF activity. Drosophila cytoplasmic, egg, and embryo extracts differ in their ability to support splicing in vitro (D.Rio, 1988 and personal communication). Cytoplasmic extracts (Kc S100) and egg extracts do not efficiently splice pre-mRNA in vitro. In contrast, 0-12 h embryo extracts do splice pre-mRNA efficiently. Anti-U2AF antibodies detected only a small amount of 55 and 37 kd proteins in the KC S100 (Figure 4B), which contains a low level of U2AF activity (Figure 4D). Egg extracts, which lack U2AF activity (Figure 4D), did not contain either the 55 kd or the 37 kd proteins (Figure 4B). In contrast, embryo extracts, which support in vitro splicing and reconstitute splicing in the HeLa AU2AF-NE (Figure 4D), contained the 55 and 37 kd proteins (Figure 4B). (Egg and embryo extracts also contain an abundant 45 kd protein which cross-reacted with the anti-pep D antibody but not anti-pep A. In contrast, the D. melanogaster 55 kd polypeptide was recognized by both the anti-pep A and anti-pep D antibodies.)

Based on these results, the 55 and 37 kd proteins are likely to be Drosophila U2AF. Like human U2AF, Drosophila U2AF appears to consist of two subunits because the 55 and 37 kd proteins co-distribute in extracts and fractions that contain Drosophila U2AF activity. The 65 kd U2AF polypeptide has an unusual intranuclear localization Using indirect immunofluorescence antibody staining, various investigators have found that snRNP polypeptides [the Ul 70K protein (Spector, 1984; Nyman et al., 1986; Verheijen et al., 1986), sm-epitope-bearing snRNP proteins (Mattioli and Reichlin, 1971; Northway and Tan, 1972; Deng et al., 1981; Spector, 1984, 1990; Smith et al., 1985), and the U2-specific B" protein (our unpublished data)] and a splicing factor termed sc-35 (Fu and Maniatis, 1990) are localized in 20-50 'speckles' in the nucleoplasm. We examined the intranuclear distribution of U2AF 65 kd. Figure 5 shows the distribution of U2AF 65 kd in HeLa cells as detected with the anti-pep A antibody by indirect

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Fig. 3. A: Reverse-phase chromatography of purified U2AF. One hundred 1.1 purified U2AF was injected onto a 2.1 mm x 150 mm Vydac C4 column equilibrated in 5% acetonitrile, 0.06% TFA. The column was developed with a gradient of acetonitrile (1.67%/min) in 0.06% TFA at a flow-rate of 200 p3/min. Forty-five min corresponds to 80% acetonitrile. Absorbance at 210 nm was monitored. The peak at 32 min represents NP40 present in the input U2AF sample. B: Reverse-phase chromatography peaks 1 and 2 represent isolated U2AF 35 kd and U2AF 65 kd, respectively. Immunoblot analysis of purified U2AF and peaks 1 and 2 was performed with anti-U2AF 35 kd (anti-pep C) and anti-U2AF 65 kd (anti-pep D) antibodies. C: Only U2AF 65 kd and not U2AF 35 kd is needed to reconstitute splicing in the AU2AF-NE. The individual HPLC peaks were renatured before testing their function (see Materials and methods). Splicing reactions (20 Al) contained 8 1 AU2AF-NE plus: 2 11 buffer (lane 4); 1 p3 isolated U2AF 35 kd and 1 Al buffer (lane 3); 1 41 U2AF 65 kd and 1 Al buffer (lane 2); or 1 p3 U2AF 35 kd and 1 141 U2AF 65 kd (lane 1). Lane S shows a splicing reaction with 2 p3 U2AF but no AU2AF-NE. Lane 6 is a standard splicing reaction containing 10 p3 NE.

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immunofluorescence microscopy. U2AF 65 kd is distributed throughout the nucleoplasm. It is also present in a small number (1-5 per cell) of intensely stained 'centers' (Figure 5, panels A, B and D). Neither the bright U2AF centers nor the diffuse U2AF coincide with nucleoli. The distinct intranuclear localization of U2AF 65 kd is unlikely to be an artifact of fixation or staining. First, cells stained with monoclonal Y12 anti-sm antibody (panel C) display the previously reported speckled pattern. When these same cells are stained with the anti-pep A antibody (panel

D), U2AF 65 kd is detected in a few intense centers and diffusely throughout the nucleoplasm. Second, CarmoFonesca et al. (1990) have shown that the areas of concentrated U2AF 65 kd are the same sites at which U2, U4, U5, and U6 snRNAs are localized.

Discussion The experiments described here show that U2AF is an essential, highly conserved splicing factor; that the two

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Fig. 4. A: Vertebrate cell lines contain a 65 kd protein antigenically related to U2AF 65 kd. Mini-extracts (Schreiber et al., 1989) prepared from F9 (mouse), CHO (hamster), or QT6 (quail) cells were analyzed by immunoblotting with anti-peptide antibodies against U2AF 65 kd. B: Drosophila melanogaster extracts and chromatography fractions contain 55 kd and 37 kd proteins antigenically related to U2AF 65 kd and 35 kd, respectively. Immunoblot analysis of D.melanogaster proteins was performed using anti-peptide antibodies against U2AF 65 kd (anti-pep A and anti-pep D) and U2AF 35 kd (anti-pep D). C and D: D.melanogaster extracts and chromatography fractions that contain proteins antigenically related to U2AF also contain U2AF activity. (C) Micrococcal-nuclease treated Kc NE added to HeLa AU2AF-NE reconstitutes splicing. One Al MN-KC NE (lane 1) or MN-HeLa NE (lane 2) was incubated with 9 1d1 AU2AF-NE in a standard splicing reaction with MINX pre-mRNA. In lanes 3 and 4 the MN-HeLa NE or MN-KC NE was incubated with 9 Al buffer instead of AU2AF-NE. (D) One ltl of various Drosophila extracts or chromatography fractions was added to 9 1l AU2AF-NE in a standard 20 Al splicing reaction. Lane 1, Kc NE; lane 2, Kc S100; lane 3, egg extract; lane 4, embryo extract. In lanes 5, 6 and 7, KC NE that had been chromatographed on DEAE-Sepharose was tested for U2AF activity when added to AU2AF-NE: proteins that flowed through at 0. M KCI (lane 5) or eluted at 0.25 M (lane 6) or 0.5 M KCI (lane 7). In lanes 8, 9, and 10, Kc-Ne that was chromatographed on heparin-Sepharose was tested: proteins that flowed through at 0.1 M (lane 8) or eluted at 0.3 M (lane 9) or 0.6 M (lane 10). Splicing products of MINX pre-mRNA in (C) and (D) are displayed on 13% polyacrylamide gels. 211

P.D.Zamore and M.R.Green

Fig. 5. U2AF 65 kd is both present in the nucleoplasm and concentrated in a small number of nuclear centers. The intranuclear distribution of U2AF 65 kd in HeLa cells was analyzed by indirect immunofluorescence using the anti-pep A antibody (A, B, and D) A represents a detail from the field of cells in B. C shows the same cells as in D stained with the Y12 monoclonal anti-sm antibody. Magnification = 320x (B) and 800x (C,D).

U2AF polypeptides are antigenically distinct; that all U2AF activity in vitro resides in the 65 kd polypeptide, and that U2AF has a novel nuclear distribution. The amount of U2AF needed to produce maximal splicing of various pre-mRNAs is inversely related to its affinity for each pre-mRNA's polypyrimidine tract. This suggests that U2AF initiates spliceosome assembly and splicing by binding directly to the polypyrimidine tract (Zamore and Green, 1989). When the concentration of U2AF is limiting, a particular 3' splice site's affinity for U2AF will determine its strength. Proteins which bind to sequences within the 3' splice site might block U2AF binding and repress use of that splice site. In particular, we note that the D.melanogaster protein, sex-lethal (sxl), binds to a pyrimidine-rich sequence at the 3' splice site of several pre-mRNAs (Inoue et al., 1990; for review see Baker, 1989). By binding to these sites, the sxl protein could prevent U2AF binding, blocking use of that 3' splice site and promoting use of a 3' splice site bearing a lower-affinity U2AF binding site. Several proteins similar in size to U2AF polypeptides have been implicated in pre-mRNA splicing. One such protein, p62, binds the polypyrimidine tract and is present in spliceosomes (Garcia-Blanco et al., 1989). Although p62 and U2AF 65 kd have similar RNA binding specificities, our preliminary experiments suggest that purified p62 cannot substitute for U2AF. Another protein, the U5-associated protein IBP, may bind the 3' splice site during spliceosome assembly (Gerke and Steitz, 1986; Tazi et al., 1986). IBP has been shown to be distinct from U2AF (Ruskin et al., 212

1988). A third protein, the mammalian homologue of the yeast PRP8 protein binds pre-mRNA (Garcia-Blanco et al., 1990), associates stably with U5 snRNP (Anderson et al., 1989), and is present in a U4/5/6 particle (Pinto and Steiz, 1989). Binding of this protein requires ATP and is likely to occur following binding of U2 snRNP (Garcia-Blanco et al., 1990). The PRP8 homologue, like p62 and IBP, may recognize the 3' splice site. As previously suggested (Ruskin et al., 1988), it seems probable that the 3' splice site/polypyrimidine tract may be read multiple times during a single splicing event. A group of 30-35 kd polypeptides, named ASF (Ge and Manley, 1990) or SF2 (Krainer et al., 1990), contains an activity essential for 5' exon cleavage and lariat formation. We have examined by immunoblotting a preparation of ASF (kindly provided by H.Ge and J.L.Manley) with our anti-pep C antibody against U2AF 35 kd. This antibody does not recognize any of the ASF polypeptides (data not shown), suggesting that ASF/SF2 is not U2AF 35 kd. Since the U2AF 65 kd is sufficient for U2 snRNP binding and pre-mRNA splicing in vitro, what function does U2AF 35 kd perform? The 35 kd protein co-purifies with U2AF 65 kd (Zamore and Green, 1989) and is evolutionarily conserved, suggesting it has an important cellular function. One possibility is that U2AF 35 kd may be required to splice some pre-mRNAs other than those used in this study. Alternatively, U2AF 35 kd may play a role in vivo that is not evident in our in vitro splicing system. For example, U2AF 35 kd could: (i) interact with U2AF 65 kd in the

U2 snRNP auxiliary factor

cytoplasm and send it to the nucleus; (ii) target U2AF 65 kd to specific sites within the nucleus (such as the centers); or (iii) direct spliceosomes to sites of RNA export. Using anti-peptide antibodies against U2AF, we have detected U2AF polypeptides in three non-human, vertebrate cell lines and have identified in Drosophila cells 55 kd and 37 kd proteins antigenically related to human U2AF. All of these cell lines contain U2AF activity. Thus, the role of U2AF in pre-mRNA splicing appears widely conserved in evolution. We have not detected U2AF activity in yeast or cauliflower nuclear extracts (data not shown). This result is not surprising: pre-mRNA splicing in plants and yeast does not appear to require a polypyrimidine tract, the U2AF binding site. However, these extracts do contain polypeptides that cross-react with anti-U2AF antibodies. The role of these cross-reactive proteins in splicing remains to be established. The intranuclear distribution of U2AF is unlike that previously reported for protein splicing factors. U2AF is present throughout the nucleoplasm and in a small number of nuclear centers. It is difficult to reconcile our results, and those of Carmo-Fonseca et al. (1991), with the previously reported speckled distribution of other splicing-related proteins (Mattioli and Reichlin, 1971; Northway and Tan, 1972; Deng et al., 1981; Spector, 1984, 1990; Smith et al., 1985; Nyman et al., 1986; Verheijen et al., 1986; Fu and Maniatis, 1990). However, these speckles are unlikely to be fixation or staining artifacts because the same cells that display the reported speckled distribution of sm-epitopes show the novel distribution for U2AF. Fu and Maniatis (1990) have shown that depletion of the protein sc-35 from nuclear extracts blocks splicing in vitro. Sc-35 has a speckled intranuclear distribution. Thus, antibodies against different protein components of the spliceosome (sc-35 and U2AF 65 kd) produce different immunofluorescence staining patterns. Perhaps a subset of the nuclear sites of sc-35 are involved in splicing or the two proteins act at different stages of spliceosome assembly. U2AF is both concentrated in discrete centers and distributed throughout the nucleoplasm. We do not understand the relationship of these two pools of U2AF. Because U2, U4, U5, and U6 snRNAs have been localized to these same regions (Carmo-Fonseca et al., 1991), the centers may represent sites of spliceosome assembly. In any case, these centers reveal new aspects of nuclear structure and function.

Immunoblot analysis Proteins were electrophoresed in an SDS-10% polyacrylamide gel, transferred to PVDF (Millipore), and blocked in 5 % (w/v) non-fat dry milk, 10 mM Tris-HCI, pH 7.4, 0.9% (w/v) NaCl, 0.5% Tween-20. All antibody incubations and washes were performed with this buffer. Antibodies were detected with [1251]protein A (1 ACi/ml; New England Nuclear). Antibody concentrations: anti-pep A and C, 2 ytg/ml; anti-pep D, 10 ytg/ml.

U2AF-depletions Nuclear extract (2.5 ml; Dignam et al., 1983) was adjusted to 20 mM HEPES-KOH, pH 7.9, 1 M KCI, 3 mM MgCl2, 0.05% Nonidet P40 (NP-40), 1 mM dithiothreitol (DTT), 20% glycerol and applied to a 5 ml poly(U) -Sepharose column (1.5 cm diameter; Pharmacia) equilibrated in this buffer containing 10% glycerol. The column was run at a flow-rate of 10 ml/cm2/h. One ml fractions were collected. Peak fractions were pooled and dialyzed into this buffer containing 100 mM KCI and 20% glycerol. U2AF was purified as described (Zamore and Green, 1989) except that the initial centrifugation step was omitted.

Splicing reactions Splicing reactions (20 1d) contained 200 000 c.p.m. (-4 fmol) 32P-labeled MINX (run-off at the BamHI site; Zillmann et al., 1988) or A9 pre-mRNA (run-off at the BamHI site. previously referred to as SP64-H,BminiEl in Bindereif and Green, 1987), 40 U RNasin (Promega), 20 mM creatine phosphate, 50 Ag/ml rabbit skeletal muscle creatine kinase (BoehringerMannheim), 0.1 mM ATP, 2.67% polyvinyl alcohol (PVA), 3 mM MgCI2, 50 mM KCI, 0.025% NP-40, 0.5 mM DTT, 10 mM HEPES-KOH, pH 7.9, 10% glycerol. PVA was omitted for native gel analysis. Buffer added to splicing reactions or used to dilute U2AF contained 20 mM HEPES-KOH, pH 7.9, 3 mM MgCI2, 1 mM DTT, 100 mM KCI, 0.05% NP-40, 20% glycerol. To assay peaks from reverse-phase chromatography, fractions corresponding to each peak were pooled and evaporated to dryness. The resulting pellet was dissolved overnight at 4°C in 2 ytl buffer (above) containing 6 M guanidine-HCI, then diluted 50-fold with buffer lacking the chaotropic agent. For micrococcal nuclease digestion (MN), extracts were incubated with 600 U/mi MN (USB) in 1 mM CaC12 at 30'C for 20 min. Nuclease digestion was stopped by adding EGTA (2 mM final concentration). Immunofluorescence microscopy HeLa cells were grown to - 50% confluency on glass coverslips in DMEM (Gibco) containing 10% fetal bovine serum (Hyclone) and antibiotics. Cells were washed three times with phosphate-buffered saline before fixing in ice-cold 3% paraformaldehyde/0.2% Triton X-100 in PBS for 10 min followed by -20°C acetone for 5 min. Cells were then washed three times with PBS and blocked with 20% fetal bovine serum in PBS containing 0.5% Tween-20. All subsequent antibody incubations and washes were in this blocking buffer. Primary (10 Ag/ml anti-pep A or 1:1000 Y12 cell culture supernatant) and secondary antibodies (1:300 affinity-purified FITC goat anti-rabbit or rhodamine-goat anti-mouse; Jackson) were incubated with cells for 1 h at room temperature followed by three 10 min washes. After a rapid water wash, coverslips were mounted in 90% glycerol containing 1 mg/ml p-phenylenediamine (Johnson and Araujo, 1981). Stained cells were observed with a Zeiss Axiophot epifluorescence microscope equipped with 40 x and 100 x objectives. Ektachrome 400 film (Kodak) was used for photographs.

Acknowledgements Materials and methods Anti-U2AF antibodies Antibodies were raised in rabbits against synthetic peptides (kindly provided by Maurice Green or synthesized on an ABI peptide synthesizer) coupled to maleimide-activated keyhole limpet hemocyanin (Pierce) via an aminoor carboxy-ternminal cysteine residue not present in U2AF. Peptide sequences were determined by microsequencing tryptic and Staphylococcus aureus protease V-8 fragments of SDS-PAGE-isolated U2AF polypeptides. Microsequencing was performed by William Lane of the Harvard microchemistry facility. Pep A corresponds to the sequence

CELLTSFGPLK; pep C was VEMQEHYDEFFC; pep D was SAHKLFIGGLPNYLNDDQVKEC. Initial injections were in complete Freund's adjuvant (Difco); subsequent injections used incomplete Freund's adjuvant. Antibody-containing rabbit serum was purified using the appropriate peptide coupled to CNBr-activated Sepharose 4B (2-4 mg/ml resin; Pharmacia). Antibodies were bound to the column in 20 mM NaPO4, pH 7.2. The column was washed with 20 mM NaPO4 containing 0.5 M NaCl and 0.05% Tween-20, then eluted with 0.1 M Triethylamine, pH 11.5.

The authors are grateful to Don Rio and Chris Seibel for providing Drosophila extracts and column fractions; to William Lane for expert assistance with microsequencing and HPLC; to Maurice Green and Tom Ellenberger for help with peptide synthesis and purification; to Nandini Nair for providing QT6 cells; to Ruth Padmore for help and advice with immunofluorescence microscopy; to Angus Lamond for communicating results and helpful discussions; and to members of the Green laboratory for advice and critical comments on the manuscript. P.D.Z. was supported by an NSF

fellowship.

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