Assemblage of the prespliceosome complex with separated fractions isolated from HeLa cells.

The first ATP-dependent complex formed in pre-mRNA splicing is the prespliceosome, a 30 S complex. This reaction was investigated using partially purified fractions isolated from nuclear extracts of HeLa cells. Previous studies (Furneaux, H. M., Perkins, K. K., Freyer, G. A., Arenas, J., and Hurwitz, J. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 4351-4355) have shown that DEAE-cellulose chromatography of nuclear extracts yielded two fractions (fractions I and II, eluted at 0.2 and 1 M NaCl, respectively) which carried out pre-mRNA splicing only when combined. Fraction II, alone and in the presence of ATP, supported the formation of the 30 S complex. In this report, we have separated fraction II into ribonucleoprotein and protein-rich fractions by isopycnic banding in CsCl. The combination of these two fractions completely replaced fraction II in prespliceosome formation; when supplemented with fraction Ib (1 M NaCl Biorex fraction derived from fraction I), the preparations supported spliceosome formation; when supplemented with fraction I, they yielded spliced products. The CsCl fractions, like fraction II, efficiently converted pre-mRNA to the 30 S complex with high yields (30-70%). The 30 S complex was shown to contain pre-mRNA complexed to U2 small ribonucleoproteins and small amounts of U1 small ribonucleoproteins. The 30 S complex protected a 50-nucleotide region at the 3'-end of the intron from T1 RNase attack. This region included sequences spanning the branch site, the polypyrimidine stretch and the AG dinucleotide of the 3'-splice site. When the 30 S complex was first generated with partially purified fractions, followed by the addition of a large amount of poly(U) or unlabeled pre-mRNA, the 30 S complex could be chased into a 55 S spliceosome complex by the addition of fraction Ib. These results support the conclusion, initially derived from kinetic data, that the 30 S complex is a precursor of the 55 S complex.

When the 30 S complex was first generated with partially purified fractions, followed by the addition of a large amount of poly(U) or unlabeled pre-mRNA, the 30 S complex could be chased into a 55 S spliceosome complex by the addition of fraction Ib. These results support the conclusion, initially derived from kinetic data, that the 30 S complex is a precursor of the 55 S complex.
The splicing of pre-mRNA in uitro is preceded by the accumulation of a 55-60 S spliceosome complex (l-4). This complex, formed only in the presence of ATP, contains pre-* This work was supported in part by American Cancer Society Grant ACS NP-89s and National Science Foundation Grant DMB 8415369. Tbe costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "uduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
$ Supported by a postdoctoral fellowship from The American Cancer Society. mRNA splicing intermediates and a number of snRNPs ' (5, 6). The function of the spliceosome is to juxtapose exon ends so that they can be ligated together. This process must depend upon intermolecular reactions between proteins, ribonucleoprotein complexes, and the pre-mRNA.
A smaller ATP-dependent complex of 30-35 S (prespliceosome), containing unaltered pre-mRNA, has also been observed. This complex, which is formed rapidly and accumulates prior to the formation of the spliceosome, is thought to be a precursor of the 55 S complex. However, altered pre-mRNAs that are either incapable of forming spliceosomes or support the formation of low levels of the spliceosomes can yield increased levels of the pre-spliceosome complex (7,8,24). Formation of 55 S complex requires the presence of Ul, U2, U5, and the U4-U6 snRNPs. While there is general agreement that U2, U5, and U4-U6 snRNPs are found in the spliceosome complex, there is controversy about the presence of Ul snRNP, although the integrity of Ul snRNP is essential for spliceosome formation and splicing (5,6,9). The 30 S complex has been shown to contain U2 snRNP (6,9, lo), but like the 55 S complex, there is controversy about the presence of Ul snRNP in the smaller complex (5, 11).
A variety of components interact with pre-mRNA prior to the formation of the 30 S complex. The first complex that is formed results from the interaction of Ul snRNP with the 5' end of the intron-exon border (12,13). This reaction occurs at 0 "C and is formed in the absence of ATP. A number of reactions at the 3' end of the intron-exon region have been reported. A poorly characterized protein, U2 snRNP auxiliary factor (U2AF), binds to pre-mRNA recognizing sequences around the 3'-splice site (14). This protein is required for the binding of U2 snRNP to the branch site of the pre-mRNA. Another protein, intron-binding protein also binds to the 3'end of the intron recognizing the same sequence, required for U2 snRNP binding (15,16). The binding of intron-binding protein occurs at 0 'C and does not require ATP. The role of this interaction in prespliceosome formation is presently unclear.
Since the prespliceosome is the first ATP-dependent reaction in a series of discernible partial reactions that lead to splicing, we have focused on this reaction as a more systematic way of examining the more complex overall splicing reaction. We have previously shown that nuclear extracts of HeLa cells can be separated by DEAE-cellulose chromatography into two fractions that are both required for splicing (fractions I and II) (17). Fraction II, which is eluted from DEAE-cellulose at high salt concentrations, contains all of the snRNPs required for splicing. This fraction alone can convert pre-mRNAs to the prespliceosome complex (4). In the present communica-Formation of Prespliceosome Complex tion, we have utilized this fraction as the starting point for a study of the syntheiis of the prespliceosome complex. We have examined CsCl isopycnic gradient cenkifugation as a strategy for separating snRNPs from proteins. It was reported earlier that snRNPs retained their structural integrity on CsCl gradients (18). We have found that fraction II can be further resolved by this procedure into a RNP fraction and a fraction which is a mixture of "light" snRNPs and proteins. A combination of these fractions can replace fraction II in a variety of splicing reactions. The two fractions alone form the nresnliceosome comnlex. and when sunnlemented with frac- tion Ib (a derivative of fraction I (4)), support spliceosome complex synthesis. Furthermore, in conjunction with fraction I, the t,wo CsCl fractions fully replace fraction II in the overall splicing reaction. The experimerks presented here explore the fractions required for 30 S complex formation. We have detected protein fractions in addition to snRNPs that are essential for prespliceosome complex. Similar studies have been reported by Kramer (19) and by Krainer (20), and our results are in complete accord with their findings.

MATERIALS AND METHODS
Preporotion of pre-rnRNA-The plasmid pKT1 and its construction were previously described (17  Fig. 1, this procedure resulted in the separation of protein, snRNPs, and free RNA. Some resolution of the snRNPs was also observed, most likely due to differences in their ratio of protein to RNA. Thus, U2 and U4-U6 snRNPs banded at a higher density than Ul and U5 snRNPs (Fig. lA).
The material that banded at a density of 1.42 gm/ml (fraction IIR) supported the splicing of pre-mRNA when supplemented with nuclear extract pretreated with micrococcal nuclease (Fig. 1B). The activity observed with this nucleoprotein complex (1.42 gm/cm) was relatively stable. When subjected to a second CsCl isopycnic gradient centrifugation, it banded at the same density and showed an identical ability to support splicing when supplemented with nuclear extracts pretreated with micrococcal nuclease (results not presented).
Reconstitution of splicing activity with fraction IIR peaked at a density that contained little Ul RNA, suggesting that either the amount of Ul snRNP in this fraction was augmented by the was obtained by measuring splicing activity of each fraction supplemented with fraction I (DEAE-cellulose step) as described under "Materials and Methods." micrococcal nuclease-treated crude extract or that the low levels present in this fraction were ample to support splicing.
A second peak of splicing activity (IIL), banding at a density of 1.35 g/ml, was detected when fraction IIR and fraction I pretreated with micrococcal nuclease were added to each CsCl gradient fraction (Fig. 1B). This second peak of splicing activity corresponded to a region enriched in Ul and U5 RNAs, as well as in proteins. This peak of activity did not coincide with the protein profile, suggesting that the activity contained in this fraction may not only be due to a protein factor.
When fraction I was pretreated with micrococcal nuclease, both fractions IIR and IIL were required for splicing (Fig. 2). Previous studies showed that fraction I treated with micrococcal nuclease supported splicing when combined with fraction II. This suggested that the snRNPs and additional protein factors essential for splicing in fraction II, were distributed between fractions IIR and IIL.
We believe that a protein component(s) that functions in splicing is present in the protein-enriched CsCl fraction 11 or 12 (Fig. 1). This component(s) supported splicing when added to nuclear extracts that were heated at 45 "C for 15 min Fraction I was pretreated with micrococcal nuclease (200 units/ml for 30 min at 30 'C) to eliminate cross-contaminating snRNPs.
Splicing reactions were carried out for 2 h (as described under "Materials and Methods") after which time the RNA was extracted and separated on an 18% denaturing polyacrylamide gel. All reactions were carried out in the presence of micrococcal nuclease-treated fraction I (60 pg of pratein). In lane 1, fraction II (23 pg of protein) was added in addition to micrococcal nuclease-treated fraction L Zunes 2 and 4 were assayed with fraction IIL (8.5 pg of protein) or IIR (1 pg of protein), respectively, while in he 3, fractions IIL, IIR, and fraction I were all combined. The positions of the different RNA products formed in the splicing reactions are schematically presented next to that individual species.
(result not shown). Such heated extracts alone did not support splicing. In addition, preincubation of fraction IIL with Ca*+ alone, for 15 min at 30 'C, inactivated its ability to support any of the reactions involved in splicing. This inactivation most likely represented proteolysis rather than an activation of an endogenous RNase. After incubation with Ca*+, the Urich RNAs present in fraction IIL were intact (result not shown). Fraction II Can Be Replaced by the CsCl Fractions for the Synthesis of the 30 S Prespliceosome Complex-Fraction II alone, incubated with ATP and pre-mRNA, yielded a 30 S complex (4). This complex, first identified by sucrose gradient centrifugation, can be assayed by its gel mobility (8). The complex was isolated following sucrose gradient centrifugation and was shown to migrate with the same mobility as the complex detected by gel electrophoresis (Fig. 3). Two fractions (IIR and IIL), isolated by CsCl banding, were required to form the prespliceosome complex (Fig. 4, A-C). As shown, the amount of the prespliceosome formed was proportional to the concentration of both IIR and IIL and required ATP. The rate of prespliceosome complex formed ( Fig. 4D) with both fractions, showed a lag of 5 min followed by a linear reaction for about 30 min at which time, approximately 40% of the input pre-mRNA had been converted to the 30 S complex.
We have previously shown that after incubation of fraction II with fraction Ib (which was isolated by Biorex 70 chromatographic separation of fraction I to yield fraction Ia and Ib), the 55 S spliceosome containing 5'-exon and the intron-exon lariat accumulated (4). As shown in Fig. 4E, the CsCl gradient fractions, supplemented with fraction Ib, supported the accumulation of a 55 S complex. It was noted that with increasing concentration of fraction Ib, the 30 S complex migrated significantly slower than the complex formed in the presence of the CsCl fractions alone. This change in mobility of the 30 Fraction II (23 fig of protein) was incubated with pre-mRNA (as described under "Materials and Methods") for 90 min at 30 OC. An aliquot (0.2 ml) was applied to a 5-ml lo-30% sucrose gradient which was centrifuged at 48,000 rpm for 195 min in an AH-650 rotor. Fractions were collected from the bottom of the tube and the entire fraction was counted (Cerenkov). The inset above the graph shows an autoradiogram of a gel run with sucrose gradient fractions (native, 3% polyacrylamide (1:80), 0.5% agarose composite gel in 50 mM Tris, 50 mM glycine). Electrophoresis was continued until the xylene cyanole dye reached the bottom of the gel which was dried and then exposed for autoradiogmphy.
S complex was also noted in reactions containing large amounts of CsCl fractions (data not shown).
Both fractions were tested for their sensitivity to N-ethylmaleimide (NEM) treatment, heat, and micrococcal nuclease in order to characterize the components present in each fraction (Table I). When each fraction was treated with NEM, and then supplemented with the untreated fraction, prespliceosome formation was either unaffected (IIR) or was reduced by only 30% (IIL). In contrast, when both CsCl fractions were treated with NEM, their combination resulted in a marked decrease in 30 S complex formation (lo-fold). This observation suggests that a minimum of three components are necessary to form the 30 S complex and that an NEM-sensitive component is distributed between IIL and IIR. We have established by chromatographic separation that fraction IIL contains two components, one of which is NEM sensitive as described below. While both fractions showed some heat lability, fraction IIR was more stable than the lower density fraction (IIL); this RNP-rich fraction was partially resistant to boiling for 5 min. If fraction IIR, pretreated with NEM (which had almost no effect), was used to complement fraction IIL which was heated for 5 min at 45 'C, virtually no 30 S complex was formed (Table I). This indicated that the heat labile component in fraction IIL was also present in fraction Reactions were incubated for 1 h at 30 'C and then loaded directly onto a 3% polyacrylamide (1:80), 0.5% agarose gel in 50 mM Tris, 50 mM glycine. Electrophoresis was carried out until the xylene cyanole dye reached the bottom of the gel. In punel A, 1une.s l-7, 20 ~1 of fraction IIR (0.57 pg/pl protein) was incubated with pre-mRNA and ATP; in lanes 2-7, 0.1, 0.2, 0.5, 1, 2, and 5 ~1 of fraction IIL (12.6 fig/p1 protein) were added, respectively. In lorzcs 8-12, 2 ~1 of fraction IIL was added to reaction mixtures and 1, 2, 5, and 10 ~1 of fraction IIR were added in addition to .!nnes 9-12, respectively. In lone 13, the reaction mixture was identical to lane 12 with the exception that ATP was omitted. The quantitation of 30 S complex formed is presented in pane/s B and C. Panel B represents the titration of fraction IIR, in the presence of a constant amount of IIL while punel C presents the variation of IIL addition in the presence of a fixed amount of RR. In punel D, the rate of 30 S complex formation was measured using saturating amounts of fraction IIL (4 ~1) and 20 ~1 of fraction IIR. In Punel E, lanes l-4 contained CsCl fractions IIL (4 ~1) and IIR (20 ~1); lunes 2-4 contained 7.5, 15, and 37.5 pg of protein of fraction Ib, respectively. Fraction Ib (37.5 pg) alone or in combination with either fraction IIL or IIR did not lead to a detectable band in the 30-55 S region (data not presented).
IIR and was NEM-sensitive. Micrococcal nuclease treatment of fraction IIR prevented the synthesis of prespliceosome complex. Since fraction IIL was inactivated by incubation in the presence of 1 mM CaC12 without the addition of micrococcal nuclease, it was not clear whether this fraction contained an essential RNP or only essential proteins. Subsequent studies indicated that an essential protein component(s) was present in fraction IIL that was required for 30 S complex formation.
Determination of Sequences of pre-mRNA in the 30 S Complex Protected against RNase Attack-The sequestered regions of the pre-mRNA in the 30 S complex were determined using Tl RNase digestion. For this purpose, 30 S complex was formed with the CsCl fractions IIL and IIR (Fig. 5). After synthesis of the 30 S complex, reaction mixtures were digested with increasing amounts of Tl RNase and then subjected to gel-electrophoresis (Fig. 5A). A significant portion of the labeled pre-mRNA was resistant to Tl RNase digestion, and this material, complexed to RNA-binding components, migrated to the same position as observed with the parental 30 S complex. The RNase-resistant material was eluted from the 2aoa Formation of Prespliceosome Complex Reaction mixtures (as described under "Materials and Methods"), containing the fractions treated as indicated above, were incubated with pre-mRNA at 30 "C for 1 h. Where indicated, 10 gl of fraction IIR (0.57 mg protein/ml) and 2 ~1 of fraction IIL (12.6 mg protein/ ml) were used. The 30 S complex was separated by electrophoresis on a native composite gel, and the band corresponding to the prespliceosome complex was excised and counted. NEM (5 mM) treatment involved incubation on ice for 20 min followed by the addition of DTT (10 mM). Prior to the heat treatment, fractions were centrifuged at 12,000 X g for 2 min. Following heat treatment they were again centrifuged before they were assayed. Treatment with micrococcal nuclease was for 30 min at 30 'C using a final concentration of 200 units of micrococcal nuclease/ml. The amount of complex obtained with fraction IIL alone (6%) has been subtracted from reactions in which this fraction was added to IIR. 100% activity represented 30 fmol of pre-mRNA incorporated into the 30 S complex. gel and again digested with RNase Tl after proteinase K and phenol treatment. Prior to the second RNase Tl treatment, a 50-nucleotide fragment was detected as the major species present in the eluted complex (Fig. 5B). After the second Tl RNase digestion, oligonucleotides of 18, 14, and 8 nucleotides in length were observed. These oligonucleotides were derived from the region surrounding the branch point, the polypyrimidine stretch and the AG dinucleotide at the 3'-splice site, all at the 3'-end of the intron (Fig. 5C). These results are analogous to the results obtained by Kramer (19) and Konarska and Sharp (8) (24). As previously reported by us (7) and others (5, 6), the 5'end of the intron appears to play no role in the formation of the 30 S complex since pre-mRNAs containing alterations in the 5'-end support 30 S formation. In contrast, regions involving the polypyrimidine stretch and the 3'-end of the intron appear to be essential for the accumulation of this complex. Alterations of these regions (7) result in no complex formation with the two CsCl fractions (see Fig. 8

) (24).
Nucleoside Triphosphate Requirement for the Synthesis of 30 S Compkx-The formation of the prespliceosome complex was dependent upon the addition of ATP (Fig. 6A). The rate of complex formation was maximal between 0.4-0.8 mM. The presence of an ATP regenerating system substantially reduced the concentration of ATP required in the reaction to 0.05 mM. In addition, the yield of complex formed in the presence of the regenerating system was 2.5 times greater than that observed with ATP alone. While dATP could completely substitute for ATP, no other nucleotide worked in its place. The non-hydrolyzable ATP analogues (AppNp, AppCp, adenosine 5'-(3-0-thio)triphosphate did not support 30 S complex formation in place of ATP (data not presented). These experiments were carried out in the absence of an ATP regenerating system as was the control. Identification of the products formed from ATP during the synthesis of the prespliceosome complex was hampered due to the hydrolysis of ATP to ADP UC~U~CUUAUCCUgUCCCuuuuuuUuCCacag CUCGlCG/G/uuG/ 12 9 AGKVACAAACUCUUCGICG~GLICUUUCCAG~U DKT~ ore-mRNA (5800 cpm/fmol of RNA) and other reagents a; described under "Materials and Methods" for 1 h at 30 'C. The reaction mixture was then incubated for 10 min at 30 "C with RNase Tl, as indicated, and loaded onto a native gel and electrophoresed as described under "Materials and Methods." & analvsis of RNase Tl-resistant RNA. The RNase Tl-resistant 30 S complex was excised from the native gel (krne 5 of panel A ) and the RNA eluted with a solution containing TBE, 0.1% SDS, 0.5 mM CaCl?, and 0.5 mg/ml proteinase K. The mixture was incubated for 15 min at 37 'C and then extracted with phenol-chloroform, chloroform, and ethanol precipitated. This RNA was loaded onto an 18% polyacrylamide, 50% urea gel and electrophoresed in TBE. A 50-nucleotide band, the dominant band detected on the gel, was eluted with a solution containing 0.5 M ammonium and P, in the absence of pre-mRNA. Although CsCl fraction IIR was free of ATPase activity, fraction IIL contained a substantial amount of this activity. The synthesis of prespliceosome complex required Mg'+ and concentrations between 1 and 2 mM were optimal (data not shown). The formation of the 30 S complex was reduced 50% by the presence of 0.05 M NaCl and virtually abolished at 0.15 M (Fig. 6B). In contrast, once formed, the product was resistant to high NaCl concentrations. Approximately 80% of the product remained after incubation with 0.4 M NaCl for 20 min on ice.
snRNPs Associated with Prespliceosome Complex-In order to assess which U-rich RNAs were associated with the prespliceosome complex, reactions were carried out in the presence of fractions IIR and IIL and unlabeled pre-mRNA. The position of the prespliceosome was determined using labeled 30 S complex as the marker. When the complex was eluted from the gel, and isolated RNA was run on a denaturing gel, transferred to a nylon membrane, and probed for the various U-RNAs, only U2 RNA was detected. However, control experiments lacking pre-mRNA, also lead to the detection of U2 RNA in the region corresponding to the 30 S complex. The experiment was repeated using biotinylated pre-mRNA as the substrate. This technique has been previously used to obviate the isolation of endogenous complexes in the analysis of the U-rich RNAs present in splicing complexes (5, 6). We have used immobilized anti-biotin in order to select complexes containing the biotinylated RNA substrate. This technique resulted in lower backgrounds than observed with streptavidin and permitted the elution of the complex using mild conditions (presence of biotin in the elution buffer). In the experiment described in Fig. 7, prespliceosome complex was first isolated by sucrose gradient centrifugation in order to obtain large amounts of this product. Complexes from the 30 S peak as well as the adjacent fraction (containing the ATP-dependent complex) were incubated with immobilized anti-biotin. After washing the immobilized anti-biotin several times, the RNA was eluted using an excess of free biotin at 4 "C. The RNA was isolated, run on a denaturing gel, transferred to a nylon membrane, and probed for the presence of Ul and U2 RNA (Fig. 7). As shown, U2 RNA was detected in the prespliceosome complex as well as small amounts of Ul RNA. RNA Binding Reactions with Pre-mRNA-In an attempt to develop a rapid assay for the synthesis of intermediates in the splicing reaction, we have explored a number of approaches. One such treatment, the resistance to Tl RNase, was used to isolate the 50-nucleotide oligomer complexed to RNA binding activities (Fig. 5). The procedure was altered so that after incubation of the CsCl fractions with wild-type pre-mRNA, Tl RNase was added, and after a short incubation, the reaction mixture was then passed through a nitrocellulose filter. The bound material, representing protein associated   C. RNase Tl effect on other me-mRNAs. The pre-mRNAs used were those previously described (7). These included pKT1, wild-type pre-mRN& pGT3, the 5'splice site mutant in which wild-tvpe GUGAGU was changed to CUGACU: nPY1. the mutant in which-42% of the pyrimidines in the polypyrimidine tract were changed to G residues; pIEC, the 3'splice site mutant in which the 3'-end of the intron AG was changed to AC. In all cases, 100 fmol with Tl RNase-fragments, was eluted from the filter and then subjected to polyacrylamide-urea gel electrophoresis (Fig. 8). In the presence of ATP, a 50 nucleotide fragment was the dominant labeled product isolated (Fig. 8A, lunes 9 and 11). This 50-nucleotide oligonucleotide was hydrolyzed by RNase Tl and the products were determined.
As was the case with the material eluted from the 30 S complex (Fig. 5B), oligonucleotide of 18, 14, and 8 nucleotides in length were detected (data not shown). This indicated that the region surrounding the branch site and the 3'-splice site were complexed and inaccessible to RNase Tl. In the absence of ATP, however, there was a marked increase in fragments that bound to the filter (Fig. 8A). This suggested that ATP decreased the nonspecific binding activity. The nucleotide effect was specific since none of the other common ribonucleoside triphosphates tested replaced ATP (Fig. 8B). The ATP-mediated decrease in binding of Tl RNase fragments was not dependent on the formation of the 30 S complex. In Fig. 8C, pre-mRNAs formed from plasmid pGT3 (where the 5'-end of the intron, GU-GAGU, was mutated to CUGACU), which does support the formation of the 30 S complex (7), yielded the 50-nucleotide fragment after RNase Tl digestion. The other two mutated pre-mRNAs, pPY 1 (a polypyrimidine tract mutant) and pIEC (where the 3'-end of the intron was changed from AG + AC), do not support 30 S complex formation and did not yield the 50-nucleotide fragment (24). All four of the RNAs examined in Fig. 8C showed a marked ATP-dependent decrease in the binding of fragments to the nitrocellulose filter. The decreased binding of the RNA fragments to nitrocellulose was not dependent on RNase Tl. Incubation of pre-mRNAs with ATP and the CsCl fractions reduced the amount of RNA bound to nitrocellulose 3-4-fold compared with similar reactions lacking ATP (data not presented). The relationship between splicing and the ATP-dependent alteration of the RNA binding reaction is now under investigation.
Further Resolution of Fractions Required for 30 S Complex Form&ion-As described in Table I, the activity with NEMtreated CsCl fractions suggested that more than two fractions are required for 30 S complex formation. Further fractionation of both IIR and IIL resulted in the resolution of the components required (Fig. 9). For this purpose, fraction IIR was treated with NEM and then used to monitor the synthesis of 30 S complex in reactions supplemented with purified fractions derived from IIL. Fraction IIL was subjected to the purification procedures outlined in the legend to Fig. 9 which resulted in the isolation of fraction IILl and IIL2. It was evident that fraction IILl contained one NEM-sensitive component while IIL2 contained a different NEM-sensitive component which was also present in fraction IILl. Further fractionation of both fraction IIR and fractionation of IILl and IIL2 has revealed that at least three other components plus fraction IIR are required for 30 S complex formation (data not presented).
The 30 S Complex Can Be Chased in& the 55 S Complex-The proposed pathway of spliceosome assembly suggests that the 30 S complex is first formed and then converted to the 55 S complex. However, the direct precursor-product relationship between these complexes has not been demonstrated.
In order to determine whether the 30 S complex is a functional intermediate of the 55 S complex (i.e. spliceosome), the following experiments were carried out. The 30 S complex was generated by the incubation of pre-mRNA pkT1 (wild-type) with ATP and fractions IIL and to IIR. The further synthesis of RNA labeled with [cr"'P]GTP (specific activity, 5800 cpm/fmol) was used and the reactions were as described in panel A. of the 30 S complex was almost completely blocked by the addition of poly(U). Fraction Ib was then added and the synthesis of 55 S complex was followed (Fig. lOA). As shown, 50% of the 30 S complex was converted to the 55 S complex after 15 min (Fig. lOA, lune 3). When the reaction was repeated with pre-mRNA pGT3 (where the 5'-end of the intron was mutated), the 30 S complex formed was not converted to the 55 S complex after the addition of fraction Ib. This observation is in keeping with the requirements for spliceosome formation. When the polypyrimidine mutant pre-mRNA (pPY1) was used in place of the wild-type pre-mRNA (pkTl), as expected, no 30 S or 55 S complex was detected.
Other experiments have been carried out which indicate that the 30 S complex can be directly converted to the 55 S complex. In these experiments, after 30 S complex formation, labeled pkT1 pre-mRNA was diluted lOO-fold with unlabeled pKT1 pre-mRNA or reaction mixtures were adjusted to 0.13 M KCl, conditions which block synthesis of 30 S and 55 S complexes. The addition of fraction Ib in either case resulted in the rapid accumulation of the 55 S complex (data not presented). The chase of the 30 S complex to the 55 S complex also resulted in the formation of the 5'-exon and the intron-A pre-mRNA pKT1 pPY1 pGT3 pKT1 polyoJI ' ------.+ + + + +cz~~~~-polyW~ t++ttt-----t+tt+ttttttt---Ib +t+ttttt+tt+t+tttt-----t+t T,m.dm,d 0 5 ,5306Ol200 l5~60l2060120 0 l5306Ol200 153060120060l20 exon lariat (Fig. 1OB). In this experiment, the chase was carried out in the presence of 0.13 M KC1 and the 55 S complex was eluted from the gel, digested with proteinase K, and then subjected to urea-acrylamide gel electrophoresis (Fig. lO&  he 2). A control, in which the pre-mRNA was incubated with crude nuclear extract (Fig. lOB, lane 1 ), served as markers for the expected products generated in the overall splicing system. Similar results were obtained with reaction mixtures in which poly(U) was used to inhibit 30 S complex formation. These results support the conclusion that the 30 S complex is a precursor of the 55 S complex.

DISCUSSION
The fractions isolated from nuclear extracts of HeLa cells and their activities in the splicing reactions are summarized in Scheme 1. Nuclear extracts fractionated on DEAE-cellulose yielded two fractions required for splicing (1'7). Materials eluted at high salt (fraction II) contained all snRNPs essential for the irz vitro splicing but lacked other protein components necessary for splicing. This fraction alone was sufficient for the formation of the ATP-dependent 30 S prespliceosome complex. Based on the observations of Sri-Wadada et ul. (16), we used isopycnic C&l gradient centrifugation to resolve the snRNPs in fraction II from protein-rich components, While U2 and U4-U6 snRNPs banded at a density distinct from the protein (1.44 g/ml), Ul and U5 snRNPs overlapped with the protein rich-peak (1.37 g/ml). This snRNP profile is similar to that observed by  who demonstrated that the various snRNPs band at different bouyant densities in the presence of high concentrations of MgC& (15 mM). The presence of MgCl* had no influence on the snRNP profile in our experiments.
Material that passed through DEAE-cellulose at 0.2 M NaCl (fraction I) was insensitive to treatment with micrococcal nuclease (17), suggesting that all of the snRNPs required for splicing were in fraction II. The converse experiment could not be performed since fraction II was sensitive to incubation with Ca*+ in the absence of micrococcal nuclease (data not shown). When the CsCl gradient fractions were assayed for their ability to complement fraction I in the splicing reaction, it was found that fraction IIL was the only other component required. The requirement for fraction IIR was only observed when fraction I was treated with micrococcal nuclease. This suggested that the difference between fractions I and II was not due to the distribution of snRNPs but most likely reflected the presence of different protein factors in these fractions.
U2 snRNP is an essential component of fraction IIR, since blotting experiments detected U2 RNA in the 30 S complex. In the presence of fraction IIL purified U2 RNA supported the 30 S complex reaction (at 30% efficiency) when added in addition to micrococcal nuclease-treated fraction IIR. However U2 RNA alone did not replace fraction IIR in the splicing reaction (data not shown). Differences were also observed with fraction IIL in splicing and in 30 S complex formation. Splicing activity was distributed over a broad peak (Fig. l), near the top of the gradient, while the 30 S complex activity peaked in the top fraction. The activity at the top of the C&l gradient may include the U2 AF protein factor previously described by Ruskin et al. (14), which was reported to be required for U2 snRNP binding to pre-mRNA. However, as described in Fig. 9 and in unpublished experiments carried out in our laboratory, at least three additional components isolated from the top of the CsCl gradient are involved in 30 S complex formation.

HeLa nuclear extract
In order to determine which snRNPs are part of the 30 S prespliceosome, we analyzed the complex isolated from sucrose gradients or from native gels. The RNA present in this complex was extracted, run on denaturing gels, blotted to nylon membranes, and then hybridized to probes for the various U-RNAs. Using this direct analysis, Ul and U2 RNAs were detected. However, these RNAs were also detected in reactions carried out in the absence of pre-mRNA. The use of biotinylated pre-mRNA and its selective adsorption to immobilized streptavidin (5, 6) resulted in a high background of Ul and U2 RNA. We have altered this technique by using immobilized antibiotin as the affinity reagent to select biotinlabeled pre-mRNA complexes. Besides improving the background, the complex was readily displaced with free biotin. Using this procedure, U2 RNA was detected as a stable component of the 30 S complex. Ul RNA was also detected as a component of the 30 S complex but present in much lower amounts than U2 RNA (lo-fold). Ul snRNP may be weakly associated and thus easily dissociated from the 30 S complex or alternatively may function catalytically in the generation of the 30 S complex. Ul RNA was not detected by blotting native gels (8, 9), but we have found this technique to be less sensitive than the procedure we have used (data not shown). Our efforts to completely free fraction IIL of Ul snRNP failed. Attempts to cleave the 5'-end of Ul RNA with RNase H and complementary oligonucleotides were also unsuccessful. A population of Ul snRNPs resistant to this treatment was consistently observed. Bindereif et ul. (5), concluded that Ul snRNP is involved in prespliceosome complex formation since Ul snRNP binds immediately to pre-mRNA and was detected in the 55 S spliceosome complex. The direct isolation of Ul snRNP in the prespliceosome complex was inferred but not directly demonstrated. The initial complex formed between Ul snRNP and pre-mRNA is more pronounced at 0 'C than at 30 "C (which is optimal for splicing) and does not require ATP.
The binding of Ul snRNP to pre-mRNA has also been examined in the yeast system by Ruby and Abelson (26). They observed that the binding of Ul snRNP to immobilized pre-mRNA required both the 5'splice site as well as the UAC-UAAC intron sequences. The latter sequence is uniquely essential for splicing in yeast. They also showed that RNase H degradation of Ul snRNP blocked subsequent binding of U2 snRNP and other snRNPs. These results indicate that Ul snRNP is essential for complex formation.
Our results do not define the role played by Ul snRNP in forming the 30 S splicing complex. We and others (7,19) have found that pre-mRNAs containing altered 5'-intron sequences support 30 S complex formation as efficiently as do wild-type pre-mRNAs. Such altered structures do not form the 55 S spliceosome complex. Thus, the role of Ul snRNP in the accumulation of the 30 S complex in the HeLa splicing system is unclear at present.
We have shown that labeled 30 S complex can be converted to the 55 S complex upon addition of fraction Ib. The synthesis of the 55 S complex depended upon the presence of functional 5'-and 3'-intron sequences previously shown to be essential for prespliceosome formation (7). The experiments described here suggest that the conversion of the 30 S complex to the 55 S complex most likely involves the interaction of snRNPs U4-U6 and U5 with the nucleoprotein-pre mRNA complex rather binding only to free pre-mRNA. The rapid synthesis of 55 S complex after the addition of poly(U) or the addition of a larger excess of unlabeled pre-mRNA supports this consideration.
The results presented here suggest that the prespliceosome complex contains at least, UZ-snRNP, proteins, and pre-mRNA. The roles played by the multiple protein fractions and fraction IIR in this ATP-dependent reaction remain to be elucidated.
The synthesis of the 30 S complex could be exploited to define the structure and biological activity of the U2 snRNP in a reaction critical for splicing but less complex than the overall reaction. Similar observations have been reported by Kramer (19) and by Krainer (20). They have also developed procedures for the separation of various fractions involved in the accumulation of intermediates in the splicing system. They, as well as we, have not as yet determined whether the isolated protein components which are free of the snRNPs are nuclear proteins or represent proteins weakly associated with snRNPs and other ribonucleoprotein complexes and thus easily dissociated. The further purification and characterization of these protein fractions should help resolve this problem.