the u1 small nuclear rna-protein complex selectively binds a 5′ splice site in vitro

Cell, Vol. 33, 509-518, June 1983, Copyright0 1983 by MIT 0092.8674/83/060509-lOSO2.00/0

The Ul Small Nuclear RNA-Protein Complex Selectively Binds a 5’ Splice Site In Vitro

Stephen M. Mount, lngvar Pettersson, Monique Hinterberger, Aavo Karmas, and Joan A. Steitz Department of Molecular Biophysics and Biochemistry Yale University New Haven, Connecticut 06510

Summary

The ability of purified Ul small nuclear RNA-protein complexes (Ul snRNPs) to bind in vitro to two RNAs transcribed from recombinant DNA clones by bacteriophage T7 RNA polymerase has been studied. A transcript which contains sequences corresponding to the small intron and flanking exons of the major mouse @-globin gene is bound in marked preference to an RNA devoid of splice site sequences. The site of Ul snRNP binding to the globin RNA has been defined by T, ribonuclease digestion of the RNA41 snRNP complex. A 1517-nucleotide region, including the 5’ splice site, remains undigested and complexed with the snRNP such that it can be co-precip- itated by antibodies directed against the Ul snRNP. Partial proteinase K digestion of the Ul snRNP abolishes interaction with the globin RNA, indicating that the snRNP proteins contribute significantly to RNA binding. No RNA cleavage, splicing, or recognition of the 3’ splice site by Ui snRNPs has been de- tected. Our results are discussed in terms of the probable role of Ul snRNPs in the messenger RNA splicing of eucaryotic cell nuclei.

Introduction

Ul RNA is an abundant small nuclear RNA (snRNA) which was first observed in mammalian cell nuclei over a decade ago (Hodnett and Busch, 1968; Weinberg and Penman, 1968). The sequences of rat, human, chicken, and fruit fly Ul RNA are known @eddy et al., 1974; Branlant et al., 1980; Roop et al., 1981; Mount and Steitz, 1981). Like the related RNAs U2, U4, U5, and U6, Ul exists not as a free RNA, but as a small nuclear RNA-protein complex (snRNP). The proteins of the Ul snRNP are recognized by two classes of antisera from patients with autoimmune disease (Lerner and Steitz, 1979). Antibodies of one class, known as antiSm, also bind snRNPs containing the small nuclear RNAs U2, U4, U5, and U6. As expected, these RNPs share some, but not all, of the eight identified Ul snRNP proteins (Hinterberger et al., 1983; Kinlaw et al., 1982). A second class of antisera recognizes only Ul snRNPs; the historic name of this specificity is RNP, but we will use the name anti-(Ul)RNP in order to avoid confusion.

The 5’ terminus of Ul RNA is maGpppA,U,- AC#J/ACCUG. This sequence, which is unchanged between fruit flies and vertebrates (Mount and Steitz, 1981), displays complementarity to the consensus sequence for

5’ splice sites, ~AG/GT$AGT, and that for 3’ splice

sites, T ’ AG/G (see Mount, 1982, Sharp, 1981, and Breath-

nach and Chambon, 1981, for reviews of splice site consensus). The proposal (Lerner et al., 1980; Rogers and Wall, 1980) that Ul snRNPs play a role in the splicing of RNA transcripts of nuclear protein-coding genes is supported by 1) the parallel conservation of Ul RNA, splice site sequences, and Ul snRNP antigens; 2) the abundance of Ul snRNPs in metazoan cells; 3) their salt-labile asso- ciation with hnRNP (Zieve and Penman, 1981; Lerner et al., 1980); and 4) their exclusively nuclear location. In addition, the inhibition of splicing in isolated nuclei by anti- (Ul)RNP antibodies (Yang et al., 1981), the cross-linking of Ul RNA to large RNA molecules (Calvet and Pederson, 1981), and a demonstration that the 5’ end of protein-free Ul RNA is available for base-pairing with DNA (Lazar et al., 1982) have been reported.

The original proposals for the involvement of Ul RNA in splicing (Lerner et al., 1980; Rogers and Wall, 1980) suggested that its 5’ end might base-pair with the pyrimidine-rich region which lies adjacent to 3’ splice sites as well as with the consensus sequences which surround the 5’ and 3’ splice junctions. Modification of this detail of the model was required by the observation that the stretch of eight purines found in positions 14-21 of vertebrate Ul is not conserved in the Drosophila Ul sequence (Mount and Steitz, 1981). Thus, it seemed reasonable to suppose that the Ul snRNP might function primarily in the recognition of 5’ splice sites. However, biochemical evidence for such an interaction has been lacking. The in vitro results presented here further strengthen the hypothesis that Ul snRNPs play a role in messenger RNA splicing and suggest that their role does indeed involve 5’ splice site recognition.

Results

The experiments described in this paper were motivated by the expectation that it should be possible, if the in vivo function of Ul snRNPs is the recognition of splice sites, to reproduce this interaction in vitro. Such an in vitro experiment would minimally require 1) purified Ul snRNPs, 2) an RNA which contains splice sites, and 3) a means of detecting any interaction between them.

We have recently described (Hinterberger et al., 1983) the isolation of snRNPs containing Ul, U2, U4, U5, and U6 RNAs from mouse Friend erythroleukemia and human HeLa cells. HeLa cell Ul snRNPs purified through all but the final hydrophobic column chromatography step have been used routinely here. This snRNP fraction is free of RNA other than Ul , and snRNP proteins constitute about 40% of the protein present (Hinterberger et al., 1983).

Natural splicing substrates are generally long (several kilobases), short lived, and difficult to isolate as pure RNA species. To obtain an RNA which contained splice site sequences, was of moderate length, and could be readily

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radiolabeled, we constructed a template for in vitro transcription of an RNA which contains primarily /3-globin sequences. Since T7 RNA polymerase displays extreme specificity in promoter utilization and synthesizes long RNAs without premature termination (Rosa, 1979; Studier and Dunn, 1983) we chose to clone a block of the major mouse fl-globin gene downstream of a T7 RNA polymerase class III promoter. (See Figure 1 and Experimental Proce- dures for a detailed description of the construction of pAK105.) The 508nucleotide pAK105 transcript has the triphosphate 5’ end characteristic of T7 transcripts and a nonpolyadenylated (“run-off”) 3’ end, but it also contains the entire small intron from the major mouse P-globin gene together with 118 nucleotides of mouse sequence before the 5’ splice site and 208 nucleotides of mouse sequence following the 3’ splice site (Figure 1 B).

We used pAR864 to generate control transcripts. This plasmid, a gift of Dr. F. William Studier, contains a T7 RNA polymerase class Ill promoter inserted at the BamHl site of pBR322 (see Studier and Rosenberg [ 19811 for the details of pAR864 construction). Transcription of pAR864 pro- ceeds counterclockwise around the standard map of pBR322 and runs off, in this case, at the unique Hindlll site. The resulting RNA contains no sequence with a match of six or more nucleotides including the invariant GT to the nine-nucleotide 5’ splice site consensus. A typical transcription of these plasmids yielded generous amounts (normally 2-5 pg at 2 X lo6 cpm =P/pg.) of a relatively pure RNA species. This RNA was purified further by gel electrophoresis before use in binding studies.

In Vitro Binding of RNA by Ul snRNPs Binding reactions were routinely carried out at 0°C with concentrations of approximately 5 nM Ul snRNP and 2 nM transcript in a buffer containing 12.5 mM magnesium and 100 mM salt. Binding of the 3zP-labeled RNA to Ul snRNPs was assayed by immunoprecipitation of the RNPs with human autoimmune sera of the anti-Sm or anti- (Ul)RNP type, or with mouse monoclonal antiSm antibodies (Lerner et al., 1981). Antibodies were added just after the addition of a lOOO-fold excess (15 rg) of unlabeled competing RNA (E. coli 16s rRNA), which served to pre- vent further binding of the labeled RNA to snRNPs.

Figure 2 compares Ul snRNP binding to the run-off transcript from pAK105 with that from pAR864. In this experiment, 13% of the pAK105 transcript and 2% of the pAR864 transcript were bound after 2 hr. About half of the pAK105 binding occurred within the first 5 min, while pAR864 binding after 5 min was negligible. Thus, despite the fact that the two RNAs used in this experiment are of approximately equal length and were present in approximately equal concentrations, the transcript which contains the mouse j3-globin intron was bound at least 5-fold better and considerably more rapidly than the control transcript.

The addition of 20 pg of competing 16s RNA to the pAK105 transcript before the addition of Ul snRNPs, the use of control serum, or the omission of either Ul snRNPs

or antibodies resulted in the precipitation of only background amounts of =P (data not shown). In other experiments, binding was found to be surprisingly insensitive to both ionic strength and magnesium concentration. Fur- thermore, the use of a fraction containing purified U2, U4, U5, and U6 snRNPs (Hinterberger et al., 1983) rather than Ul snRNPs likewise failed to produce evidence of any binding interaction. Note (Figure 2) that the background in these binding reactions, which probably represents trapping of RNA in the Pansorbin pellet (see Experimental Procedures), is the same for pAK105 and pAR864 and varies less than 5% from sample to sample. On a number of occasions, the pAK105 transcript was examined by electrophoresis following incubation with the Ul snRNP. Even when incubation was performed at elevated temper- atures (25, 30, 33, or 37°C) in the presence of ATP and GTP, no cleavage or splicing was ever observed (data not shown, but see Figure 5); these experiments only served to demonstrate the absence of any nuclease in the Ul snRNP fractions.

In experiments like that shown in Figure 2, between 7 and 16% of the pAK105 transcript was observed to be bound by Ul snRNPs. This apparently inefficient binding can be shown to result from the experimental protocol. When identical binding reactions (but lacking 16s rRNA) were assayed on 5-20% sucrose gradients (instead of by Pansorbin precipitation), greater than 80% of the pAK105 transcript was displaced to the bottom of the gradient (data not shown). This displacement was dependent upon the presence of both Ul snRNPs and antibodies, and was not observed with control serum. Thus, in an optimal binding assay, the majority of the pAKl05 transcript can be shown to interact with Ul snRNPs, but sucrose gradients are not suitable for time course or other experiments involving many samples.

Ul snRNPs Selectively Bind the 5’ Splice Site Localization of the Ul snRNP binding site on the pAK105 transcript was accomplished by ribonuclease treatment of the snRNP-RNA complex, immunoprecipitation of the resulting trimmed complex, and fingerprinting of the immunoprecipitate. The T, ribonuclease fingerprint of the entire Barn run-off transcript from pAKl05 is shown in Figure 3A. Secondary analyses using pancreatic ribonuclease allowed identification of most of the oligonucleotides in this fingerprint and revealed no discrepancies with the sequence previously reported (Konkel et al., 1978; Konkel et al., 1979; Dunn and Studier, 1983) which is presented in Figure 1B. Figure 38 shows a fingerprint of the immunoprecipitate obtained after T, ribonuclease digestion of the Ui snRNP-pAK105 RNA complex. The three oligonucleotides present, CAG, UUG, and UAUCCAG, can be found together in a single region which has the sequence GGGCAG/GUUGGUAUCCAGGU and encompasses the 5’ splice site. Thus, an RNA fragment of 15-17 nucleotides, indicated by underlining in Figure 1, can be recovered associated with the Ul snRNP after nuclease

Splice Site Recognition in Vitro 511

I3

1 2n AAGGT CCCTA AATTA ATACG ACTCA CTATA GGGAG AT/'&G GGCCT TTACG

ATTAT TACTT TAAGA TTTAA CTCTA AGAGG AATCT TTATT ATGTT GAC:: -- -- ' Hint II

ACAAC CCCAG AAACA GACAT CATGG TGCAC CTGAC TGATG CTGAG AA;;;

170 TGCTG TCTCT TGCCT GTGGG GAAAG GTGAA CTCCG ATGAA GTTGG TGGTG

l 220

AGG~~ CTGGG CA(;CT TGGTA TCCAG GTTAC AAGGC AGCTC ACAAG AAGAA --

GTTGG GTGCT TGGAG ACAGA GGTCT GCTTT CCAGC AGACA CTAAC TT:;:

l 320

GTGTC CCCTG TCTAT GTTTC CCTTT TTAGG CTGCT GGTTG TCTAC CCTTG

370 GACCC AGCGG TACTT TGATA GCTTT GGAGA CCTAT CCTCT GCCTC TGCTA

TCATG GGTAA TGCCA AAGTG AAGGC CCATG GCAAG AAGGT GATAA CT:::

470 TTTAA CGATG GCCTG AATCA CTTGG ACAGC CTCAA GGGCA CCTTT GCCAG

508 -- CCTCA GTGAG CTCCA CTGTG ACAAG CTGCA TGTGG ATCCA CAGGA CGGGT

Ernr

0 30 60 90 120 I50 MINUTES OF BINDING

Figure 2. Ul snRNP Binding to Globin and Control Sequences

The binding of Ul snRNPs to globin (pAK105, filled diamonds) and control (pAR864, empty diamonds) transcripts was measured as described in Experimental Procedures.

trimming of the RNARNP complex. (Note that T, ribonuclease has been used twice in this experiment: originally in the digestion of the RNPRNA complex and again in the fingerprinting of the immunoprecipitate.) Note also that the large oligonucleotide which immediately abuts the 3’ splice site is not recovered in immunoprecipitated material. The position of this oligonucleotide (UUUCCCUUUUUAG) in the T, fingerprint is indicated by an arrow in Figure 3A; the 3’ splice site is indicated by an asterisk in Figure 1 B.

To demonstrate that the three oligonucleotides present in the fingerprint shown in Figure 38 were contained within a continuous RNA fragment, the immunoprecipitated RNA obtained from the nuclease-treated snRNP-RNA complex was first run on a 10% polyacrylamide gel (Figure 3C). Individual RNA bands were then excised and fingerprinted. The major band (starred in figure 3C) has a T, fingerprint containing all four of the oligonucleotides expected from a complete T, ribonuclease digest of the 15-17.nucleotide region: G, CAG, UUG, and UAUCCAG (Figure 3D). (The transcript used in this experiment was made using a-%P- UTP in addition to (u-32P-GTP. Consequently, an additional nucleotide, Gp, appears in fingerprint 3D due to nearest neighbor transfer of label.) The smaller of the two less abundant larger fragments (arrows) contained the addi-

Frgure 1. Structure of the Template pAK105

A, In order to place globrn rntron sequences under the control of a T7 RNA polymerase promoter $65, the Hincll fragment of the mafor mouse fl-globrn gene (given to us by Dr. Charles Weissmann) was ligated to the unique Hpal sate of ~06 (grven to us by Dr. Margaret Rosa), generating pAK103. The large Sau3A fragment of pAK103 was then ligated into the unique BamHl site of pBR322, generating pAK105. 8, the sequence of pAK105 surrounding the transcribed region is taken from Konkel et al. (1979) and Dunn and Studrer (1983). Numbering beings with the Initiating nucleotide of the T7 RNA polymerase transcript (Rosa, 1979). The Hincll site at nucleotide 65 represents the boundary between T7 and mouse sequences; Its structure was confirmed by$equence analysis using pAK105 DNA (see Experimental Procedures). The BamHl site at nucleotide 508 specifies the 3’ end of the (run-off) transcript used fn the experiments described here. Asterisks over nucleotrdes 184 and 299 designate the 5’ and 3’ termfni of the small P-globrn intron. The underlinrng of nucleotides 181 through 195 Indicates the regron which remains associated with the Ul snRNP after T, ribonuclease digestion

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C

n

Figure 3. Ul snRNPs Selectively Bind the 5’ Splice Site

A. a Tr RNase fingerprint of the intact pAK105 transcript was obtained as described by Barrel1 (1971). pH 3.5 electrophoresis was from right to left and homochromatography was from bottom to top. The small discrete spot in the upper right is an ink spot. The oligonucleotide UUUCC- CUUUUUAG, which represents the extreme 3’ terminus of the @globin intron, is indicated by an arrow. Individual oligonucleotides were identified by mobility and secondary pancreatic RNase digestion (Barrell. 1971). 6, a T, RNase fingerprint was made from pAKlOS-derived RNA which remained bound to the Ui snRNP following partial digestion of the transcript-U1 snRNP complex with T, RNase. pH 3.5 electrophoresis was from right to left and homochromatography was from bottom to top. Individual oligonucleotides were identified by mobility and secondary pancreatic RNase digestion (Barrell, 1971). C, pAK105-derived RNA which remained bound to the Ul snRNP following partial digestion of the transcript-U1 snRNP complex with T, RNase was electrophoresed through a 10% polyacrylamide (29:i acrylamide:N,N’- methylene bisacrylamide), 7 M urea, 90 mM Tris- borate (pH 8.3). 1 mM EDTA gel and vrsualized by autoradicgraphy. “ori” indicates the top of the gel. “XC” and “BP” indicate the positions of the xylene cyanol FF and bromophenol blue marker dyes. Individual bands (arrows and star) were eluted and fingerprinted. D, a T, RNase fingerprint of the RNA in the starred band (see C) was obtained as described by Barrel1 (1971). pH 3.5 electrophoresis was from right to left and homochromatography was from bottom to top. Individ- ual oligonucleotides were identified by mobilrty and secondary pancreatic RNase digestion (Barre4 1971).

tional T, oligonucleotide UUACAAG, showing that it ex- tends at least eight nucleotides further into the intron than the predominant protected fragment. In addition to all of the above oligonucleotides, the yet larger minor fragment contained CUCACAAG, which occurs another 12 nucleotides into the intron. Thus, the 5’ splice site can be isolated following ribonuclease digestion of the snRNP-RNA complex as a nested set of intact fragments extending different distances into the intervening sequence. However, the use of lower concentrations of T, ribonuclease in the protection experiment (not shown) yielded the additional oligonucleotide CCCUG, which occurs 5’ to the splice site.

To determine if any sequence other than that in the protected region itself is necessary for the initial binding but does not remain associated with the Ul snRNP, we studied the interaction of the Ul snRNP with a pAK105 transcript partially fragmented by alkali. The rationale for this experiment is that if sequences (such as the 3’ splice site) which are remote from the 5’ splice site are required for the Ul snRNP to bind the 5’ splice site, then these sequences should be present on all of the fragments selected by the Ul snRNP from a random pool. Accord- ingly, a collection of randomly terminated fragments with a broad size distribution were generated and allowed to


bind the Ul snRNP. Complexes were immunoprecipitated and pAKl05-derived RNA from both the immunoprecipitate and supernatant were displayed on the 10% polyacrylamide gel shown in Figure 4. It is clear that Ul snRNPs preferentially bound fragments which, as a population, are longer (averaging about 120 nucleotides) than both the bulk of the starting material (less than 40 nucleotides) and the 15-17nucleotide protected fragment. RNA from regions of the gel designated A, 8, and C was eluted and subjected to T, ribonuclease fingerprint analysis. As expected, each region comprised a group of RNAs rather than a single species. Each fingerprint also showed marked over-representation (1 O-fold or greater) of oligonucleotides occurring within the length of the RNA from the 5’ splice site. For example, the mixture of RNA molecules from the region of the gel marked A (all roughly 135 nucleotides in length) had a fingerprint which included ACUCACAACCCCAG (nucleotides 67-80) but not AAUC- UUUAUUAUG (nucleotides 51-63) and UUUCCCUU- UUUAG (nucleotides 285-299) but not ACCCAG (nucleotides 322-327). Those molecules which contributed an oligonucleotide beginning at nucleotide 67 could have extended no further than nucleotide 202 in the 3’ direction and those which contributed an oligonucleotide ending at 299 could have extended no further than nucleotide 164 in the 5’ direction. The simplest explanation for these results is that fragments containing the 5’ splice site are bound by the Ul snRNP without regard to the relative position of the splice site within the fragment. Collective data from the three RNA populations identify a region no longer than 25 nucleotides, including the 5’ splice site, as present on all selected fragments. Thus, it appears that sequences at or near the 5’ splice site itself are sufficient for Ul snRNP binding.

snRNP Proteins Contribute to Splice Site Binding To determine whether Ul snRNP proteins are required for RNA binding, we first examined the ability of purified protein-free Ul RNA to compete for binding. In two experiments, addition of Ul RNA at the beginning of a standard binding reaction did not lead to inhibition of pAK105 binding by the Ui snRNP particle. For example, in an experiment in which each measurement was made in triplicate, and the background precipitation was 883 f 10 cpm, a standard binding led to 1992 rf: 127 cpm while a reaction in which Ul RNA was present in 2-fold molar excess over Ul snRNP led to 1988 f 170 cpm. Thus, we can conclude that Ul RNA does not effectively compete with Ul snRNP for binding to the pAK105 transcript.

It has been observed that partial proteinase K digestion of Ul snRNPs does not destroy their immunoprecipitability (M. Rosa, unpublished experiments). We have used this observation to confirm that Ul snRNP proteins are essen- tial for binding of the pAK105 transcript. Ui snRNPs were treated with proteinase K for 1 hr on ice and the protease was inactivated by addition of PMSF. In a control preparation, PMSF was added prior to proteinase K. Both of

these Ui snRNP fractions, as well as untreated Ui sn- RNPs, were then tested for their ability to bind the pAK105 transcript. The results, shown in Figure 5A, reveal that partial proteinase K treatment abolishes the rapid interaction of Ul snRNPs with the pAKlO5 transcript and reduces the binding at 2 hr by 80%.

To demonstrate that a loss of immunoprecipitability by the Ul snRNP was not responsible for the results obtained in the protease experiment (Figure 5A), a single time point (30 min of binding) was repeated. RNAs from the immunoprecipitates and supernatants from both the treated and control RNPs were examined by electrophoresis on a 7.5% polyacrylamide gel followed by 1) visualization by ethidium bromide staining of the gel (not shown), 2) transfer to diazotized paper and autoradiography (lanes 1 through 4) and 3) probing with the nick-translated Ul DNA HRMp8 (lanes 5 through 12). Autoradiography prior to probing located the radioactive pAK105 transcript and confirmed the counting data of Figure 5A, indicating that the transcript was bound by the controlltreated but not the protease- treated snRNPs. Note that these lanes also show that the size of the pAK105 transcript is not altered during the procedure, thereby confirming our previous observation that no cleavage or splicing reactions were catalyzed by the isolated Ul snRNPs themselves under these conditions. Autoradiography after probing revealed that the immunoprecipitates, shown in lanes 5 through 8, contain roughly equal amounts of apparently undegraded Ul RNA while the corresponding supernatants, shown in lanes 9 through 12, do not contain detectable Ul RNA. This demonstrates that the failure of protease-treated RNPs to bind the pAK105 transcript is not due to degradation of the Ul RNA or loss of immunoprecipitability by the Ul snRNPs.

An experiment complementary to the demonstration that intact snRNP proteins are required for splice site binding involves nuclease treatment of the Ul snRNP to destroy its RNA moiety. Here we used micrococcal nuclease, which has an absolute dependence on calcium, which in turn is specifically chelated by EGTA. Ul snRNPs were treated with micrococcal nuclease in the presence of calcium or calcium plus EGTA. After EGTA was added to the digested snRNPs (so that nuclease activity would not continue beyond the defined time of digestion), the nuclease-treated and control RNPs were compared in binding experiments (not shown) identical to those presented in Figures 2 and 5A. Surprisingly, the two sets of RNPs showed identical ability to bind the pAK105 transcript. However, while the control snRNPs (like untreated RNPs; Figure 2) did not bind the pAR864 RNA, the nuclease- treated RNPs bound the pAK105 and pAR864 transcripts equally. Furthermore, protection experiments like those in Figure 3 revealed that the control RNPs behaved like untreated RNPs in protecting specific oligonucleotides around the 5’ splice site, while the nuclease-treated sn- RNPs “protected” all oligonucleotides of the pAK105 transcript. An attractive explanation for these results is that Ul

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ori-

BP

SUP M PPT

IA

IB

IC

Frgure 4. Binding and lmmunoprecipitation of pAK105 Transcript Frag- ments

T7 RNA polymerase transcripts of pAK105 were fragmented with alkali and assayed for brnding to Ul snRNPs as described in Experimental Proce- dures. Unbound pAK105-dewed RNA is displayed in the lane marked “SUP” and bound RNA is displayed in the lane marked “PPT.” Molecular weight markers (HeLa cell RNA) are displayed in the lane marked “M” and

RNA functions in the snRNP by conferring specificity upon an RNA-binding domain comprised largely of snFlNP proteins. However, an alternative possibility is that nuclease treatment removes Ul RNA from binding sites on the snRNP proteins which then interact nonspecifically with the pAK105 transcript.

Discussion

5’ Splice Site Recognition in Vitro We have demonstrated the ability of purified Ul small nuclear RNA-protein complexes to bind a 5’ splice site selectively in vitro. We take this result to be strong support for the hypothesis that Ul snRNPs act to identify 5’ splice sites in vivo.

The observed specificity of binding indicates that the most 5’ region of Ul RNA is unique in its capacity for direct interaction with other RNAs. A computer-generated sequence comparison of Ul RNA and the pAKl05 transcript reveals that the transcript has seven stretches ca- pable of forming seven or eight contiguous Watson-Crick base pairs with portions of Ul RNA not at the 5’ end. (Approximately this number of long complementary stretches is expected on a random basis for two RNAs of this length.) However, none of these other potential interactions leads to protection and immunoprecipitation of the nucleotides involved, suggesting that the regions of Ul RNA involved are less available in the snRNP than is the 5’ end.

It is also interesting to note that several sequences within the pAKlO5 transcript could base-pair with the 5’ end of Ul RNA but are not protected. One of these, CCAGGU (nucleotides 192-197) should, according to the base- pairing rules of Tinoco et al. (1973) have a higher affinity for the 5’ end of Ul than does the bona fide 5’ splice site which is protected. The lack of snRNP binding to the CCAGGU sequence is clear from the absence of the oligonucleotide UUACAAG in the fingerprints shown in Figure 3, B and D. (This oligonucleotide runs very close to UAUCCAG, which is in the protected fragment, but can be shown to be absent by the lack of AAG in secondary pancreatic digests of the spot; see,Experimental Proce- dures.) Thus, Ul snRNP binding in vitro appears to mimic splice site use in vivo faithfully despite the failure of such splice site selection to follow easily understood rules.

lmmunoprecipitation of the 3’ splice site as a complex with the Ul snRNP has not been observed. However, it is not possible to conclude beyond doubt that Ul snRNPs do not recognize 3’ splice sites. The immunoprecipitation protocol used for both quantitative transcript binding and T, ribonuclease protection experiments involves the use of conditions (such as extreme dilution and the presence

the length in nucleotides of prominent bands IS rndrcated at left “XC’ and “BP” rndrcate the positions of the xylene cyanol FF and bromophenol blue marker dyes. Thus gel was identical in composition to that described rn the legend to Figure 3C.


A

MINUTES OF BINDING

precipitate hybridized

precipitate hybridized supernatant

I234 5678 9 IO II 12

ori - - ori

pAKIO5 RNA- - pAKIO5 RNA

UI RNA- -UI RNA

Frgure 5. Inhibitron of Binding by Pretreatment of Ul snRNPs wrth Proteinase K

A, the binding of protease-treated (empty dra- monds) and control (filled diamonds) Ul snRNPs to the pAKIO5 transcript was measured as described in Experimental Procedures. B, the immu- noprecipitabrlrty of Ul snRNPs pretreated with proteinase K was examined by electrophoresrs. Protease-treated (lanes 3, 4, 7, 8. 11, and 12) and control (lanes 1, 2, 5, 6, 9. and 10) Ul snRNPs were allowed to bind the pAK105 transcript for 30 min (even-numbered lanes) or prevented from binding by pre-addrtion of E. coli 16s rRNA (odd- numbered lanes). RNA from the immunoprecrpl- tates (lanes 1-8) and supematants (lanes 9-12) was then electrophoresed through a 7.5% polyacrylamide (29.1 acrylamide,N,N’-methylene brs- acrylamrde). 7 M urea, 90 mM Tris-borate (pH 8 3), 1 mM EDTA gel. Transfer to diazobenzyloxymethyl paper was followed by autoradrography (lanes I- 4) or hybridization to nick-translated Ul DNA and autoradiography (lanes 5-12). The material at the ongrn in lanes 9-12 was observed on autoradrography before probing (not shown) and therefore must represent pAK105 transcript. Thus RNA pre- sumably failed to enter the gel because of the overloadrng of 16s rRNA rn these supernatant lanes (see Experrmental Procedures).

voteinase - - + + - - + + - - + l

pre-binding + - + - l - + - + - + -

of a large excess of competing RNA) which preclude the detection of any binding which has a half-life of less than about 10 min. It is also possible that Ul snRNP binding to 3’ splice sites blocks the antibodies used to detect binding. Furthermore, these studies have yet to be extended to RNAs containing introns other than the one studied here. Nevertheless, our failure to detect 3’ splice site binding strengthens a pre-existing idea that the Ul snRNP interacts primarily with 5’ splice sites. This tentative con- clusion was based on the nature of 3’ splice site consensus and the lack of conservation of those nucleotides within Ul which were originally proposed to recognize the pyrimidine-rich stretch which precedes 3’ splice sites (Mount and Steitz, 1981). What might recognize the conserved pyrimidine-rich sequence preceding 3’ splice sites remains an interesting question.

Binding Requires Ul snRNP Proteins The fact that intact RNP proteins are required for Ul snRNPs to bind the pAK105 transcript argues that the RNP as a whole is designed to bind sequences complementary to the 5’ end of Ul RNA. Although we have not proven that the 5’ end of Ul RNA is specifically involved in binding this 5’ splice site, other alternatives seem unlikely. The 5’ maG of Ul RNA is accessible to antibodies while in the RNP (Bringmann et al., 1983) the first 11

nucleotides of Ul RNA are almost certainly single-stranded (Branlant et al., 1981; Mount and Steitz, 1981) and this region is complementary to the 5’ splice site consensus. A most attractive idea is that there exists a protein surface or pocket on the RNP with general affinity for RNA, to which the 5’ end of Ul RNA contributes specificity.

Specific versus Nonspecific Binding In addition to their capacity for highly specific recognition of a particular sequence, Ul snRNPs have a significant ability to bind RNA nonspecifically. In a titration experiment (not shown), the binding of Ul snRNPs to the pAK105 transcript (at a concentration of roughly 0.7 pg/ml) was reduced to half by the presence of either E. coli 16s rRNA or brewer’s yeast tRNA at approximately 5 pg/ml. This roughly IO-fold weaker binding by nonspecific RNA is consistent with the comparison of binding between the pAK105 and pAR864 transcripts shown in Figure 2. We do not know whether this nonspecific binding represents binding at secondary sites or truly nonspecific binding. Such competition by nonspecific RNA for Ul snRNP binding, weak binding of Ul snRNPs to RNA free of splice site sequences (the pAR864 transcript), and enhancement of this nonspecific binding by predigestion of the RNPs with micrococcal nuclease all suggest that the Ul snRNP pos- sesses a general RNA-binding capacity.

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Splice Site Recognition and the Complete Splicing Reaction

Splicing patterns observed in a number of artificially constructed genes indicate that splice sites are autonomous rather than coupled signals for splicing in vivo. Deletion of material to within a few nucleotides of either 5’ or 3’ splice sites usually has no effect on splicing (see Elder et al., 1981, and Mount and Steitz, 1983, for reviews of these data). Furthermore, 5’ splice site sequences are functional when paired with 3’ splice sites from other genes or other regions of the same gene (Khoury et al., 1980; Chu and Sharp, 1981; Young et al., 1982). The in vitro results obtained here are consistent with these observations. In particular, the experiment shown in Figure 4 strongly sug- gests that the splice site itself is sufficient for Ul snRNP binding. Thus, the Ul snRNP-5’ splice site complex probably serves as the true substrate for further stages in splicing. In particular, we would like to propose a splicing mechanism in which the RNA-RNA helix formed between the 5’ splice site and the 5’ end of Ul RNA is subsequently recognized by a splicing nuclease or nick ligase.

There also exists a number of examples in which either sequences some distance from splice sites affect the efficiency or fidelity of splicing (see, for example, Spence et al., 1982; Khoury et al., 1979) or the loss of a natural splice site leads to the use of “cryptic” splice sites (Felber et al., 1982; Treisman et al., 1982; Weiringa et al., 1983). Thus, superimposed on the ability of a particular sequence to act as a splice site are other criteria which dictate how efficiently that splice site will be used and with which splice site of the opposite type it will pair. Our results strengthen the hypothesis that the Ul snRNP is responsible for 5’ splice site recognition in vivo, but do not attempt to address the questions of splice site choice and pairing.

In vitro systems that carry out complete splicing reactions, such as those described by Kole and Weissman (1982), Goldenberg and Raskas (1981), and Padgett and Sharp (personal communication), are needed to explore mRNA splicing in its totality. We hope to build on our present system by adding preformed snRNP-RNA complexes to such extracts and by supplementing the snRNP- RNA complex with other purified components. In this way, the reaction described here may provide the basis for a more detailed understanding of messenger RNA splicing.

Experimental Procedures

Plasmids, Antisera, and Enzymes pAKlO5 was constructed by standard cloning methods as depicted in Figure IA. Starting materials were pC6, a gift from Dr. Margaret Rosa, and pM@G, a gift from Dr. Charles Weissmann. The structure of pAK105 was partially confined by a single Maxam and Gilbert (1980) sequencing run using DNA labeled at the Avail site (position -25). pAR864 was obtained from Dr. F. William Studier. HRMp8 is a clone of human genomic Ui DNA provided by Dr. Ian Eperon and originally derived from pHSD2 (Mansar and Gesteland, 1982). E. coli 16s rRNA was a gift from Dr. Peter Moore.

Sera from patients with systemic lupus erythematosus were provided by Dr. J. Hsrdin. Antibodies were prepared from sera, and their specrfrcrtres were determined, as described by Hendrick et al. (1981). The monoclonal

anti-Sm antibody was prepared from ascttes fluid as described (Lerner et al., 1981).

Restriction enzymes and DNA polymerase were purchased from New England Biolabs and used as directed by the supplier. DNA ligase was purchased from Boehringer Mannheim. Proteinase K was obtained from Beckman. DNase I and micrococcal nuclease were from Worthington. T, and pancreatic RNases were obtained from Calbiochem. T7 RNA polymerase was a gift from Dr. Margaret Rosa.

Ui snRNPs were prepared from HeLa cells as described by Hinterberger et at. (1983) through the second DEAE cdumn. These snRNPs were sometimes concentrated further using a third DEAE cdumn. In such cases, the Ul snRNPs were loaded in buffer A (15 mM MgClp, 20 mM Tris (pH 7.0). 5 mM 2-mercaptoethanol) plus 50 mM NH&I, eluted with buffer A plus 200 mM NH&I and dialyzed against buffer A plus IOU mM NH&I. Ui snRNP concentrations were estimated by the intensity of ethidium bromide staining of extracted Ui RNA which was electrophoresed on a 5% poly- acrytamide gel adjacent to a series of concentrations of a 16s rRNA standard. Ul snRNPs were stored up to 1 month at 4’C.

Preparation of the pAK105 and pAR884 Transcripts Transcription was performed as described by Rosa (1979) except that each reaction contained 3 pg of DNA and 100 &i of a-?-GTP. Following incubation for 30 min at 37°C the transcription reactions were adjusted to contain 200 mM EDTA, 1 M urea, 0.025% xylene cyan01 FF, and 0.025% bromophenol blue, were heated to 65°C for 2 min, and were efectropho- resed through a 5% polyacrylamide (29~1 acryfamide:N,N’-methylene bisacrylamide). 7 M urea, 90 mM Tris-borate (pH 8.3) 1 mM EDTA gel. RNA was eluted by soaking a slice of the OX-mm gel as described by Maxam and Gilbert (1980).

Analysis of Binding between Ul snRNPs and pAKlO5 or pAR884 Transcripts Fortymicroliter mixtures of 5 nM Ul snRNPs, 2 nM transcript (24,ooO Cerenkov cpm in the experiment shown in Figure 2; 11,009 cpm in Figure 5) 12.5 mM MgCI,, 15 mM Tris (pH 7.1) 50 mM NH&I, 50 mM NaCI, and 2.5 mM 2-mercaptoethanol were incubated on ice. Fifteen micrograms of 16s rRNA in 1.5 ~1 of HZ0 were added to terminate binding; points at zero time correspond to the addition of 16.5 rRNA before initiation of the reaction by addition of Ul snRNPs. In time course experiments, incubations were staggered such that all samples could be processed together. Next, IO pl of antiserum specific for the Ui snRNP were added. Unless stated other- wise, the antibody used in these experiments was a particular patient antiserum (AM) which displays both anti-(Ul)RNP and anti-Sm activities. Two hundred microliters of Pansorbin (formalin-fixed S. aureus, Calbiochem) in 150 mM NaCI, 50 mM Trts (pH 7.5). 0.05% NP40 were added and after 30 additional min the mixture was spun for 60 set in Brfnkmann Microcen- trifuge 5412. The supernatant was carefully removed and Cerenkov radia- tion was measured with an Intertechnique scintillation spectrometer. A single centriiugation of formalinfixed S. aureus results in significant nonspecific trapping of radiolabeled RNA. Accordingly, the procedure just described produces a high, but reproducible, background. This background could be eliminated by repeated washings of the Staphylococcus pellet. However, such washings were found to introduce variability and were not generally performed.

In the case of T, ribonuclease protection experiments, 10 pg of T, RNase were added to a 206~1 binding reaction after the Pansorbin pellet had been washed five times wfth 0.5 ml of 150 mM NaCI, 50 mM Trfs (pH 7.5) and 0.05% NP40. After 15 min of digestion on ice, the precipitate was washed an additional four times, extracted with phenol:chloroform:isoamyi alcohol (5050:1), and fingerprinted.

T, Ribonuclease Fingerprints Frngerprinting was performed as described by Barrel1 (1971) with thin layer chromatography on PEI 300 (Brinkmann) for the second dimension. Oligo- nucleotides were subsequently eluted and analyzed by digestion with pancreatic nuclease. followed by separation on DEAE paper at pH 3.5 (Barrell, 1971).

Splice Site Recognition in Vttro 517

Computer Analysis Regions of potential complementarii between Ul and the pAKlO5 transcript were identified by use of the OVRLAP program described by Staden (1979).

Alkaline Digestion Conditions To generate the RNA used as starting material in the experiment shown in Figure 4, 2 x 106 cpm of the pAK105 transcript were equally divided into four 50.pl aliquots of 10 mM NaHC03, pH 10.6. These were heated at 90°C for 20 set, 2 min. 12 min, and 22 min, recombined, and brought to 300 mM sodrum acetate, pH 6.0. Three volumes of ethanol were added and the precrpitate was used directly in a 400.pl binding reaction.

Proteinase K Treatment of Ul snRNPs Ui snRNP fractions in buffer A plus 100 mM NH&I were digested with proteinase K at a concentration of 1 mg/ml for 1 h on ice, at which time the protease inhibitor PMSF was added to 3 mM. PMSF was added just before proteinase K in the control digest.

Transfer and Hybridization Transfer of RNAs to diazobenzyloxymethyl paper was performed according to Alwine et al. (1979) as modified by H. Hottinger, C. Berg, J. Lapointe, D. Pearson, and D. Soll (unpublished data). Hybridization of nick-translated plasmid to RNA on paper was performed as described by Alwrne et al. (1979). Nick translatron was performed as described by Rigby et al. (1977).

Micrococcal Nuclease Treatment of Ul snRNPs Ul snRNP fractions In buffer A plus 100 mM NH&l were drgested with 0.28 mg/ml micrococcal nuclease at 30% for 60 min in the presence of 3 mM CaCI. To terminate the digest, EGTA was added to 9 mM. In the control digest, EGTA was added before the nuclease and was present during the incubation.

We thank Dr. James Stefano for discussions; Ellen Gottlieb. Sandra Wolrn, Dr. Ian Eperon, and Dr. James Stefano for their critical reading of the manuscript; Martha Krikeles for help with sequencing; and Dr. Ian Eperon for providing the Staden programs and HRMp8. Dr. J. Hardin (Yale Univer- sity) kindly provided us with patients’ sera. We are grateful to Dr. Margaret Rosa for providing Tr RNA polymerase and ~06, Dr. Peter Moore (Yale University) for providing us with 16s rRNA, Dr. F. William Studier (Brook- haven Natronal Laboratory) for providing pAR864, and Dr. Charles Weiss- mann (Universrty of Zurrch) for provrding pM/3G. Drs. C. Weissmann, T. Manratrs (Harvard Unrversity). J. Rrnke (Max Planck Instrtute), and J. Dunn (Brookhaven) are appreciated for their communication of results prior to publication. Thus work was supported by grants from the National Science Foundation and National Institutes of Health.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to Indicate this fact.

Received March 20. 1983

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the u1 small nuclear rna-protein complex selectively binds a 5′ splice site in vitro

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