cell, vol. 45, 869-877, june 20, 1986, copyright 0 1986 by ... · onto a deae membrane. the deae...

Cell, Vol. 45, 869-877, June 20, 1986, Copyright 0 1986 by Cell Press

Specific Small Nuclear RNAs Are Associated with Yeast Spliceosomes

Claudio W. Pikielny’ and Michael Rosbashl ‘Department of Biochemistry TDepartment of Biology Brandeis University Waltham, Massachusetts 02254

Summary

Two different methods have been devised for the analysis and purification of spliceosomes formed in a yeast in vitro splicing system. The first method relies on the electrophoretic separation of ribonucleoprotein particles in composite acrylamide-agarose gels. A large fraction of added substrate is located in spliceosomes, the formation of which can be shown to be dependent on the presence of both a yeast 5’ splice junction and a TACTAAC box on the RNA substrate. The second method relies on oligo(dT)-cellulose chromatography of spliceosomes formed with a polyadenylated substrate. Purification of spliceosomes by either method indicates that at least three small nuclear FlNAs, approximately 160, 165, and 215 nucleotides in length, are specifically associated with yeast spliceosomes.

Introduction

During the last several years, a two step splicing pathway for introns of RNA polymerase II transcripts has been de- fined, both in the yeast Saccharomyces cerevisiae and in higher organisms (Ruskin et al., 1964; Padgett et al., 1984; Domdey et al., 1984; Rodriguez et al., 1984; Zeitlin and Ef- stratiadis, 1984; Konarska et al., 1985; Newman et al., 1985; Lin et al., 1985). The first step consists of cleavage at the 5’ splice site and formation of a lariat intermediate containing a 2’-5’ phosphodiester bond between the 5’ end of the intron and an internal site, the branchpoint, close to the 3’ end of the intron. In yeast, the branch occurs at the last adenosine residue of a highly conserved heptanucleotide, the”TACTAAC box”(Domdey et al., 1984; Rodriguez et al., 1984; Newman et al., 1985; Lin et al., 1985). The second step consists of cleavage at the 3’ splice site and exon ligation.

More recent studies have demonstrated that in vitro splicing of pre-mRNAs, both in yeast and metazoa, takes place in large structures (4OS-60s) called spliceosomes (Brody and Abelson, 1985; Grabowski et al., 1985; Fren- dewey and Keller, 1985; Bindereif and Green, 1988). As- sembly of these large ribonucleoprotein (RNP) particles has the same requirements as the splicing reaction itself, although partial complexes may form on RNAs containing only a 5’ splice junction (Grabowski et al., 1985) or only a branchpoint (Frendewey and Keller, 1985), or they may form even in the absence of added ATP (Frendewey and Keller, 1985). In HeLa cell nuclear extracts, U small nuclear RNPs (U snRNPs) are required for spliceosome as-

sembly (Frendewey and Keller, 1985), and they can be shown to be an integral part of the spliceosome by precipitation with different anti-snRNP antibodies (Grabowski et al., 1985). The final complexes, the mature spliceosomes, are characterized by the presence of the RNAs produced by the first step of the splicing reaction (free exon 1 and intron-exon 2 in a lariat configuration) as well as the lariat intron produced by the second step. The other product of the second step, mRNA, seems to be released or to be more loosely associated with spliceosomes (Brody and Abelson, 1985; Frendewey and Keller, 1985; Bindereif and Green, 1988).

More generally, in the case of higher eukaryotes, a large body of data now supports the role of snRNPs, and in particular the Ul and U2 snRNPs, in the splicing of this class of pre-mRNAs. The most compelling evidence comes from the inactivation of in vitro splicing in extracts where either snRNA has been specifically cleaved by RNAase H in the presence of a complementary oligonucleotide (Kramer et al., 1984; Blacket al., 1985; Krainer and Mania- tis, 1985).

In contrast, no yeast snRNA has yet been assigned a specific function (Tollervey et al., 1983; Tollervey and Guthrie, 1985). In particular, efforts to find the homo- logues of the U class of snRNAs either by immunoprecipitation with sera from Lupus patients (Lerner et al., 1981) or by cross-hybridization with cloned genes have failed, perhaps because of the high degree of evolutionary diver- gence or the low abundance of yeast snRNAs (Wise et al., 1983).

By purifying spliceosomes from a yeast in vitro splicing system, we have obtained evidence that at least three specific snRNAs are present in yeast spliceosomes.

Results

Detection of Spliceosomes on RNP Gels To analyze splicing complexes that form after addition of substrate RNA to an in vitro splicing reaction (Brady and Abelson, 1985; Grabowski et al., 1985; Frendewey and Keller, 1985; Bindereif and Green, 1988), we adapted an electrophoretic separation method first devised for the analysis of native polysomes, ribosomes, and ribosomal subunits from Escherichia coli (Dahlberg et al., 1989). La- beled RNA was synthesized in vitro (Melton et al., 1984) from a plasmid containing an SP6 promoter followed by the 400 nucleotide rp5lA intron and flanking exon sequences (pSPrp51A). This RNA substrate was added to a splicing reaction containing whole-cell yeast extract prepared as described previously (Newman et al., 1985; Lin et al., 1985). Splicing was initiated by the addition of ATP and, at various times thereafter, aliquots were removed, the reaction was quenched, and samples were analyzed on both RNA and RNP gels (Figures 1A and lB, respectively). As a control, reactions were carried out in the absence of added ATT? In this experiment and others described below, the RNAs generated during incubation

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A I 77 400 320

+ATP -ATP B--m B +ATP -ATP

15” 2’ 5’ 10’ 20’ 30’ 15” 30’ Ilr *‘.

SP

U

Figure 1. Time Course of Appearance of Spliceosomes on RNP Gels

(A) A splicing reaction was carried out, in the presence or absence of 2 mM ATP, with an RNA substrate containing the full-length rp51A intron (generated from pSPrpBlA, see Experimental Procedures). At the indicated times, 10 ~1 aliquots were taken from the reaction mixture and were quenched by mixing with an equal volume of 0 buffer. Part of the reaction mixture was deproteinized and analyzed on a 5% polyacrylamide gel. The structure of the pSPrp51A transcript is shown at the top right; lengths in nucleotides are indicated. The different bands were identified by their mobilities relative to DNA size standards (data not shown), and their structures are shown schematically on the right. Exons are boxed, introns are lines. e,, exon 1; es, exon 2. (8) The rest of each sample was loaded directly on an RNP gel (see Experimental Procedures). The two RNP bands, U and SP, are discussed in the text.

were identified by their apparent molecular weights relative to RNA and DNA standards on polyacrylamide gels of various percentages (data not shown). They correspond to the expected intermediates (77 nucleotide exon 1 and 720 nucleotide intron-exon 2 lariat) and final products of a splicing reaction (400 nucleotide exon 1-exon 2 and 400 nucleotide lariat intron; see Figure 1A).

The lariat intermediate is first visible 5 min after the addition of ATP, and it reaches a maximum level at 10 min; production of the 5’exon shows similar kinetics. The products of the second step (exon 1-exon 2 and lariat intron) are first visible at 10 min and accumulate with time. RNP gel analysis shows that the addition of ATP has two dis- tinct effects. First, there is a very rapid disappearance of some slowly migrating material of heterogeneous mobility and a decrease in background radioactivity in the lane (compare the 15 set time points in the presence or absence of ATP). Second, there is the time-dependent appearance of a prominent RNP band (SP, standing for spliceosome) that requires 2 to 5 min to accumulate to its maximum level (Figure 16). Paralleling the appearance of the SP band is the disappearance of a band of higher mo-

bility, the U band (U stands for unspecific; this band is clearly retarded in comparison with naked RNA [data not shown]). The complexes formed immediately after the addition of RNA to the extract in the absence of ATP are likely to be unspecific, since they form on any RNA and can be dissociated by the addition of carrier (data not shown). Moreover, they disappear rapidly after the addition of ATl? Although the addition of ATP has complicated and multiple effects, it is required for rapid formation of a prominent SP band.

We tested whether RNA splicing signals (the 5’ splice junction and the TACTAAC box) are necessary for the appearance of this relatively homogeneous ATP-dependent band (SP), as might be expected of a splicing complex. We employed two mutant genes derived from the wild- type described above by small deletions: Y-0, a 67 nucleotide deletion including part of the 5’ splice junction; and A36, a 29 nucleotide deletion including the TACTAAC box. Transcripts from these genes are completely defective as splicing substrates, both in vivo (Pikielny et al., 1983) and in vitro (our unpublished data). Similarly, RNP gel analysis shows that neither transcript is incorporated into the SP

snRNAs in Yeast Spticeosomes 871

5'-0 W.T.

I

A3B

ATP - +I- +I- +

SP

U

Figure 2. Both the 5’ Splice Junction and a TACTAAC Box Are Neces- sary for the Formation of Spliceosomes

The three RNAs used in this experiment were run-off transcripts from the following plasmids: pSPrp51A (W. T); pSPrpSlA(5’-0) (identical to pSPrp51A except for a 87 bp deletion including part of the 5’ splice junction); and pSPrpSlA(ASl3) (identical to pSPrp51A except for a 29 bp deletion including theTACTAAC box [Pikielnyet al., 19833). Splicing reactions were carried out for 20 min in the presence or absence of 2 mM ATP as indicated and were analyzed on an RNP gel.

band or any other ATP-dependent band, indicating that both the 5’ splice site and the TACTAAC box are required for the generation of this complex (Figure 2). This is in agreement with the observation that the 40s complex does not form if the substrate contains a point mutation at the branchpoint (TACTACC instead of TACTAAC; Brody and Abelson, 1985). The small amount of radioactivity present in the SP region of the lanes corresponding to the mutants is not due to slow, correct complex formation; rather, it is almost certainly due to nonspecific interactions, since it does not increase with time and can be completely eliminated by the addition of competitor RNA prior to electrophoresis, in contrast to the SP band formed with the wild-type substrate (data not shown).

To analyze the RNA content of the various RNPs, an RNP gel was electroblotted in the presence of detergent onto a DEAE membrane. The DEAE membrane was autoradiographed (Figure 3A), and the RNAs were then eluted and analyzed on a denaturing urea-polyacrylamide gel (Figure 38). The SP band contains all the RNAs present in the unfractionated sample except for the exon 1-exon 2 product, which is almost entirely absent. The ratio of lariat intermediate to lariat intron in the SP band is

increased, implying that some of the lariat intron has been released from the particle. The U band, in contrast, contains precursor RNA exclusively.

In conclusion, the complexes present in the SP band seem to behave according to the criteria previously established for spliceosomes. Their rapid formation is ATP-dependent and requires specific sequences on the substrate; in addition, they form before any covalent modi- fication can be detected on the substrate RNA, and, at later times, they contain the intermediates of the reaction and one of the two products (the lariat intron). The U band, in contrast, contains a variable fraction of the RNA (compare Figure lA, Figure 2, and Figure 3A). It seems to correspond to a nonspecific complex in light of the criteria discussed above as well as its sensitivity to added competitor RNA prior to electrophoresis (data not shown). It may correspond to a bona fide but nonspecific RNP or to an artifact.

Enrichment for Specific Spliceosomes on Oligo(dT)-Cellulose To develop an additional assay for spliceosome formation, we took advantage of the fact that polyadenylated RNAs present in an RNP particle are available for hybridization to oligo(dT)-cellulose (Lindberg and Sundquist, 1974). This property has been used for the characterization of poly(A)-binding proteins (see, for example, Manrow and Jacobson, 1988) as well as general mRNP components (Dreyfusset al., 1984; Choi and Dreyfuss, 1984). By an ap- propriate construction in an SP6 vector, pSP65AT (Baum and Wormington, unpublished data; see Experimental Procedures for the construction of pSPA2pA), an active splicing substrate with a 100 nucleotide poly(A) tail was generated. The construction used in these experiments (A2 in Pikielny et al., 1983) has an intron of 137 nucleotides from which unnecessary regions have been deleted. It was used in these experiments because it generates fewer degradation products than are obtained with a substrate containing the full-length intron. After incubation of this RNA for 20 min in an in vitro splicing reaction, the RNPs were fractionated by affinity chromatography on oligo(dT)-cellulose, and the RNAs corresponding to the total reaction (T), the flow-through (FT), and the bound fraction (Bd) were analyzed on a denaturing urea-acrylamide gel (Figure 4). All the expected products and intermediates are detectable (the bands corresponding to pre- mRNA and spliced exon I-exon 2 are distinguishable on lower exposures of the same gel; data not shown). If the initial substrate is poly(A)+, a fraction of the substrate, lariat intermediate, and mature exon I-exon 2 bind specifically to oligo(dT)-cellulose, whereas the intron species do not (Figure 4). The bound fraction also contains the 5’ exon; since the 5’ exon lacks a poly(A) tail, it may be retained because it is bound in a complex that also contains the lariat intermediate. With a poly(A)- control substrate, the bound fraction contains only very small amounts of RNAs (see Figure 4); in particular, no 5’ exon can be detected, even after long exposures (data not shown).

Visible in the flow-through fraction are several new bands, most of which presumably are discrete degrada-

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A

SP

U

B Total I I

SP U

Figure 3. The Intermediates of the Splicing Reaction and the Lariat lntron Are Present in the SP Band

(A) A splicing reaction was carried out with a pSPrp5lA transcript. After 5, 10, and 30 min, an aliquot was taken and quenched in an equal volume of Q buffer. A fraction of each aliquot was analyzed on an RNP gel. The gel was electroblotted onto a DEAE membrane (see Experimental Proce- dures), which was then autoradiographed at 4OC. (B) The RNA was then eluted from the relevant bands and analyzed on a denaturing 5% polyacrylamide gel. The structures of the RNAs are shown schematically at left with the same symbols as in Figure 1

tion products generated during incubation in the presence of oligo(dT)-cellulose. These species are not retained on oligo(dT)-cellulose. The observation that much of exon 1 appears in the flow-through fraction is probably due to the fact that any cleavage of the RNA in exon 2 would result in a poly(A)- RNP containing an intact exon 1. The RNA pattern of the bound fraction is very similar to that observed prior to fractionation except for the absence of intron species and the marked decrease in background, indicating that this fraction is relatively free of poly(A)- species. The fact that the ratio of 5’exon to lariat intermediate in the bound fraction is similar to that in the unfractionated reaction suggests that both products of the first step are present in spliceosomes, which remain relatively intact under these conditions (in particular, the presence of 10 mM EDTA), consistent with the results obtained with the RNP gels. The absence of intron species from the bound fractions confirms the presence of these RNAs in a different particle from those containing the poly(A)+ mRNA, at least under the conditions employed.

Three snRNAs Are Specifically Associated with Yeast Spliceosomes To analyze small nuclear RNAs that might be present in yeast spliceosomes, RNA was extracted from the SP band and from an identical region of a parallel lane containing the RNP products of a mock reaction (i.e., with no added substrate). The RNA was then labeled by the addition with RNA ligase of radioactive pCp to the 3’ ends of all RNA species (England et al., 1980). To improve the visualiza- tion of snRNAs, immunoprecipitation was carried out with an antibody (Liihrman et al., 1982; Bringmann et al., 1983) directed against the trimethylguanosine (TMG) cap structure present at the Vend of most mammalian (Reddy and Busch, 1978) and yeast (Wise et al., 1983) snRNAs (see Figure 5A). As a control, [32P]pCp-labeled, immunoprecipitated yeast nuclear RNA was analyzed in parallel (this particular immunoprecipitation eliminated most, but not all, 5s and 5.8s RNAs). The pattern of total snRNAs obtained by immunoprecipitation (Figure 5A) shows an even greater number of species than has been reported previ-

snRNAs in Yeast Spliceosomes a73

PA- I PA+

Figure 4. Isolation of Poly(A)+ Spliceosomes through Their Interac- tion with Oligo(dT)-Cellulose

The two RNAs used in this experiment were run-off transcripts from pSPrp51AA2pA (see Experimental Procedures). The structure of the pSPrp51AA2pA transcript is shown at the top right; lengths in nucleotides are indicated. The two RNAs were identical to the wild-type transcript described in Figure 1 except for the intron, which was only 137 nucleotides long; most of the unnecessary sequences were deleted (see A2 in Pikielny et al., 1983). The poly(A)+ (PA+) transcript also contained a poly(A) stretch of approximately 100 nucleotides at its 3’ end. A poly(A)- (PA-) transcript served as a control. A splicing reaction in a total volume of 50 ul was carried out for 20 min with each RNA; mix- tures were then fractionated into flow-through (FT) and bound (Bd) fractions. The fractions were deproteinized, and an aliquot of each, as well as an aliquot of the total reaction, was analyzed on an 9% acrylamide gel. The structures of the various RNAs are shown schematically on the right as in Figure 1.

ously for the postlabeling of yeast nuclear RNA (Wise et al., 1983) probably because of the greater sensitivity af- forded by the immunoprecipitation, and it is similar to what has been observed by others (Zagorski and Fournier, personal communication).

The SP band contains many small RNA% comparison with the mock reaction suggests that most of these correspond to snRNPs that are present in the whole cell extract (Figure 5A). Three snRNAs are significantly enriched in the SP band relative to the mock reaction, suggesting that these three snRNAs are specifically associated with spliceosomes containing the exogenously added RNA. The sizes of these three species, as calculated by comparison with RNA markers, are 160, 185, and 215 nucleotides.

To confirm by an independent method that these three snRNAs are genuine components of yeast spliceosomes, we analyzed the snRNAs present in spliceosomes col- lected by oligo(dT)-cellulose chromatography. We compared four substrates: a wild-type RNA containing a poly(A) tail; a wild-type RNA lacking a poly(A) tail; and the

two deletion mutants described above (lacking either the TACTAAC box or the 5’ splice junction), both containing poly(A) tails. lmmunoprecipitation of [32P]pCp-labeled RNAs from the bound fractions shows that, in all four cases, many snRNAs are present, raising the interesting possibility that they are associated with poly(A)+ RNAs present in the extract; alternatively, their presence could be due to nonspecific binding of snRNPs to the oligo (dT)-cellulose under the conditions employed (see Figure 5B). More importantly, only the poly(A)+ wild-type template causes an enrichment of the same three snRNAs (160, 185, and 215 nucleotides), as compared with the poly(A)- control or the two defective substrates (Figure 56). The slight differences visible between the lane for the poly(A)- substrate and the two lanes corresponding to the mutants are most likely due to the fact that two different extracts were used to test the substrates (lanes pA+ and pA- come from one experiment, lanes 5-O and A38 come from another; see Figure 58). The results confirm that the three enriched snRNAs are almost certainly components of yeast spliceosomes. In addition, we have con- sistently found an immunoprecipitable species of high molecular weight (ml,000 nucleotides) in the bound fraction from oligo(dT)-cellulose chromatography (Figure 58). This may constitute a fourth spliceosome-associated snRNA (the other high molecular weight band, visible in lane 5’-0, Figure 58, is most probably the 5’-0 transcript itself, and its presence in that sample is due to a low level of contaminating RNA lacking a TMG cap). However, since this large, TMG-capped species is not visible in the SP band (Figure 5A), perhaps because of a high background level in the SP region of the RNP gel, its assign- ment to the assembled spliceosome is at present tenta- tive. Experiments to resolve this conflict are currently in progress.

Discussion

Analysis of Yeast Spliceosomes on Native Gels The gel assay described here enables us to analyze splicing complexes that correspond to spliceosomes by every criterion previously used for their definition (Brody and Abelson, 1985; Grabowski et al., 1985; Frendewey and Keller, 1985; Bindereif and Green, 1986). However, these complexes are not identical to those previously observed, since our analysis is carried out in solutions of somewhat higher ionic strength (200 mM KCI after quenching as op- posed to 100 mM KCI as in Brady and Abelson, 1985), which also contain high concentrations of EDTA (10 mM after quenching and 1 mM in the gel). EDTA completely in- hibits splicing, since the splicing reaction has a strict re- quirement for Mgs+ ions (Hardy et al., 1984; Krainer et al., 1984; Lin et al., 1985). We have found that similar gels, electrophoresed in the presence of 2.5 mM MgC12 (which is the concentration used in the in vitro reactions), tend to show aggregates and artifactual bands in addition to real splicing complexes (data not shown). To test the effect of our conditions (EDTA and 200 mM KCI) on the structure of the spliceosomes, we studied their sedimentation (after quenching the reaction) in glycerol gradients containing

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:a.lK

185

160

-+M 6

. 1K

5.8s

5s

W.T.

pA+pA-S’O A3B

215

160

Mg2+ (as described in Brody and Abelson, 1985) or 0.5x TBE (which is the running buffer used for the RNP gels; see Experimental Procedures). In the presence of Mg2+, spliceosomes had a sedimentation coefficient of 4OS, as described previously (Brody and Abelson, 1985); EDTA and 200 mM KCI shifted the sedimentation coefficient to slightly lower values. However, cosedimentation of the splicing intermediates in a large structure, clearly separate from naked RNA, still takes place (data not shown). This suggests that the chelation of Mg*+ by the addition of EDNA affects the structure of spliceosomes either by removing some factors or by “unfolding” its structure (compare the effect of EDTA on ribosomal subunits; see Van Holde and Hill, 1974, for review). Nonetheless, these

Figure 5. Analysis of snRNAs Associated with Yea.9 Spliceosomes

(A) A IO pl splicing reaction was carried out using saturating (50 ng) concentrations of a short, spliceable transcript (the A2/Ddel RNA is generated by using as a template pSP- rp51A[AZ] cut with Ddel, and it has the following structure: 77 nucleotide exon 1; 137 nucleotide intron; and 33 nucleotide exon 2). After quenching, the reaction was loaded on two lanes of an RNP gel as described in Ex- perimental Procedures. A mock reaction containing no substrate was treated in an identical manner. After electroblotting, the RNA was extracted from the SP band and from the identical regions of the mock reactions as described in Experimental Procedures except that salmon sperm DNA was used as a carrier instead of 1RNA. The RNA was then labeled with [32P]pCp, immunoprecipitated, and analyzed on a denaturing 7% polyacrylamide gel. Mark- ers were yeast nuclear RNAs (a generous gift of John Zagorski and Skip Fournier) similarly labeled with (32P]pCp and RNA ligase and then immunoprecipitated. Lanes: mock reaction (-); bona fide reaction (+); yeast snRNAs (M). Molecular sizes in nucleotides were calculated by comparison with 5s (121 nucleotide) and 5.8s (156 nucleotide) RNAs and a 242 nucleotide SP6 run-off transcript (data not shown). (B) Splicing reactions were carried out for 10 min with saturating amounts of cold RNAs tran- scribed from the following plasmids: pSPrp- 51A(5’-0)pA; pSPrp51A(A3B)pA; pSPrpSlApA, cut with EcoRl to generate poly(A)- RNA; and pSPrpUApA, cut with BamHl to generate poly(A)+ RNA. The reactions were then sepa- rated into bound and flow-through fractions as described in Experimental Procedures except that salmon sperm DNA was used as a carrier. The bound fraction from each reaction was labeled with [3*P]pCp, immunoprecipitated, and analyzed on a denaturing, 7% polyacrylamide gel. The lanes corresponding to poly(A)- (PA-) and poly(A)+ (PA+) substrates show a different experiment (with another extract preparation) than the lanes corresponding lo the two mutants 5’-0 and A38, which almost certainly ac- counts for the slight differences visible between the pA- lane and the lanes for the two mutants.

EDTA-resistant particles still correspond to spliceosomes if one accepts as the main operational criterion the presence of both products of the first cleavage step in a single particle.

Three Specific snRNAs Are Associated with Yeast Spliceosomes The data presented above show that three specific snRNAs are associated with yeast spliceosomes formed in an in vitro splicing reaction. However, for several rea- sons, it is possible that we missed one or more additional RNAs. First, they may be very poor substrates for RNA ligase (which is not unprecedented; see England et al., 1980). Second, their background levels may be so high

snRNAs in Yeast Spliceosomes 875

that any enrichment caused by the addition of splicing substrate is obscured (indeed, this may well be the case for the high molecular weight species that is enriched by oligo(dT)-cellulose chromatography but not by the gel assay). Third, they may not have TMG caps at their 5’ ends (for example, the mammalian U6 snRNA is not immunoprecipitated by anti-TMG antibody [Bringmann et al., 19831). Fourth, they may be released from spliceosomes by EDTA. Fifth, a single band on our gels may correspond to two or more comigrating RNAs. And finally, one of the species may correspond to a 5’4erminal degradation product of a larger RNA. This last possibility seems un- likely because of the reproducibility of the pattern in different experiments; however, it will only be rigorously ex- cluded once the sequence of each species is known. The three identified bands have a similar intensity in the immunoprecipitated nuclear RNA; likewise, they appear enriched to a similar extent in the spliceosome (within a factor of two or three in both cases). However, the first and fourth caveats above and the possibility of differential loss of snRNAs from nuclei during the preparation of nuclear RNA preclude an assessment of relative or absolute abundance of the three snRNAs, either within the nuclear RNA population or within the spliceosome.

Although one or more of the three snRNAs may correspond to the specific subset of yeast snRNAs previously characterized genetically and biochemically (Wise et al., 1983; Tollervey et al., 1983; Tollervey and Guthrie, 1985), differences in the gel systems make this correlation diffi- cult at present.

Comparison with Mammalian In Vitro Splicing The powerful combination of an in vitro splicing system with a battery of antibodies able to discriminate between different U snRNPs has recently led to a rather detailed picture of the interaction of U snRNPs with a pre-mRNA during splicing in a mammalian system. The most striking characteristic is that each of the three snRNPs seems to recognize one of three splicing signals. The Ul snRNP interacts with the 5’splice junction (Mount et al., 1983; Black et al., 1985), the U2 snRNP interacts with the branchpoint (Black et al., 1985) and a third snRNP (probably U5) interacts with the 3’splice junction (Chabot et al., 1985). Appar- ently, the initial interactions at the 5’and 3’splice junctions occur in an independent manner, since in each case an oligonucleotide containing a single binding site is effi- ciently recognized by its respective snRNP (Mount et al., 1983; Chabot et al., 1985). The interaction of U2 at the branchpoint may take place only after the 3’splice site has been recognized, since branchpoint protection from nu- cleases is only detectable in transcripts containing a poly- pyrimidine stretch that is not itself part of the protected region (Ruskin and Green, 1985). Subsequently, a second level of complex formation occurs that probably involves interactions between the different elements, in particular U2 and Ul. This has been deduced from the presence of common Tl fragments after immunoprecipitations with anti-U1 or anti-U2 antibodies (Black et al., 1985) as well as from the appearance of a protected region around the 5’ splice site only if a branchpoint is present in the same

transcript (Ruskin and Green, 1985). This second stage seems therefore to involve not only an interaction between Ul and U2 snRNPs already bound to the pre-mRNA, but also a tighter interaction with the pre-mRNA itself.

Against this background, it is attractive to speculate that the situation in yeast may be similar: the three snRNAs described in this study may correspond to the three above-mentioned mammalian snRNAs, and they may in- teract independently with three yeast splicing signals. If so, then, it is somewhat surprising that both a 5’ splice junction and a TACTAAC box are required for the detection of spliceosomes or any specific complex formation. One can postulate that individual interactions occur at both sites in an independent manner but that these initial interactions are not visible with this assay, perhaps because of the presence of EDTA. However, this possibility is not exclusive, since recent protection studies, similar to those carried out with mammalian extracts (Ruskin and Green, 1985) and performed under normal splicing conditions (i.e., in the presence of Mgz+), indicate that protection at the Ysplice junction occurs only in the presence of a TAC- TAAC box, and vice versa (Rymond and Rosbash, unpublished data). These data support the interpretation that individual snRNA-splice signal interactions, of the precise kind described for mammalian cells, may not exist in yeast.

A higher resolution gel assay shows that the SP band can be resolved into three separate bands that correspond to kinetic intermediates (Pikielny and Rosbash, unpublished data). The analysis of the snRNAs present in each of these complexes may provide evidence for an or- dered binding of snRNPs and perhaps for some kind of interaction of individual snRNAs with individual sites of the pre-mRNA. We would also like to point out that the fact that these snRNAs are part of spliceosomes does not necessarily indicate that they are directly involved in the splicing reaction itself, as is probably the case for Ul and U2. One could imagine, for example, that these large complexes are involved in several aspects of pre-mRNA me- tabolism, such as splicing, polyadenylation, etc., and that the individual snRNAs in the complex have specialized functions. These alternative possibilities should now be directly testable.

These future studies, and the sequences of the snRNAs themselves, should give us insight into the molecular ba- sis for any mechanistic differences between the yeast and mammalian systems (see Rymond and Rosbash, 1985; Ruskin et al., 1986; and references therein), as well as shed light on the much higher conservation of the yeast 5’ splice junction and branchpoint sequence (TACTAAC box) in comparison with mammalian introns. The use of gene-replacement technology, now standard in Sac- charomyces cerevisiae, should make it possible to study in detail the roles of these three snRNAs in pre-mRNA splicing.

Experimental Procedures

Plasmids and RNAs The rp51A gene and the deletion derivatives used here have been pre- viouslydescribed (Teem and Rosbash, 1983; Pikielny et al., 1983). For

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each of the four constructions (pSPrp5lA, pSPrp51AA2, pSPrpSlA[S’- 01, and pSPrp51A[A3B]), a Hindlll-Pvull fragment (from pHZlfJ, pA2, p5’-0, and ~836, respectively) was cloned into pSP62 cut with Hindlll and Smai. The poly(A) derivatives were generated by cloning the Hind- Ill-EcoRI fragment from pSP65AT (Baum and Wormington, unpublished data) into pUC8, thereby generating pUCpA. An EcoRI-Seal fragment from pUCpA was then cloned into pSPrp51A and the three other deletion derivatives, each cut with Seal and EcoRI.

Run-off RNAs were generated in vitro with SP6 polymerase as described previously (Melton et al., 1984). In all cases the 5’ exon is 77 nucleotides. The wild-type intron is 400 nucleotides, and the A2 derivatives have a 137 nucleotide intron. The 3’exon is 320 nucleotides if the piasmid has been cut with EcoRl and 33 nucleotides if it has been cut with Ddel. For the constructions derived from pSP65AT, cutting with BarnHI includes a poly(A) stretch of approximately 100 nucleotides 3’ of the 320 nucleotide 3’ exon. The RNA synthesized under our conditions has a specific activity of approximately 10,000 cpmlng.

Splicing Reactions, pCp Labeling, and lmmunopracipitations Splicing reactions were carried out as described previously (Newman et al., 1985; Lin et al., 1985); a standard reaction of 10 pl contained about 1 ng of substrate. For analysis of RNAs, samples were deproteinized by incubation in lx PK buffer (0.1 M Tris-HCI, pH 7.5; 12.5 mM EDTA; 0.15 M NaCI; 1% SDS; and 0.2 mg/ml proteinase K) at 3pC for 15 min, followed by extraction with an equal volume of phenol-chlo- roform (l:l), precipitation with ethanol in the presence of 2.5 M NHdAc, and electrophoresis on a (2O:l :: acrylamide:bisacrylamide) poiyacrylamide gel containing 7 M urea (see legends to figures for the percentage of acrylamide). Gels were dried and then autoradiographed with an intensifying screen at -7OOC.

In vitro labeling of RNA with pCp and RNA ligase was adapted from England et al. (1980). A 5 ul reaction contained 10% DMSO, 50 mM Hepes (pH 7.5), 3 mM OTT, 20 mM MgClz, 5 uM ATP, 40 uCi of 13*P] pCp (at 7000 Cilmmol), and 5 units of RNA iigase (Pharmacia), and the reaction was incubated at O°C overnight. The labeled RNA was then extracted with phenol and precipitated with ethanol. Immunoprecipita- tions were carried out with anti-TMG antibodies (which were a generous gift of R. Luhrmann) with a scaled-down procedure based on the method described by Liihrmann et al. (1982). Briefly, the RNA was resuspended in 20 ul of Ix PBS (130 mM NaCI, 7 mM Na2HP04, 3 mM NaHzP04 [pH 7.01) 0.2 ul of IgG (at 20 mglml) was added, and the mixture was incubated for 2 hr at 4OC. Approximately 20 ul of protein A-Sepharose (Sigma), which had been previously equilibrated with lx PBS and extensively washed, was added, and the mixture was then incubated for an additional 2 hr at 4OC with gentle shaking. The Sepharose beads were then washed five times with 200 ul of NET (50 mM Tris-HCI, pH 7.4; 150 mM NaCI; 0.05% NP40) at room temperature. The bound RNA was recovered by treating the beads with proteinase K and extracting with phenol as described above.

RNP Gels The splicing reactions were quenched by mixing with an equal volume of 0 buffer (400 mM KCI. 2 mM Mg(Ac)z, 20 mM EDTA, and 100 mM Hepes-NH.,OH [pH 7.51) on ice. Just before loading, 2.5 ul of loading buffer (50% glycerol, 2.5x TBE [see below], and a trace of bromo- phenol blue) was added to each 10 PI sample, which was then loaded directly on a 3% polyacrylamide (6O:l :: acrylamide:bisacrylamide), 0.5% agarose gel in 0.5x TBE (50 mM Tris-borate [pH 8.51, 1 mM EDTA). The gels were prepared as described previously (Goodwin and Dahiberg, 1982) and they were poured into a horizontal gel apparatus (Hendrickson and Schleif, 1964). They were run submerged in 0.5x TBE for 12 hr at 100 V in a BRL apparatus in the cold (4OC). The gel was then dried and exposed to film at -70°C with an intensifying screen. Alternatively, to anaiyse the RNA composition of the various RNPs, the gels were eiectrobiotted onto an NA45 DEAE membrane (Schleicher and Schueil) in 0.5 x TEE, 0.1% SDS for 4 hr at 250 mA (the cross section of the electrobiotter was approximately 30 cm x 20 cm). The membrane was exposed to film at 4°C and the relevant bands were excised. The RNA was then eiuted by incubation at 65OC for 15 min in 200 ui of 55% formamide, 1.8 M Na acetate, 2 mM EDTA, 0.2% SDS (pH 6.5) with 10 ug of tRNA; extracted with phenol; and precipitated with ethanol.

Ollgo(dT)-Cellulose Chromatography of RNPs The oligo(dT)-cellulose binding protocol is a scaled-down adaptation of the methods described in Manrow and Jacobson (1986). Splicing reactions were carried out in a 50 ui final volume for 20 min, and 50 ul of Q buffer was added along with approximately 100 ul of oligo(dT)-cellulose (type 3, Collaborative Research), which had been previously treated with 0.1 M NaOH and equilibrated with 0.5x Q buffer. The mixture was then incubated for 45 min at 4OC with gentle agitation. A low-speed spin at 4OC in a pierced 0.5 ml tube, itself contained in a 1.5 ml microcentrifuge tube, allowed for the easy separation of the oligo(dT)-cellulose from the liquid (FT fraction). Two additional washes were carried out in the same way with 200 ul of cold 0.5x 0 buffer, and the wash liquid was discarded. Elution was obtained by washing the oligo(dT)-cellulose twice with 200 ul of E buffer (1 mM EDTA; 1 mM Mg(Ac)z; 50 mM Hepes-NHIOH, pH 7.5) at room temperature; these two washes were then pooled (Bd fraction).

Acknowledgments

We are grateful to Albrecht Bindereif and Michael Green for help with glycerol gradients, Ellen Baum and Michael Wormington for providing pSP65AT Allan Jacobson for advice on oligo(dT)-cellulose chromatography, Paul Mitsis for help with the horizontal gel system, Reinhard Liihrmann for gifts of anti-TMG antibodies, David Adams for advice on immunoprecipitations, and John Zagorski and Skip Fournier for the gift of yeast nuclear RNA. We thank members of the Rosbash laboratory and in particular Alain Jacquier for their ideas and support, and members of Michael Green’s and Skip Fournier’s laboratories for helpful dis- cussions. The following persons were kind enough to read and com- ment on the manuscript: H. V. Coiot, B. Ruskin, A. Bindereif, M. Wormington, and P Mitsis. This work was supported by a predoctorai Dretzin fellowship to C. W. P and a National Institutes of Health grant (GM23549) to M. R.

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 7, 1986; revised April 11, 1966.

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Note Added in Proof

Several more recent experiments bear on the data presented in this paper. First, purification of spliceosomes from acrylamide gels after the addition of carrier RNA confirms that the ca. 1.0 kb RNA is a bona fide spliceosome-associated species (Pikielny, Rymond, and Ros- bash, unpublished data). Second, a yeast gene coding for a 1300 nucleotide RNA with extensive homology to vertebrate U2 snRNA has been identified and characterized by Dr. Manny Ares of Yale University (M. Ares, personal communication).

cell, vol. 45, 869-877, june 20, 1986, copyright 0 1986 by ... · onto a deae membrane. the deae...

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