u2 as well as u1 small nuclear ribonucleoproteins are involved in premessenger rna splicing

Cell, Vol. 42, 737-750, October 1985, Copyright 0 1985 by MIT 0092-86741851100737-14 $02.0010

U2 as well as Ul Small Nuclear Ribonucleoproteins Are Involved in Premessenger RNA Splicing

Douglas L. Black, Benoit Chabot, and Joan A. Steitz Department of Molecular Biophysics and Biochemistry Yale University New Haven, Connecticut 06510

Summary

Two different experimental approaches have provided evidence that both U2 and Ul snRNPs function in pre- mRNA splicing. When the U2 snRNPs in a nuclear extract are selectively degraded using ribonuclease H and either of two deoxyoligonucleotides complementary to U2 RNA, splicing activity is abolished. Mixing an extract in which U2 has been degraded with one in which Ul has been degraded recovers activity. Use of anti-(U2)RNP autoantibodies demonstrates that U2 snRNPs associate with the precursor RNA during in vitro splicing. At 60 min, but not at 0 min, into the reaction intron fragments that include the branch-point sequence are immunoprecipitated by anti-(U2)RNP At all times, Ul snRNPs bind the 5’ splice site of the pre- mRNA. Possible interactionsof the U2 snRNP with the Ul snRNP and with the pre-mRNA during splicing are considered.

Introduction

Most protein-coding genes of higher eukaryotes contain intervening sequences that are precisely excised from their transcripts by a nuclear process known as pre- mRNA splicing. Our understanding of the biochemical mechanisms involved in splicing has been greatly facili- tated by the recent development of active in vitro systems (Padgett et al., 1983a; Hernandez and Keller, 1983; Krainer et al., 1984). These have allowed the identification and characterization of a novel splicing intermediate, an RNA lariat structure that contains a unique branched nucleotide (see Keller, 1984, for a review). The first observ- able step in the in vitro splicing reaction is a cut at the 5’ splice junction accompanied by formation of a 2’-5’ phos- phodiester bond between the Vterminal G residue of the intron and an A residue located about 30 nucleotides up- stream of the 3’ end of the intron (Krainer et al., 1984; Grabowski et al., 1984; Ruskin et al., 1984; Padgett et al., 1984). In the second step, splicing is completed by cleavage at the 3’ splice site followed by ligation of the 5’ and 3’ exons to produce a spliced RNA and an excised intron still in the form of a lariat.

Less progress has been made toward elucidating which nuclear components contribute to the pre-mRNA splicing process and how they achieve the high degree of ac- curacy observed both in vivo and in vitro. It was proposed some time ago that the Ul small nuclear RNA might be involved in splicing because of the striking complementarity its conserved 5’terminus exhibits to both the 5’and the 3’ splice junction consensus sequences (Lerner et al.,

1980; Rogers and Wall, 1980). Ul exists in cells not as a naked RNA molecule, but in stable association with at least 8 different proteins comprising a small nuclear ribonucleoprotein (snRNP) (Hinterberger et al., 1983; Kin- law et al., 1983). Experimental evidence that the Ul snRNP is indeed an essential component of the mammalian pre-mRNA splicing apparatus includes the following. First, splicing in vitro (Yang et al., 1981; Padgett et al., 1983b; Kramer et al., 1984; DiMaria et al., 1985) and in vivo (Bozzoni et al., 1984; Fradin et al., 1984) is inhibited by addition of anti-(Ul)RNP or anti-Sm autoantibodies (which recognize different subsets of Ul snRNP proteins) or of anti-m,G cap antibodies (which bind the 5’ terminal cap common to most U RNAs). Second, isolated Ul snRNPs bind the 5’(but not the 3’) splice site of a /3-globin pre-mRNA-like transcript; their selective protection of a 15-17 nucleotide region from RNAase is dependent on the integrity of both the RNA and protein components of the Ul snRNP particle (Mount et al., 1983). Finally, splicing is abolished when just the 5’end of the RNA in Ul snRNPs is removed by treatment of an in vitro system with a complementary deoxyoligonucleotide and RNAase H (Kramer et al., 1984).

U2snRNPs are nearly as abundant as Ul snRNPs (both about lo6 molecules/mammalian cell) and likewise consist of one RNA molecule tightly bound by several (at least six) different proteins (Mimori et al., 1984). Someof these poly- peptides are unique to the U2 particle and react specifically with anti-(U2)RNP autoantibodies, while others (rec- ognized by anti-Sm antibodies) are also present on the Ul and other related snRNPs. Like the Ul snRNP the U2 snRNP has been found associated with hnRNA (Lerner et al., 1980; Zieve and Penman, 1981; Calvet and Pederson, 1981; Calvet et al., 1982). A number of proposals implicat- ing U2 RNA in the splicing process have been made (Oh- shima et al., 1981; Fradin et al., 1984; Keller and Noon, 1984).

Here, we have directly tested the idea that U2 snRNPs are involved in splicing. We find that selective removal of certain U2 sequences using complementary deoxyoligonucleotides and RNAase H abolishes splicing in vitro. In addition, we detect an association between U2 snRNPs and the pre-mRNA substrate by probing a splicing extract with anti-(U2)RNP antibodies. The immunoprecipitated fragments protected from Tl RNAase digestion include the intron branch point, suggesting that the U2 snRNP might be the component responsible for branch-point recognition during pre-mRNA splicing. Use of two different pre-mRNA transcripts gave comparable results.

Results

Specific Regions in the U2 snRNP Are Susceptible to RNAase H Digestion An effective way to inactivate a single type of snRNP particle in a complex mixture (such as a splicing extract) is to

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Figure 1. Oligonucleotides Complementary to U2 RNA Diagrammed on a Possible Second- ary Structure

Oligonucleotides (see Table 1) are indicated by solid lines showing the sequences in U2 to which they are complementary. The U2 secondary structure is a composite of those proposed by Rsddy et al. (1961) and Branlant et al. (1962). The sequence is that of rat U2 RNA (Reddy et al., 1961). The sequence of human U2 DNA has been determined (Van Arsdell and Weiner, 1964) and is the same as that shown here except at 5 positions: 106 (G in human), 110 (A), 111 (G), 116 (A), and la8 (C).

Table 1. Sequences and Effects of Oligonucleotides

Oligonucleotide Complementary Length of Digestion Inhibition Abbreviation Length Sequence RNA Nucleotides Complementarity of RNA of Splicing

El5 L15 s15 s9 5% 5-19 R5S

3 Y 15 AGGCCGAGAAGCGAT u2 l-15 15 + + 15 CAGATACTACACTTG u2 28-42 15 + + 15 AAATCTTAGCCATTT u2 i a-27 10 ?

9 CTGATAAGA u2 44-52 9 16 TTCAGGTAAGTACTCA Ul 2-11 10 + + 19 CTTTGGTGAAAGGCGAAAG U5 33-51 19 16 TAGATCAGACGAGATA 5s 32-45 14 -

The sequence in each oligonucleotide complementary to a particular small RNA is underlined, and the corresponding RNA region is listed in the fifth column. We have also tested another 9 base oligonucleotide (CAGGTAAGT), which is complementary to the B’end of Ul RNA and also allows efficient degradation of Ul in the extract (similar to YC). The RNA sequence references are as follows: U2, Van Arsdell and Weiner, 1964; Ul, Branlant et al., 1960; U5, Krol et al., 1961; and 5S, Forget and Weissman, 1967.

use RNAase H, as first demonstrated for the Ul particle by Kramer et al. (1984). RNAase H selectively degrades the RNA strand of an RNA:DNA duplex. Yet, a complementary deoxyoligonucleotide will induce cleavage of an RNA only in the regions that are both available for base pairing and accessible to the enzyme (Rinke et al., 1984). Hence, in a native snRNP particle, either bound proteins or RNA structure might prevent digestion.

We therefore synthesized and tested oligonucleotides complementary to four different regions of U2 RNA. These are diagrammed in Figure 1 along with a possible U2 secondary structure (Reddy et al., 1981; Branlant et al., 1982). As indicated, the four oligonucleotides could theo- retically base pair with 15 residues at the 5’end of the RNA (E15), with 15 residues within the first loop (L15), with 10 residues on one side (S15), or with 9 residues on the other side (S9) of the first stem in the U2 molecule. As controls, we also synthesized oligonucleotides of similar length complementary to other RNAs. These include an oligonucleotide complementary to the 5’ end of Ul RNA (5%) (similar to, but longer than, that used by Kramer et al. [1984]) and oligonucleotides complementary to putative single-stranded regions of ribosomal 5s RNA (R5S) and of U5 RNA (5-19). The sequences of all these oligonucleotides are presented in Table 1.

A HeLa cell nuclear extract (Dignam et al., 1983) was treated with each of these oligonucleotides and with E. coli RNAase H. This was followed by examination of the small RNAs on a polyacrylamide gel (Figure 2). The oligonucleotides were added in about lo- to 40-fold molar excess over the estimated concentration of Ul or U2 snRNPs in the extract (0.1-0.2 PM). Specific degradation of U2 RNA was observed in two cases. The oligonucleotide complementary to the 5’ end of U2 (E15) produced nearly complete conversion to a slightly shorter product denoted U2* (Figure 2, lane h). The oligonucleotide complementary to the loop in U2 (L15) likewise induced the disappearance of full-length U2 RNA, but the yield of expected product (U2**, Figure 2, lane k) was low. Neither oligonucleotide complementary to stem sequences (S9 and S15) induced degradation of U2 RNA in the extract (Figure 2, lanes d and f), although S9 caused some degradation of Ul RNA in this experiment (see below). The Ul oligonucleotide 5% induced digestion of Ul RNA, yielding the expected slightly shorter product denoted Ul* (Figure 2, lane b). Surprisingly, the other two oligonucleotides, 5-19 (complementary to 05 RNA) and R5S (complementary to 5S RNA), had no noticeable effect on their respective RNAs or on any other RNAs in the extract (Figure 3A, lanes d and e).

U2 SnRNPs Function in Splicing 739

Oligo: 5’c 5’c s9 S9 515 515 El5 El5 Ll5 L15 ATP: ++-•+-+-+--+

u2 - UP-

IJI - u1*-

u4-

5s -

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o b c d e f g h i j k l m

Figure 2. Oligonucleotide-Induced RNA Degradation in the Presence and Absence of ATP

The nuclear extract was incubated with an oligonucleotide (see Table 1) in the presence (+) or absence (-) of 0.8 mM ATP 20 mM creatine phosphate, in 25 ~1 reactions containing 4.4 mM MgCI,, 1 unit RNAase H, plus other reagents as listed in Experimental Procedures. The reactions also contained: lane a, no oligonucleotide; lanes b and c, 10 rglml5’C; lanes d and e, 20 pglml S9; lanes f and g, 20 rglml S15, lanes h and i, 10 Kg/ml E15, lanes j and k, IO rglml L15 (note the inversion of the - and + lanes). The reactions in lanes I and m each contained RNA that had been PCA-extracted from 12 ~1 of nuclear extract and ethanol-precipitated. This deproteinized RNA was incubated for 1 hr at 30% in a 25 ~1 reaction containing 15 ~1 of buffer D, 2 mM MgCI,, 15 units of RNasin, 1 unit of RNAase H, and 10 pglml of oligonucleotide (L15). Lane I also contained 1 mM ATR After incubation each reaction was treated with proteinase K, extracted with PCA, and precipitated with ethanol as described in Experimental Procedures. The total ethanol precipitates were run on a denaturing gel and were visualized under UV after staining with ethidium bromide.

The digestion products of 5%, E15, and L15 are denoted Ul*, UZ*, and U2**, respectively. The presence of ATP seems to stabilize a number of RNAs in the extract, most notably the tRNA. The identity of a new band migrating at the top of the gel in the -ATP lanes is not known, but its presence or absence has no effect on splicing activity.

In the nuclear extract, ATP is necessary for efficient RNAase H digestion of U2 by the loop oligonucleotide L15 (Figure 2, lanes j and k). A slight enhancement is also seen for the U2 5’ end oligonucleotide El5 (Figure 2, cf. lanes h and i). Digestion of the U2 loop does not require ATP if the RNA is first phenol-extracted (Figure 2, lanes I and m), suggesting that ATP in some way induces a change in the availability of this region only when the RNA is packaged with proteins. No other oligonucleotide reproducibly induced RNAase H cleavage in an ATP- dependent fashion (Figure 2, lanes b-g).

Other parameters of the incubation conditions were

oligonucleotide S9 gives some digestion of the 5’ end of Ul RNA (Figure 2, lanes d and e), with which it exhibits limited complementarity. This does not occur at 2.2 mM MgCI, and at lower amounts of added RNAase H (see Ex- perimental Procedures), as in Figure 3A, lane c, which were the conditions used in all later experiments. The nuclear extract itself contains substantial amounts of endogenous RNAase H, which often gives nearly complete digestion of an RNA after addition of a complementary oligonucleotide. Results are more consistent, however, when E. coli RNAase H is added to each reaction.

also found to affect the extent of RNAase H cleavage in- Specific Degradation of U2 RNA duced by the various oligonucleotides. At a magnesium Inhibits Splicing In Vitro chloride concentration of 4.4 mM and somewhat larger Samples of nuclear extract were preincubated with vari- amounts of added RNAase H (Figure 2), the U2 stem ous oligonucleotides and RNAase H for 1 hr. Portions of

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a b c d e f g h

160-

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abcdefghij

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labeled RNA unlabeled cDNA -

unspliced

El E2

spliced

Figure 3. Extracts Treated with Various Oligonucleotides and Assayed for Excision of the First lntron of the Adenovirus Major Late Transcript

(A) The small RNAs from the nuclear extract after incubation with: lane a, no added DNA; lane b, 16 rglml mp9 (a derivative of the single stranded DNA phage M13) DNA; lane c, 16 &ml S9; lane d, 16 rglml 5-19; lane e, 16 Hg/ml R5.S; lane f, 16 pgglml 5%; lane g, IO rglml E15; and lane h, 16 rg/ml L15. The RNAs were then analyzed as in Figure 2. (B) Tl protection assays of the splicing products from each of the extracts shown in Figure 3A. The substrate used was a 3ZP-labeled transcript from pSPAd. Reaction conditions and gel fractionation were as described in Experimental Procedures. Lane a contained unincubated precursor and lane b marker DNA& some of the sizes of which are indicated on the left side of the gel. Other lanes contain products from nuclear extracts preincubated with: lane c, no DNA; lane d, mp9 DNA; lane e, S9; lane f, 5-19; lane g, R5.S; lane h, 5%; lane i, E15; and lane j, L15. The Tl assay produces two sets of bands (El and E2) from unspliced RNA (see lane a) and a more slowly migrating set of bands (El-E2) from spliced product (see lanes c and d). (C) Schematic diagram of the hybridization step of the Tl RNAase protection assay. An Ml3 cDNA clone of the mature adenovirus message (pJAW 43 provided by Dr. Greg Freyer) was hybridized to RNA extracted from each splicing reaction and was treated with Tl RNAase following the procedure of Padgett et al. (1963a). Protected RNAs were not filter-selected as in the original procedure, but rather, were loaded directly on a gel after ethanol precipitation. In unspliced RNA the looped-out intron is cleaved by Tl, producing two sets of exon fragments that fractionate on the gel. The spliced product is fully protected by the cDNA, producing a more slowly migrating set of products.

each treated extract were then analyzed to confirm the ex- nick, 1985). It was synthesized from an SP8 promoter tent of selective RNA degradation (Figure 3A) and tested (Melton et al., 1984) in the presence of G(!Y)ppp(Y)G (to for splicing activity (Figure 3B). The splicing substrate provide a capped 5’ terminus; Konarska et al., 1984) and used contained the first two exons and a shortened first [(Y-~~P]CTP. The level of spliced product was assessed by intron of the adenovirus major late transcription unit (Sol- a Tl RNAase protection assay (Figure 3C; Kinniburgh et


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El-E2

abcdefgh Figure 4. Splicing Activity of Oligonucleotide-Treated Extracts Ana- lyzed by Gel Fractionation

RNA extracted from splicing reactions similar to those in Figure 38 were run out directly on a gel without being subjected to the Tl assay. Lane a shows the unincubated precursor. Lanes b-h show the products after this precursor has been incubated in extracts that have been preincubated with: lane b, no oligonucleotide; lane c, 16~g/ml5%; lane d, 10 Kg/ml E15; lane e, 16 pglml L15; lane f, 16 pglml SS; lane g, 16 pglml S15; lane h, 16 rglml 5-19.

The two reaction intermediates, the free 5’exon, and the lariat intron plus 3’exon, are marked El and IVSE2, respectively. The two reaction products, the spliced exons, and the lariat form of the excised intron, are denoted El-E2 and IVS, respectively. The products and intermediates were identified by gel mobility relative to DNA markers and by analogy to Konarska et al. (1985). The band labeled X in lane g has a mobility appropriate for the 5’terminal RNAase H digestion product of the transcript, caused by the 515 oligonucleotide.

al., 1978; Padgett et al., 1983a). Unspliced RNA produces two sets of bands (El and E2; Figure 3B, lane a), whereas the spliced product containing the two fused exons produces a more slowly migrating set of bands (ElE2; Fig- ure 38, lane c).

Figure 38 (lanes i and j) shows that extracts in which U2 RNA has been degraded at either its 5’ end or within its first loop are inactive in splicing. The extract in which Ul RNA has been degraded at its 5’end is also inactive (Fig- ure 38, lane h), in agreement with the original observations of Kramer et al. (1984). In none of the other oligonucleotide-treated extracts (Figure 38, lanes e-g) is the level of splicing significantly reduced compared with the control (Figure 38, lane c). We also observed that pretreatment with single-stranded M13mp9 DNA (and RNAase H) reproducibly enhanced splicing (Figure 38, lane d). Apparently, mp9 DNA in some way protects the extract from the substantial loss in activity usually observed during preincubation; the level of splicing in the mp9 lane (MP-9) is equivalent to that of a nonpreincubated extract. Also, because splicing activity is reduced by preincubation requiring longer exposures of the gel, a background of “spliced” products is seen (Figure 3B, cf. “unspliced” lane a with lanes h, i, and j). We believe this arises from the weak ligase activity of Tl RNAase (Mohr and Thach, 1969).

To determine which step of the pre-mRNA splicing reaction is affected by Ul or U2 degradation, we assayed splicing by another method (Grabowski et al., 1984; Ruskin et al., 1984). When RNA extracted from the reactions is run directly on a gel (Figure 4), both the products (the excised lariat intron and the spliced mRNA) and intermediates (the free 5’ exon and the lariat intron +3’ exon) of the splicing reaction are well separated from one another and from the input pre-mRNA. In extracts pretreated with oligonucleotides that degrade U2 RNA (El5 and L15), the appearance of splicing intermediates and products was virtually abolished (Figure 4, lanes d and e). Hence the block seems to occur early in the splicing reaction. After treatment with the oligonucleotide complementary to Ui RNA (5’C) the inhibition of splicing was only partial (Figure 4, lane c), but in other trials (data not shown) we observed a block in the appearance of splicing intermediates upon degradation of Ui RNA, as with U2. Oligonucleotide S15 unexpectedly reduced splicing (Figure 4, lane g) without detectably af- fecting any of the small RNAsin the extract (Figure 2, lane f). This inhibition can be ascribed to degradation of the transcript (producing a new band marked X, Figure 4, lane g) presumably because S15 exhibits a 12 nucleotide complementarity to a region 102 bases downstream from the 5’end of the RNA substrate. Degradation of the transcript has not been observed with any of the other oligonucleotides we have tested.

We next examined the relationship between the extent of digestion of Ul or U2 RNA and the level of residual splicing activity. Treating the extract with increasing amounts of oligonucleotide (5%, E15, or L15) produced increasing digestion of its target RNA (Figure 5A, lanes f-n). Examination of splicing activity by the Tl RNAase protection assay (Figure 5B) revealed that nearly all of the Ul or U2 RNA (>95%) must be degraded before a substantial inhibition of splicing (>50%) is observed. This result is perhaps not surprising since both Ul and U2 snRNPs are present in several-hundred-fold excess over the pre- mRNA substrate in the splicing reaction. It also suggests

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Jre 5. Oligonucleotide Titration and Complementation Experiments

e --- = IEWE

(A) Gel analysis of the small RNAs from extracts used in the oligonucleotide titration and complementation experiments. Lanes a-d show extracts used for the complementation experiment (Figure 5C). Extracts were incubated as above with the following oligonucleotides: lane a, 20 pg/ml R5.S; lane b, 20 pglml 5’C; lane c, IO pglml E15; lane d, 16 pg/ml L15. Lanes e-n are titrations with various oligonucleotides. Extracts were incubated for 1 hr at 30°C with the following: lane e, no oligonucleotide; lane f, 0.2 yg/ml 5%; lane g, 2 #g/ml 5%; lane h, 20 ~glml5’C; lane i, 2 rg/ml E15; lane j, 5 pglml E15; lane k, 10 pglml E15; lane I, 4 pglml L15; lane m, 10 pglml L15; and lane n, 16 pglml L15. (B) Splicing assays of the oligonucleotide titration experiment. Ti assays were performed on the splicing products after incubation of the adenovirus transcript in each of the extracts shown in lanes e-n of Figure 5A. Assay products were gel-fractionated as in Figure 38. Lane a shows the unincubated precursor, and lane b, the same DNA markers used in Figure 38. Lanes c-l correspond to lanes e-n of Figure 5A. In these reactions the extract was preincubated with: lane c, no oligonucleotide; lane d, 0.2 rglml 5%; lane e, 2 rg/ml 5’C; lane f, 20 rglml 5%; lane g, 2 pg/ml E15; lane h, 5 pglml E15; lane i, 10 rg/ml E15; lane j, 4 rglml L15; lane k, IO pglml L15; and lane I, 16 yg/ml L15. (C) Splicing assays of the complementation experiment. Ti assays were performed on the splicing products from each of the extracts in lanes a-d of Figure 5A, as well as from all pairwise mixtures of these extracts, Assay products were resolved as above. Lanes b-e show the splicing from 24 ~.tl aliquots of the extracts in the corresponding lanes of 5A (lanes a-d). The extracts were preincubated with: lane b, R5S; lane c, 5%; lane d, E15; and lane e, L15. Lanes f-k show the splicing from reactions containing 12 ~1 of one oligonucleotide-treated extract mixed with 12 PI of another. Lane f contained R5S and 5% treated extracts. Lane g contained R5S and El5 treated extracts. Lane h contained R5S and L15 treated extracts. Lanes i-k were the actual complementation experiment. Lane i was a mixture of 5’C and El5 treated extracts. Lane j contained 5% and L15 treated extracts; lane k, the El5 and L15 treated extracts. Densitometer tracings of an autoradiograph of this gel obtained without a screen and at room temperature (to produce a more linear response) were made to quantitate the levels of spliced products.

that the degradation products of snRNPs do not interfere with splicing (also see below).

To determine whether degradation of U2 RNA in the nuclear extract abolishes the excision of other introns as well, we examined the second intron of the adenovirus major late transcription unit and the first intron of human fi-globin. Splicing of the former was analyzed by the Tl protection assay (as in Figure 38) and splicing of the latter by direct gel fractionation (as in Figure 4). The results (not shown) demonstrated that degradation of either U2 or Ul RNA in the extract inhibited the excision of both these introns from their pre-mRNA transcripts.

An Extract Lacking Intact U2 RNA Can Complement an Extract Lacking Intact Ul RNA If the block to splicing in oligonucleotide pretreated extracts is due to removal of an essential component rather than generation of some inhibitory substance, then each of the extracts in which U2 has been degraded should complement the extract in which Ul has been degraded. By contrast, mixing the two extracts pretreated with the

oligonucleotides that degrade U2 would not be expected to increase splicing activity. However, if oligonucleotide- induced RNAase H cleavage continued after mixing the already predigested extracts, no complementation would be observed. Luckily, we have found that much of the deoxyoligonucleotide added to a nuclear extract has become degraded by the end of the 1 hr preincubation period (see Experimental Procedures). Thus it has not been necessary to remove the oligonucleotides by DNAase treatment to avoid substantial unwanted digestion of snRNPs after mixing. (There is, however, enough oligonucleotide remaining after preincubation to induce digestion by S15 of the adenovirus transcript, which is at least 100-fold less abundant than the Uf or U2 snRNPs; Figure 4, lane g.)

The data presented in Figure 5C demonstrate that extracts containing digested Ul or U2 RNA can indeed complement each other. Each of the four oligonucleotide pretreated extracts (Figure 5C, lanes b-e) were mixed in all possible pairwise combinations and tested for their ability to splice the adenovirus transcript. Mixing the ex-


Figure 6. Time Course lmmunoprecipitation of RNA Species Produced during the Splicing Reaction

(A) Labeled adenovirus major late precursor RNA was incubated for the times indicated in the splicing extract. The mixture was then chilled on ice, and 12 r.11 aliquots were immunoprecipitated as in Experimental Procedures. RNAs were then resolved on a denaturing gel. Lanes I-5 show l/250 of the total RNA from a 12 ~1 sample of the splicing reaction that was PCA-extracted, ethanol-precipitated, and loaded on the gel. Lanes 6-10 show the RNAs immunoprecipitated with anti-(Ul)RNP serum. Lanes 11-15 show the RNAs immunoprecipitated with anti-(U2)RNP serum. Identifica- tion of the RNA intermediates and products of the splicing reaction was based on comparison of their electrophoretic mobilities with those reported by Konarska et al. (1985). (8) lmmunoprecipitation of the RNA species produced from a human /?-globin precursor RNA incubated for various times in the splicing extract. Lanes l-6, 7-12, and 13-18 correspond to total RNA (11250 of the splicing reaction), anti-(Ul)RNP immunoprecipitations, and anti-(U2)RNP immunoprecipitations respectively. These were performed as in Figure 6A. The RNA species were identified by gel mobility relative to DNA markers and by analogy with Ruskin et al. (1984). The asterisks next to the sizes of some RNA species indicate that they contain a lariat structure and that their gel migration is abnormal. The DNA markers (M) are Yend-labeled fragments of pBR322 DNA cleaved with Msp I. The origins of unidentified bands running above the precursor and above El (in Figure 6A, lanes 2-5 and lane 12, respectively) are unknown. We also observed labeling of what are apparently endogenous tRNAs in the extract only after incubation with [o-32P]CTP-labeled transcript (see Figure 6A, lanes 2-5).

tracts in which U2 had been degraded by two different oligonucleotides (Figure 5C, lanes d and e) did not produce substantially more spliced product (Figure 5C, lane k; about 1.5-fold more as measured by densitometry of the spliced product bands). In contrast, mixing the extract treated with the oligonucleotide complementary to Ul (which retained approximately 25% splicing activity; Fig- ure 5c, lane c) with either of the extracts in ,which U2 had been degraded (Figure 5C, lanes i and j) yielded an 8- to lo-fold greater level of splicing compared with either of the extracts lacking intact U2 (Figure 5C, lanes d and e) and a 3-fold greater level compared with the extract lacking intact Ul (Figure 5C, lane c). The absolute level of splicing activity regenerated by mixing two extracts is usually not as high as that of a control undigested extract (Figure 5C, lane b) for unknown reasons. Instead, lanes i and j of Fig- ure 5C should be compared with lanes f, g, and h, where each of the extracts lacking intact Ul or U2 were mixed with an extract preincubated with RNAase H and the R5S oligonucleotide complementary to 5S rRNA.

Both Ul and U2 snRNPs Interact with the Pre-mRNA during Splicing Although isolated Ul snRNPs had previously been observed to bind the 5’splice site of a naked pre-mRNA-like transcript (Mount et al., 1983), similar experiments using purified or antibody-bound U2 snRNP particles had not revealed any specific interaction (S. Mount, B. Chabot, unpublished observations). To determine whether the situa- tion might be different in an active nuclear extract, we used autoantibodies to probe snRNP binding to the pre- mRNA at various times during the splicing reaction. Anti- (Ul)RNP or anti-(U2)RNP antibodies were added directly to the complete splicing system containing a labeled substrate, and the RNA species recovered by binding to protein A Sepharose were analyzed on gels. We first determined, by immunoprecipitation of a 32P-labeled HeLa cell extract, the amounts of antisera that were sufficient to precipitate quantitatively the Ul or U2 snRNPs, but no other snRNPs (data not shown).

Upon addition of antibodies directed against Ul

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Figure 7. Ti Ribonuclease Protection of the Human /3-Globin Precursor RNA in the Splicing Extract

(A) lmmunoprecipitation of Tl-resistant fragments with anti-(Ul)RNP (lanes 1 and 3) or anti-(U2)RNP (lanes 2 and 4) antibodies. Tl resistant fragments were immunoprecipitated from a splicing reaction incubated with the fi-globin precursor RNA for 0 (lanes 1 and 2) or 60 (lanes 3 and 4) min at 30%. After immunoprecipitation and treatment as in Experimental Procedures, the Tl-resistant fragments were resolved on a gel. The sizes of the protected fragments were estimated by comparison with DNA molecular weight markers. (B) Fingerprint analyses of the entire [a-32P]GTP-labeled human fl-globin transcript of pSP64-HB 6 (Barn HI run-off, 497 nucleotides; Krainer et al., 1984) and of the major bands containing protected fragments (A). The directions of the first dimension (electrophoresis at pH 3.5 on cellulose acetate) and second dimension (homochromatography on PEI plates) are indicated. Identification of all oligonucleotides in the total and of the darker spots in the other fingerprints was based both on their mobility and on RNAase A secondary analyses. Identification of the weaker spots (2,3,5,9,11,16, and 29) constituting the D’ and E’ species was based solely on their mobilities in fingerprints. Fingerprint and secondary analyses were also performed on the protected fragments produced from an [&*P]CTP-labeled transcript (not shown) to confirm the identity of the A, B, C, D, and E species. Note that three 7-mers of identical composition split into two spots on our fingerprints (spot 30 contains ACUCUUG, while spot 32 contains CCUAUUG and CCUUUAG), whereas for Ruskin et al. (1984), ACUCUUG and CCUAUUG comigrated. (C) Sequence of the human /I-globin transcript and the protected regions. The RNA sequence is based on the sequence of the DNA (Lawn et al., 1980), which was cloned into pSP64 by Krainer et al. (1984). Capital and small letters indicate exon and intron sequences, respectively. The numbers above the oligonucleotides correspond to their numbers in the fingerprint of the full-length transcript in B. The asterisk over the A residue in oligonucleotide #39 indicates the site of RNA branch formation. The protected regions are underlined and correspond to the Tl-resistant fragments shown in A. The protected regions corresponding to the D’and E’species are dashed to indicate that the identities of their component oligonucleotides were not confirmed by RNAase A secondary analyses.

snRNPs, approximately 1% of the input adenovirus sub-

strate was immunoprecipitated from the splicing reaction

at time zero (Figure 6A, lane 6). As the reaction proceeded and splicing intermediates and products were generated (Figure 6A, lanes l-5), the precipitability of the precursor RNA declined markedly. At late times (2 hr) a very small fraction of the released 5’ exon, spliced RNA, and the excised intron was detected in the imunoprecipitate. Three different anti-(Ul)RNP antisera, including that used by

Padgett et al. (1983b) to demonstrate inhibition of splicing, gave idenjical results (data not shown).

The use of antibodies directed against U2 snRNPs revealed a different pattern but confirmed that these particles also interact with the pre-mRNA in an active splicing extract. The anti-(U2)RNP immunoprecipitability of the input precursor RNA was lower at time zero (0.3%) than at 15 min (~0.5%) and then dropped off slowly (Figure 6A, lanes 11-15). Later in the reaction (Figure 6A, lanes 13,14,


and 15) both splicing intermediates and the excised intron were detected in the immunoprecipitate. Their levels were considerably higher than those recovered with anti- (Ul)RNP antibodies (Figure 6A, cf. lanes 9 and 10 with lanes 14 and 15).

Similar results were obtained when the human /3-globin pre-mRNA described by Krainer et al. (1984) was used as splicing substrate (Figure 6B). In this case the drop in immunoprecipitability of the transcript by antL(U2)RNP over time was greater than with the adenovirus transcript.

RNAase Protected Fragments Precipitated by Anti-(U2)RNP Include the lntron Branch Point Sites on the splicing substrate associated with Ul and U2 snRNPs were examined by adding Tl ribonuclease along with anti-(Ul)RNP or antL(U2)RNP antibodies to the splicing reaction. The immunoprecipitated RNAase-resistant fragments were fractionated on gels, and the prominent bands were fingerprinted. This analysis was performed at several different times during the splicing reaction. The data presented in Figure 7 used as splicing substrate the human fl-globin transcript used in Figure 6B.

The protected fragments associated with Ul or U2 snRNPs at 0 and 60 min in the splicing reaction are shown in Figure 7A. At time zero, anti-(Ul)RNP antibodies immunoprecipitated a fragment 15 nucleotides in length (band A; Figure 7A, lane 1). Fingerprint analysis (Figure 78) showed that band A contained the oligonucleotides CAG, UUG, and a 7-mer that could be UAUCAAG and/or UUACAAG (Figure 7C). Both the size of the Tl-resistant fragment (15 nucleotides) and RNAase A secondary analyses performed on [@P]CTP-labeled RNA (not shown) argued that the Fmer must be UAUCAAG alone. Together the three oligonucleotides in the fingerprint comprise the 5’splice site and correspond precisely to the region of this same transcript that is protected when antibody-bound Ul snRNPs are mixed with the isolated RNA (B. Chabot, unpublished observations). The yield of fragments containing the protected 5’ splice site that were immunoprecipitated from the splicing reaction at both 0 and 60 min corresponded to about 3%-4% of the input precursor RNA. Only at the later time (60 min) are other fragments immunoprecipitated by anti-(Ul)RNP antibodies, but in much lower amounts (see below).

In contrast, the pattern of pre-mRNA fragments associated with U2 snRNPs changes dramatically during the course of the splicing reaction. At the zero time point, anti-(U2)RNP antibodies do not detectably immunoprecipitate any specific fragment. This is somewhat surprising since immunoprecipitation without RNAase digestion showed that a substantial amount (mO.3%) of human fi-globin pre-mRNA associated with U2 snRNPs at time 0 (Figure 6B, lane 13). At 60 min, however, a major band (42 nucleotides) is protected and immunoprecipitated by anti- (U2)RNP antibodies (Figure 7A, band E + E’). Fingerprint (Figure 78) and RNAase A secondary analyses show this band to be a mixture of two fragments. The major species (E)containstheoligonucleotidesACUCUUG, CCUAUUG, G, UUUCUG, AUAG, CACUG, and ACUCUCUCUG, which

appear together in the intron and span the branch point (* in Figure 7C). The other species (E’) that constitutes the 42 nucleotide band is one-tenth as abundant as the E species (compare the intensities of oligonucleotides AUAG and CAG in Figure 7B) and maps to a pre-mRNA region that includes the 5’splice site (Figure 7C). A 35 nucleotide fragment (band D + D’) is the second most prominent band selected by anti-(U2)RNP antibodies. The D species corresponds to the intron branch-point region (as does the E species) but lacks the 3’ Fmer (Figure 7C), while D’ is a shorter version of the E’ species. Both the B and C bands contain fragments derived from the 3’splice site region (data not shown). Interestingly, at 60 min, all of the protected fragments immunoprecipitated by anti-(U2)RNP antibodies are also immunoprecipitated by the anti- (Ul)RNP antiserum, although at a lower level (about one- fifth; Figure 7A, lanes 3 and 4). The identity of the species immunoprecipitated by anti-(Ul)RNP (bands D + D’ and E + E’) was confirmed by fingerprint analyses (data not shown).

When the adenovirus major late leader transcript was used as a splicing substrate in the RNAase protection experiment, the results (data not shown) were consistent with those in Figure 7. Thirty minutes into the splicing reaction, antL(U2)RNP antibodies immunoprecipitated a large protected fragment (~70 nucleotides) that included both the branch-site region and the 3’splice site. The large size of the protected piece can be explained by the absence of a G residue between the putative U2 snRNP binding site at the branch point and the 3’ splice site.

Discussion

Using two different experimental strategies, we have obtained evidence that U2 snRNPs are involved in the splicing of mammalian pre-mRNAs. First, we find that targeted cleavage of either of two regions near the 5’ end of the RNA in U2 snRNPs abolishes splicing in vitro. Second, we have exploited anti-(U2)RNP autoantibodies to demonstrate an association between the U2 snRNP and the intron region where the branch forms during the course of splicing. Thus, the two most abundant mammalian snRNPs, which contain Ul or U2 RNA, are both components of the mammalian splicing apparatus. This raises further questions about their interactions with each other and with the pre-mRNA transcript.

Regions of the U2 snRNP Accessible to RNAase H Cleavage A small RNP particle is an unusually versatile cellular component in that it can use RNA-RNA, RNA-protein, or protein-protein interactions to form specific contacts with other molecules during its functioning. For instance, the Ul snRNP is believed to recognize 5’ splice junctions in part by RNA-RNA base pairing involving the 5’ end of its RNA; the single-stranded character and availability of this region of the Ul molecule are supported both by phylogenetic data (Mount and Steitz, 1981; Brown et al., 1985; Reddy, 1985) and by its accessibility to nucleases

Cell 746

in the snRNP particle (Rinke et al., 1984). In the case of the U2 snRNP however, guessing which regions might en- gage in base pairing with another RNA has been ham- pered by the lack of a well supported secondary structure model for U2 RNA. Two different folding arrangements for the 5’ end of the molecule have been proposed: one by Keller and Noon (1985) and the other (diagrammed in Fig- ure 1) first proposed by Reddy et al. (1981). Both models are compatible with the limited phylogenetic and nuclease digestion data currently available (Reddy et al., 1981; Branlant et al., 1982; Sri-Widada et al., 1983; Reddy, 1985).

The results we have obtained using complementary deoxyoligonucleotides and RNAase H are more compatible with the U2 structure shown in Figure 1 than with the alternative model. The sequences between nucleotides 1 and 15 and between 28 and 42 are accessible to RNAase H attack, while nucleotides 18-27 and 44-52 are not. This pattern is observed when the RNA is either in its native RNP form (Figure 2) or is phenol-extracted before treatment (data in part not shown). Removal of the 5’end of U2 produces a good yield of truncated RNA still packaged into an anti-Sm precipitable particle (D. Black, unpublished observations). In contrast, degradation of the loop region of U2 seems to destabilize the RNP so that most of the RNA becomes completely degraded in the extract.

An intriguing aspect of the loop digestion by RNAase H is its dependence on ATP only when the RNA is associated with proteins. The amount of cleavage obtained in the absence of added ATP varies from 0 to 40 percent, and this variation is probably due to different amounts of residual ATP in various extract preparations. Such varia- tions in the extract may also account for the somewhat different observations of Krainer and Maniatis (1985) who found that ATP markedly enhanced cleavage of the 5’end of U2. The ATP requirement for cleavage could reflect either an actual conformational change in the U2 snRNP upon ATP binding or an ATP-dependent interaction or loss of interaction of the U2 snRNP with some other component of the nuclear extract. Interestingly, the formation of large splicing complexes, also requires ATP (Grabowski et al., 1985; Brody and Abelson, 1985; Frendewey and Keller, 1985).

U2 and Ul .snRNPs Are Independently Essential for Splicing Previously it was observed that the addition of anti- (U2)RNP antibodies to an in vitro splicing system did not inhibit the splicing reaction (Padgett et al., 1983b). Here, however, we have demonstrated that intact U2 RNA is required for the appearance not only of spliced products but also of the earliest known intermediates in the splicing pathway. Apparently, anti-(U2)RNP antibodies bind to some site on the snRNP particle that does not interfere with its normal functioning during splicing. It is possible that the inhibition is due to the oligonucleotides attacking another less abundant RNP in the extract. However, this is made less likely by the fact that both oligonucleotides that cleave U2 RNA are equally inhibitory, while none of the control oligonucleotides inhibit.

We have also confirmed the results of Kramer et al. (1984), who first demonstrated the power of oligonucleotide-directed RNAase H cleavage by showing that specific removal of the 5’ end of Ul RNA inhibits splicing in vitro. In their extracts, cleavage of 50% of the Ul RNA abolished splicing. In our hands it has been more difficult to achieve complete inhibition of splicing by cleavage of Ul RNA; as little as 1% remaining intact Ul (as revealed by Northern blot analysis) sometimes allows nearly normal levels of splicing. Nonetheless, the complementation obtained upon mixing extracts lacking either intact Ui or intact U2 RNA allows us to draw several important conclu- sions. First, it rules out the possibility that the degradation products of any RNP nonspecifically inhibit splicing. (Re- call that neither of our control oligonucleotides [5-19 and R5S] degraded their respective complementary RNAs [U5 and 5S]). Second, it confirms that the inhibition of splicing cannot be ascribed to deoxyoligonucleotide- directed cleavage of the pre-mRNA transcript. Finally, it argues that Ul and U2 snRNPs either exist as indepen- dent entities at the start of the splicing reaction or readily exchange from any preexisting complex.

Krainer and Maniatis (1985) have also observed that cleavage of the 5’ termini of Ul and U2 RNAs inhibits splicing in vitro. The block occurs prior to lariat formation in their system as well.

U2 and Ul snRNPs Are Associated with Pre-mRNA Sequences Crucial for Splicing Our RNAase protection experiments have revealed that U2 snRNPs are preferentially associated with a region of the pre-mRNA that includes the intron branch point. Al- though we cannot be certain that the interaction is direct, rather than mediated by some associated component, the simplest interpretation of our results is that U2 snRNPs bind specifically to this region of the pre-mRNA. The association of U2 snRNPs with the branch point occurs only under active splicing conditions as it is not detected at time zero (Figure 7A) (although a weaker or a Tl-sensitive interaction with the pre-mRNA must be occurring [Figure 6A, lane 11 and Figure 6B, lane 13]), when ATP is omitted from the reaction mixture (data not shown), or with isolated U2 snRNPs (S. Mount and B. Chabot, unpublished observations). This suggests that some other factor must bind to the pre-mRNA or to the U2 snRNP before the more stable interaction we have observed can occur. Such a factor may be the 3’ splice site binding component, since selection of the intron branch point seems to be largely determined by its proximity to the 3’ splice site (Reed and Maniatis, 1985; Ruskin et al., 1985).

An important question not addressed by our current experiments is whether U2 snRNPs continue to bind to the same site after the RNA lariat structure is formed. We did not detect any branched oligonucleotide in the 42 nucleotide protected fragment (E species). However, intact pre- mRNA is by far the major species immunoprecipitated by anti-(U2)RNP at the time (60 min) we performed our protection experiments (Figure 6B, lane 18). At this time a protected branched oligonucleotide would be a very minor species. It is possible that later in the reaction (2-3 hr),


when the amounts of lariat-containing species increase, we might detect branched oligonucleotides within the U2 snRNP-protected region. In any case, the present data ar- gue that the U2 snRNP interaction with the branch-point region precedes lariat formation. B. Ruskin and M. Green (manuscript submitted) have observed that the same intron region becomes resistant to RNAase A attack during the splicing reaction.

Does the U2 snRNP recognize the branch point via RNA-RNA base pairing? Keller and Noon (1984) have proposed that the U2 sequence between nucleotides 40 and 52 might base-pair with the branch-point consensus sequence and with the CAG sequence at the 3’ splice site. Since we find that a part of the suggested U2 sequence is inaccessible to RNAase H in the presence of a com- lementary deoxyoligonucleotide, it seems less likely that all the base-pairing contacts predicted by Keller and Noon (1984) can be made. However, such an interaction cannot be excluded without knowing the cause of this inaccessi- bility. The length of the pre-mRNA region that we see protected by U2 snRNPs (42 or 35 nucleotides) is about twice as long as that protected by Ul snRNPs (15 nucleotides). This could reflect either a much longer pre-mRNA binding site (perhaps including both proteins and RNA) on the U2 particle or additional protection by other tightly associated components. Although there is a stretch in the first loop of U2 RNA that is complementary (by bulging the G at po- sition 33) to the loosely defined branch-point consensus sequence compiled for mammalian introns (Keller and Noon, 1984; Ruskin et al., 1984; i’eitlin and Efstratiadis, 1984; Reed and Maniatis, 1985) we cannot guess at this time whether base pairing between U2 snRNPs and the pre-mRNA is essential for splicing. It would be interesting to know the sequence of the yeast small RNA analogous to U2 in order to learn the extent of its complementarity to the strictly defined branch-point sequence (TACTAAC) in yeast introns (Langford and Gallwitz, 1983; Pikielny et al., 1983; Domdey et al., 1984; Rodriguez et al., 1984).

In contrast to U2 snRNPs, Ul snRNPs selectively bind and protect from Tl RNAase the 5’splice site of a human fi-globin pre-mRNA at all times during the in vitro splicing reaction. This is true even when the extract is kept at O°C, and therefore extends the earlier observations of Mount et al. (1983) who reported that purified Ul snRNPs protect the 5’splice site of a mouse fi-globin precursor RNA in the absence of any other components. The progressively lower efficiency with which anti-(Ul)RNP antibodies immunoprecipitate the precursor RNA as the splicing reaction proceeds is likely to be caused by the sequestration of (Ul)RNP determinants upon formation of large splicing complexes (see below). Even at time zero, only a fraction of the precursor RNA is immunoprecipitated despite the fact that the number of Ul snRNPs in the extract vastly ex- ceeds that of substrate molecules. (Comparable observations have been made by Grabowski et al., 1985.) It could be that even before incubation at 30%, the determinants of some Ul snRNPs bound to the transcript become un- available for antibody binding. This notion is consistent with our observation that at time zero a higher level of Ul snRNP interaction with the transcript is detected by Tl

RNAase protection (3%-40/o) than by immunoprecipitation without nuclease addition (1%).

Do Ul and U2 snRNPs Interact During the Splicing Reaction Recently, large complexes have been implicated as active assemblies in the pre-mRNA splicing process. When in vitro reactions are analyzed on glycerol gradients, the free 5’ exon and the lariat intron +3’ exon intermediates sedi- ment together at about 40s in the case of yeast (Brody and Abelson, 1985) or about 50s to 60s for mammalian systems (Grabowski et al., 1985; Frendewey and Keller, 1985). Formation of these large complexes requires both ATP and a complete intron. The evidence presented here makes it highly likely that both Ul and U2 snRNPs are components of these active splicing assemblies. Grabowski et al. (1985) have concluded that Ul snRNPs are present, since precursor RNA and splicing intermediates can be immunoprecipitated from the 60s region of gradients by either anti-Sm or anti-(Ul)RNP antibodies.

Whether Ul and U2 snRNPs interact directly or only in- directly (via the pre-mRNA or some other component) in these large complexes is not yet known. Consistent with (but in no way proving) a direct interaction is the observation that anti-(Ul)RNP and anti-(U2)RNP antibodies immunoprecipitate a common set of protected pre-mRNA fragments from active splicing reactions (Figure 7A). There are several regions in Ul and U2 RNAs that are complementary (e.g., Ul nucleotides 11 to 20 with U2 nucleotides 5 to 14, and Ul nucleotides 65 to 72 with U2 nucleotides 30 to 37), allowing a possible interaction of these two snRNPs by base pairing. A Ul-U2 interaction coupled with a U2 interaction at the branch point could ex- plain how the 5’ splice site and the branch point are brought together prior to lariat formation. Indeed, Y. Os- heim, 0. Miller, and A. Beyer (submitted) have visualized on nascent transcripts of Drosophila chorion genes particles bound to the regions of the 5’ and 3’splice sites that later appear to coalesce.

Another question is whether yet other snRNPs contribute to the splicing reaction and to the formation of large splicing complexes. The S value expected for one Ul snRNP (tills) and one U2 snRNP (~11s) bound to the pre-mRNA (m8S for the in vitro pre-mRNA substrates that have been examined) would be much lower than the observed sedimentation value of 60s. Also mysterious is the identity of the component that recognizes and binds the 3’ splice site of the pre-mRNA. Potential candidates include the related abundant snRNP particles containing U4/U6 (Hashimoto and Steitz, 1984; Bringmann et al., 1984) or U5 RNAs or a not yet well characterized low abun- dance Sm snRNP (R. Reddy, D. Henning, and H. Busch, submitted). So far, all Sm snRNPs to which functions have been assigned participate in some aspect of pre-mRNA processing. In addition to the involvement of Ul and U2 particles in splicing, the (sea urchin) U7 snRNP is required for the 3’ end maturation of histone pre-mRNA (Strub et al., 1984) whereas antibody inhibition data make it likely that some Sm snRNP(s) participate in the cleav- agelpolyadenylation reactions that fashion the 3’ ends of

Cell 748

most mammalian messenger RNAs (Moore and Sharp, 1984; C. Hashimoto, unpublished observations). Hence, it seems reasonable to expect that the remaining Sm snRNPs of unknown function will also play roles in the maturation of RNA polymerase II transcripts.

Experimental Procedures

Oligonucleotides Oligonucleotides were synthesized on a Biosearch SAM-I synthesizer (made available by Dr. Nigel Grindley). They were purified on denaturing 20% polyacrylamide gels followed by DEAE and Bio-gel P4 chromatography and then were lyophylized.

Sera The anti-(U1)RNP serum (AG) and the anti-(U2)RNP serum (Ya) used in most experiments were characterized as described previously (Pet- tersson et al., 1984; Mimori et al., 1984). The Ya serum was a gift of Dr. Tsuneyo Mimori and was diluted 1OO:l in PBS (130 mM NaCI; and 20 mM potassium phosphate, pH 7.4) before use.

Polyacrylamide Gels Gels shown in Figures 2-5 were 10% acrylamide, 0.38% bisacryl- amide in TBE buffer (90 mM Tris base, 90 mM boric acid, 2.5 mM EDTA) containing 7 M urea. Gels in Figure 6A, Figure 68, and Figure 7A were 10, 5, and 15% polyacrylamide, respectively, containing 8M urea and 19:l acrylamide to bis. Small RNA gels were visualized under UV after staining with ethidium bromide. Splicing reaction products, hybridization assay products, and immunoprecipitation reactions were run on gels that were dried and visualized by autoradiography at -70°C using DuPont Lightning Plus intensifying screens and Kodak XAR or XRP film.

Preparation of Splicing Substrates The plasmid pSPAd (a gift of Dr. David Solnick; Solnick, 1985) was cleaved with Bgl I (New England Biolabs) and transcribed with SP6 RNA polymerase (Boehringer-Mannheim, see Melton et al., 1984). This produces a 417 nucleotide run-off transcript that ends 35 nucleotides into the second intron of the adenovirus major late transcription unit. Transcription reactions were performed in the presence of diguanosine 5’triphosphate (G[5’]ppp[5’]G) to produce a capped RNA (Konarska et al., 1984). The plasmid pSP64-HB 6 (a gift of Dr. Tom Maniatis; Krainer et al., 1984) was cleaved with Bst I (an isoschizomer of Barn HI kindly provided by Dr. Cathy Joyce) and was transcribed in a similar manner. Substrates used in the oligonucleotide inhibition experiments were transcribed in a total volume of 50 ~1 containing 40 mM Tris-HCI (pH 7.5), 6 mM MgCI,, 4 mM spermidine, 10 mM DTT, 0.1 mM each of ATP UTR and GTP 10 FM CTR 0.5 mM G(5’)ppp(5’)G (P-L Biochemicals), plus 30 units of RNasin (Promega Biotec), 7 units of SP6 RNA polymerase, 5 pg of pSPAd DNA template, and 50 &i of [o-~~P]CTP (410 Ci/mmol, Amersham). Following incubation for 1 hr at 37oC, 1 t.rg of DNAase I and 30 units of RNasin were added, and the mixture was reincubated at 37OC for IO min. The sample was then extracted with PCA (phenol:chloroform:isoamyl alcohol, 50:49:1) and was ethanol-precipitated.

The transcripts used for the immunoprecipitation and Tl protection experiments were of higher specific activity. To produce the Tl protection substrate, reactions contained in 26.5 ~1: 40 mM Tris-HCI (pH 7.5) 6 mM MgCI,, 4 mM spermidine, IO mM DTT, 0.4 mM G(5’)ppp(5’)G, 0.25 mM each of ATR CTP and UTR 125 &i of [@P]GTP (410 Cilmmol), 30 units RNasin, 7 units of SP6 RNA polymerase, and 2.5 rg of template DNA; and were incubated at 40°C for 1 hr. Transcripts for the immunoprecipitation experiments were made under similar conditions except that the label used was [a-“‘P]CTP and the reactions contained 1 mM G(5’)ppp(5’)G. Transcripts for all reactions were gel-fractionated, eluted, ethanol-precipitated, and resuspended in water.

Nuclear Extract and Splicing Optimization The nuclear extract in buffer D (20mM HEPES, pH 7.9; 20% [v/v] glycerol; 0.1 M KCI; 0.2 mM EDTA; and 0.5 mM OTT) was prepared according to Dignam et al. (1983). For RNAase H experiments we used the in vitro splicing system described by Krainer et al. (1984), which we

reoptimized for the adenovirus substrate. Most notably we have used 2.2 mM MgCI, rather than the 3.2 mM described. We omitted the use of polyvinyl alcohol in the RNAase H experiments with the adenovirus transcript without any noticeable effect. In time course experiments with this transcript we found maximum levels of product after 1 hr of incubation; hence this is the reaction time used in most experiments, In addition, in the RNAase H digestion experiments we used 10 ~1 of nuclear extract in each 25 yl reaction rather than the usual 15 ~1. This reduced the splicing activity only slightly, while making it easier to ob- tain complete digestion of snRNAs.

The splicing conditions for the immunoprecipitation experiments and all experiments using the kglobin substrate were exactly those reported by Krainer et al. (1984).

Ribonuclease H Digestion and Splicing Inhibition Experiments For RNAase H reactions the standard 48 PI reaction contained: 20 ~1 of nuclear extract, IO ~1 of buffer D, 30 units of RNasin, 1 unit of E. coli RNAase H (Boehringer), 2.2 mM MgCI,, 20 mM creatine phosphate, and 0.5 mM ATP, plus deoxyoligonucleotide in the concentrations indicated in each figure. Reactions were incubated at 30°C for 1 hr and were then split into 24 PI aliquots. One set of aliquots was adjusted to 1 mglml proteinase K (Beckman) and 0.5% SDS and incubated for 30 min at 37OC. These aliquots were then diluted to 250 I.CI with a buffer containing 40 mM Tris-HCI (pH 7.5), 300 mM sodium acetate, 0.1% SDS, extracted with 300 ~.tl of PCA, and precipitated with 1 ml of ethanol. The total ethanol precipitate was run out on a gel to assess the extent of RNAase digestion. The other set of 24 PI aliquots was used to test splicing activity. One microliter of transcript containing 100,000 cpm (Cerenkov) (about 2 ng) was added to each, and the reactions were incubated at 30DC for 1 hr, treated with proteinase K, PCA extracted and ethanol precipitated as above. Samples from all reactions were then run on a gel to assess splicing activity and the integrity of the transcript, Some samples were also subjected to the Tl protection assay according to the procedure of Padgett et al. (1983a) (see Figure 3C).

For the complementation experiment, each RNAase H reaction was 96 ~1 rather than 48 ~1. Twenty-four microliters was used for the snRNA gel (Figure 5A, lanes a-d). To test splicing, another 24 ~1 received 1 ~1 of transcript as above. Mixtures contained 12~1 of each oligonucleotide treated reaction (Figure 5C). To assess the amount of further digestion occurring after mixing oligonucleotide-treated extracts, 5’C- and E15-treated extracts (similar to those in Figure 5A, lanes b and c) were mixed and incubated for another hour at 3OOC. Gel analysis revealed that very little further digestion of the snRNAs had occurred. However, if El5 was added to an extract that had been preincubated with 5%, incubation for another hour produced the normal amount of U2 digestion, indicating that the RNAase H was still quite active. These results indicate that degradation of the oligonucleotide during the first hour of incubation is sufficient to prevent further cleavage of snRNAs after mixing.

lmmunoprecipitation and Tl Protection Analyses Direct immunoprecipitation was performed on 12 PI aliquots of the splicing reaction containing 4 x lo5 cpm (Cerenkov) of J2P-labeled precursor RNA. Ten microliters of antiserum was added at various times, and the mixture was placed on ice. After 30 min at OOC, 50 ~1 of protein A Sepharose (4 mg, Pharmacia) in NET-2 buffer (50 mM Tris-HCI, pH 7.9; 150 mM NaCI; and 0.05% Nonidet P-40 v/v) plus 0.5 mM DTT was added. The mixture was then incubated for another 15 min on ice and spun for 8 set in a microfuge. The pellet was washed four times in NET- 2 + DTT, PCA extracted, and ethanol-precipitated. The labeled RNA species were resolved by gel fractionation.

A similar procedure was used for the Tl protection experiments except that 5 ~1 of a 29 unit/PI solution of Tl RNAase (Calbiochem) was added with the antiserum. The succeeding steps were as described above, and the protected RNA fragments were gel-fractionated. In- dividual bands were extracted from the gel in 0.3 M sodium acetate, 0.2% SDS. Each protected fragment was subjected to Tl RNAase fingerprint analysis following the procedure of Barrel1 (1971). This involved electrophoresis on cellulose acetate strips (Schleicher and Schuell) in the first dimension followed by thin-layer chromatography on PEI plates (Brinkmann) in the second dimension. RNAase A (Worthington) secondary analysis was performed using electrophoresis on DEAE pa- per (Whatman) at pH 3.5 (Barrell, 1971).


Acknowledgments

We thank Carl Hashimoto, David Solnick, Alan Weiner, and Sandy Wo- lin for discussions and helpful advice, Adrian Krainer, and Tom Mania- tis for the free exchange of unpublished information and for critically reading the manuscript, Cathy Joyce and Graham Hatfull for help in synthesizing the oligonucleotides, and Nancy Batter and Terri Moore- house for help in preparing the manuscript. Tsuneyo Mimori, John Hardin, Adrian Krainer, David Solnick, and Greg Freyer generously supplied materials. D. L. B. was supported by a fellowship from the G. E. Foundation. Research funds were provided by grants from the Na- tional Science Foundation (PCM 8215962) and the National Institutes of Health (GM 26154) to J. A. S.

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 July 15, 1985; revised August 8, 1985

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Notes Added in Proof

The work referred to throughout as R. Reddy, D. Henning, and H. Busch, submitted has been published: Reddy, R., Henning, D., and Busch, H. (1985). Primary and secondary structure of U8 small nuclear RNA. J. Biol. Chem. 260. 10930-10935.

u2 as well as u1 small nuclear ribonucleoproteins are involved in premessenger rna splicing

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