Probing the structure and function of U2 snRNP with antisense oligonucleotides made of 2′-OMe RNA

Cell, Vol. 58, 383-390,

July 28, 1989, Copyright

0 1989 by Cell Press

Probing the Structure and Function of U2 snRNP with Antisense Oligonucleotides Made of 2’1OMe RNA Angus I. Lamond, Brian Sproat, Ursula and J&g Hamm European Molecular Biology Laboratory Meyerhofstrasse 1 Postfach 102209 D6QOO Heidelberg Federal Republic of Germany

Ryder,

We have used oligonucleotides made of 2’-OMe RNA to analyze the role of separate domains of U2 snRNA in the splicing process. We show that antisense P’-OMe RNA oligonucleotides bind efficiently and specifically to U2 snRNP and demonstrate that masking of two separate regions of U2 snRNA can inhibit splicing by affecting different steps in the spliceosome assembly pathway. Masking the 5’ terminus of U2 snRNA does not prevent U2 snRNP binding to pm-mRNA but blocks subsequent assembly of a functional spliceosome. By contrast, masking of U2 sequences complementary to the pre-mRNA branch site completely inhibits binding of pre-mRNA. Hybrid formation at the branch site complementary region also triggers a specific change which affects the 5’ terminus of U2 snRNA. Introduction Splicing of nuclear mRNA precursors (pre-mRNAs) involves the organization of pre-mRNA into a spliceosome in an ordered assembly pathway (Frendewey and Keller, 1965; Pikielny et al., 1966; Konarska and Sharp, 1966; Bindereif and Green, 1967). Spliceosome assembly entails the sequential binding to pre-mRNA of specific protein factors and small nuclear ribonucleoproteins (snRNPs), including Ul, U2, U5, and U4/U6 8nRNPs. Formation of a functional spliceosome is thus preceded by formation of a number of presplicing complexes containing different combinations of snRNP particles together with unspliced pre-mRNA. Current studies on the mechanism of premRNA splicing are focused on unraveling the nature of snRNP-pre-mRNA and snRNP-snRNP interactions, which occur during the assembly of a functional spliceosome. The majority of information concerning the role of specific snRNP particles in pre-mRNA splicing has come from in vitro biochemical analyses. For example, snRNP function has been inhibited by the targeted cleavage of snRNA sequences using RNAaseH and complementary DNA oligonucleotides (Kramer et al., 1964; Krainer and Maniatis, 1965; Black et al., 1965; Berget and Robberson, 1986; Black and Steitz, 1966). This approach has demonstrated the essential roles of Ul, U2, and U4/U6 snRNPs in splicing. Nuclease protection experiments have also been used to map the binding sites for snRNPs at the 5’and 3’splice junctions (Chabot et al., 1985; Black et al., 1985; Ruskin

and Green, 1985; Konarska and Sharp, 1986; Kramer, 1987; Bindereif and Green, 1987). These data show that Ul and U2 snRNPs bind to pre-mRNAs at the 5’splice site and branch site, respectively. Compelling evidence ha8 been obtained from both yeast and mammalian splicing systems that Ul snRNP binds to the 5’ splice site of premRNA by means of direct base pairing between complementary sequences at the 5’ intron-exon junction and the 5’ end of Ul snRNA, as originally proposed by Lerner et al. (1980) and Rogers and Wall (1980). The most convincing evidence for this was provided by the demonstration that deleterious point mutations of the 5,splice site of certain pre-mRNAs could be suppressed in vivo by compensatory changes in the complementary sequence of Ul snRNA which restored the base pairing potential (Zhuang and Weiner, 1966; Seraphin et al., 1986). A similar suppressor study ha8 also shown that in Saccharomyces cerevisiae, a region of U2 snRNA base pair8 with complementary intron sequences at the conserved TACTAAC box element surrounding the branch site (Parker et al., 1967). This interaction has not yet been similarly demonstrated for mammalian U2 snRNP, where the less stringent sequence requirement for the pre-mRNA branch site renders it difficult to obtain dominant mutations that would be suppressible by simple compensatory changes in mammalian U2 snRNA. However, mutational analyses show that the sequence of the mammalian branch site is an important determinant of splicing efficiency (Reed and Maniatis, 1988). Since it is known that mammalian U2 snRNP binds at the intron branch site and since complementarity also exists between the mammalian U2 snRNA and pre-mRNA branch site sequence (Parker et al., 1967; Frendewey et al., 1967), it is quite possible that U2 snRNA-pre-mRNA base pairing also occurs in mammals and contributes to the specificity of the snRNP-premRNA interaction. In this work, we describe a novel approach for detecting snRNP particles and analyzing their function in the splicing process. We have constructed ‘antisense” oligonucleotides made of 2’-OMe RNA that are complementary to specific domains of U2 snRNA. These oligonucleotides efficiently form stable snRNA-P’-OMe RNA hybrids in HeLa cell nuclear extracts. The formation of stable hybrids that mask specific regions of U2 snRNA has been exploited to map functional domains in the U2 snRNP particle. Results Detection of U2 snRNP with Antisense 2’-OMe RNA Probes We have prepared oligonucleotides made of 2’-OMe RNA that are complementary to three separate regions of U2 snRNA: nucleotides l-20 (a); nucleotides 27-49 (b); and nucleotides 153-171 (c) (Figure 1). Each oligonucleotide was tested for binding to the mammalian U2 snRNP particle using a mobility retardation assay (Figure 2). Mi-

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Figure 1. The Sequence of Mammalian U2 snRNA, Drawn According to the Secondary Structure Model of Keller and Noon (1985) The regions of U2 snRNA complementary to the 2’-OMe RNA oligonucleotides a, b. and c are indicated in black. Oligonucleotide a is complementary to nucleotides I-20, b is complementary to nucleotides 27-49, and c is complementary to nucleotides 158-171 of U2 snRNA.

gration of the endogenous HeLa U2 snRNPs in this gel system was detected by hybridization with a UP-specific riboprobe after transfer of the complexes onto a nitrocellulose membrane (Figure 2A, lane 1). In agreement with previous studies, a major U2 snRNP band was detected together with a minor band of slower mobility (Konarska and Sharp, 1988; Lamond et al., 1987; Bond, 1988). In parallel assays, HeLa cell nuclear extracts were incubated with the 5’ end-labeled 2’-OMe RNA oligonucleotides a, b, and c, fractionated by gel electrophoresis, and complexes were detected by autoradiography (Figure 2A, lane8 2-4). The a and b oligonucleotides both show major and minor band8 comigrating with the endogenous U2 snRNP complexes detected by Northern hybridization. In each case, these are the only bands migrating in the upper region of the gel. The b oligonucleotide reproducibly gives a stronger signal for these bands than the a oligonuc&tide. The c oligonucleotide fails to detect complexes comigrating with the U2 snRNP bands. All three oligonucleotides also show a nonidentical set of rapidly migrating bands in the lower region of the gel that do not comigrate with U2 snRNl? Additional controls, including nuclease degradation and oligonucleotide competition experiments, further demonstrate that the a and b oligonucleotides bind specifically to U2 snRNP (data not shown). Such controls also confirm that the additional, rapidly migrating bands seen with all three oligonucleotides are unrelated to U2 snRNP These bands appear to result from interactions of the probe with nucleic acid binding proteins present in the nuclear extract and have not been studied further. The failure of the c oligonucleotide to bind to U2 snRNP is consistent with additional evidence indicating that the complementary sequence on U2 snRNA is covered by protein and therefore inaccessible to the probe. For example, RNAaseH fails to efficiently cleave U2 in the presence of a DNA oligonucleotide of the Same sequence as c (our unpublished data), and previous studies showed that deletion of the “c” region of U2 snRNA in Xenopus oocytes resulted in the loss of

Figure 2. Binding of 2’-OMe RNA Oligonucleotides to U2 snRNP Particles (A) Lanes l-4 show HeLa nuclear extract that has been separated on a composite agarose-polyacrylamide gel. Lane 1 shows a northern blot where the U2 snRNP particles have been detected by hybridzation with a UP-specific riboprobe. The major and minor U2 bands are indicated with arrows. Lanes 2-4 correspond to HeLa nuclear extract that has been incubated with the a, b, and c 2’-OMe RNAoligonucleotides, respectively. In each case, the bands observed result from interactions between the 5’ end-labeled oligonucleotides and components in the nuclear extract. (B) lmmunoprecipitation of 5’end-labeled oligonucleotide b from Heta nuclear extract with the following antibodies; antiSm (Y12), anti-W and anti-UPsnRNPs (V28; Habets et al., 1985) and anti-U1 (7OK). For each sample, oligonucleotide was recovered from half the reaction before immunoprecipitation (“total’? and from half the reaction after immunoprecipitation with the indicated antibody (“Ab”). Oligonucleotides were subsequently analyzed on a 20% denaturing polyacrylamide-urea gel.

binding of proteins A’and B”(Mattaj and DeRobertis, 1985). This region of U2 snRNA also shows a similar high level of phylogenetic sequence consecration to other known protein binding sites on snRNAs (Guthrie and Patterson, 1988). We note that it was not possible to label U2 snRNP by binding S’end-labeled DNA oligonucleotides of identical sequence to the 2’-OMe RNA a and b oligonucleotides. This is presumably due to the high endogenous level of RNAaseH activity in HeLa cell nuclear extracts. RNAaseH cleaves RNA in an RNA-DNA hybrid but does not cleave RNA in an RNA-2’-OMe RNA hybrid (Figure 8). As a further control for specific binding of the oligonucleotides to U2 snRNP, the ability of snRNP-specific antibodies to immunoprecipitate 5’end-labeled b oligonucleotide from HeLa cell nuclear extract was assayed (Figure 28). These data show that the b oligonucleotide is immunoprecipitated by an anti8m antibody (which recognizes all major snRNPs) and by an antibody that recognizes only Ul and U2 snRNPs, but not by an antibody that recognizes Ul snRNP alone. A similar result was obtained with oligonucleotide a (data not shown). This confirms that the a and b oligonucleotides bind to U2 snRNP and further shows that their binding does not displace the Sm or Al/B” proteins. Characteristics of Oligonucleotide Binding to U2 snRNP As expected, the a and b oligonucleotides bind to U2 snRNP through interactions with separate regions of U2

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Figure 3. Parameters Affecting Binding of the 2’-OMe RNA Oligonucleotide b to U2 snRNP The position of the major U2 snRNP band is indicated with an arrow. Complexes were analyzed on nondenaturing polyacrylamide-agarose composite gels.

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snRNA. This is deduced from the observation that oligonucleotide a does not compete with the binding of b and vice versa and from the fact that the presence of the 2’-OMe RNA a and b oligonucleotides specifically inhibit RNAaseH cleavage of U2 snRNP in the presence of corresponding DNA oligonucleotides of the same sequence (Figures 7 and 8, and data not shown). The rate, temperature, and ATP dependence of the binding reaction were analyzed, and these data are shown in Figure 3 for oligonucleotide b. Binding of oligonucleotide b to U2 snRNP is rapid, being essentially complete after 5 min of incubation, and is stable over a time course of 90 min. We also observe that binding of oligonucleotide b to U2 snRNP is significantly affected by both temperature and the presence of ATP, while binding to the nonU2-related complexes is not. Binding to U2 snRNP is strongest in the presence of ATP and at a temperature between 20°C30°C, although a low level of binding is still observed in the absence of ATP and at 0°C. The ATP analog a-8 methylene ATP was unable to substitute effectively for ATP in this assay while the 8-r methylene ATP analog was (data not shown). Other nucleoside triphosphates, including GTP, UTP, and CTP also partially stimulate binding of oligonucleotide b to U2 snRNP (data not shown). In separate experiments, the binding of the a oligonucleotide to U2 snRNP was found to be similarly rapid but showed little dependence on ATP (data not shown). It is possible that the stimulatory effect of ATP on oligonucleotide b binding is related to the ATP requirement for pre-mRNA splicing. However, this relationship is not clear as in vitro splicing specifically requires ATP and cannot be substituted by other nucleoside triphosphates (Hardy et al., 1984). Overall, these data are consistent with the 2’-OMe RNA oligonucleotide probes binding to U2 snRNP through hybridization with the complementary snRNA sequences. It is possible, however, that this hybridization may be facilitated and/or stabilized by additional interactions with protein factor(s).

Antisense Inhibition of pre-mRNA Splicing Having established that the a and b oligonucleotides form stable hybrids with specific regions of U2 snRNP, “antisense inhibition” experiments were performed to assess how such snRNA-2’-OMe RNA hybrids affect the splicing of pre-mRNA. Splicing and spliceosome assembly assays were therefore done using HeLa cell nuclear extracts that had been preincubated with either the a, b, or c oligonucleotides (Figure 4). The a and b oligonucleotides both potently inhibited splicing of two separate pre-mRNAs (Figure 4, parts A). The c oligonucleotide at an equivalent concentration did not inhibit, and in some experiments slightly stimulated, splicing. Interestingly, a parallel analysis of spliceosome assembly showed that the a and b oligonucleotides exert their inhibitory effect at different stages of the assembly pathway (Figure 4, parts 8). The a oligonucleotide allows formation of the prespliceosome complex A, which contains U2 snRNP However, this A complex is unable to subsequently associate with the additional factors, including U4/U5/U8 snRNPs, required to complete spliceosome assembly. Additional experiments show that the kinetics of presplicing complex formation in the presence of the a oligonucleotide are similar to the control (data not shown). The stable binding of U2 snRNP to pre-mRNA cannot, therefore, be strongly dependent upon the secondary structure of the Vterminus of U2 snRNA. In the presence of the b oligonucleotide, presplicing complex formation is completely blocked. Hybridization of oligonucleotide b to U2 snRNA will mask the region that is complementary to the pre-mRNA branch site. This masking appears sufficient to prevent stable binding of U2 snRNP to pre-mRNA. Addition of the a and b oligonucleotides together gave the same result as b alone, i.e., there was no formation of complex A, showing that the b oligonucleotide phenotype is dominant (data not shown). The c oligonucleotide, as expected, caused no inhibition of spliceosome assembly. Since the a and b oligonucleotides both affected the

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ability of U2 snRNP to participate in spliceosome assembly, a parallel experiment was carried out to determine the stability of preformed splicing complexes to subsequent challenge with the same oligonucleotides (Figure 5). These data show that the A and B splicing complexes are largely resistant to challenge by either the a or c oligonucleotides. However, challenge with the b oligonucleotide causes quantitative disruption of the presplicing, A complex, but does not disrupt the fully assembled spliceosome (i.e., band B, Figure 5). This underlines the importance of the “b” region of U2 snRNA for the stable association of U2 snRNP with pre-mRNA in the presplicing complex. It also demonstrates that a change must occur either in the accessibility of the U2 a and b domains to oligonucleotide probes or else in the functional requirement for these regions, at different stages of spliceosome assembly. The formation of a presplicing complex in the presence of the a oligonucleotide (Figure 4) was unexpected as RNAaseH cleavage of the 5’ terminus of U2 snRNA has been reported to prevent presplicing complex formation (Frendewey et al., 1987; Chabot and Steitz, 1987; Zillmann et al., 1988). Therefore, to extend the results shown in Figure 4, an experiment was performed to determine whether U2 snRNP particles that had been prebound to 5’end-labeled a oligonucleotide would then bind to unlabeled premRNA (Figure 8). These data show that a significant fraction of the oligonucleotide-labeled U2 snRNP particles are found in a band of slower mobility when incubated with wild-type pre-mRNA (A band, Figure 8B). This band is coincident in mobility with the prespliceosome A complex (Figures 4 and 5). The appearance of this band is ATPdependent, does not occur in the absence of exogenously

B-

Figure 4. Antisense Inhibition of Pm-mRNA Splicing The splicing products were analyzed on either 7% (globin) or 10% (adeno) denaturing polyacrylamide-urea gels. The unspliced premRNA and intron lariat bands are marked with arrowheads. Size markers are (a) end-labeled Mspl fragments of pBR322 and (b) unprocessed pm-mRNA. For both substrate RNAs, splicing complexes were analyzed on nondenaturing polyacrylamide-agarose composite gels. In each case, lanes marked a-c correspond to splicing reactions carried out using HeLa cell nuclear extracts that had been preincubated with 2’-OMe RNA oligonucleotides a, b, or c prior to addition of pm-mRNA. Lanes marked Ctrl correspond to standard splicing assays performed in the absence of oligonucleotides.

Figure 5. Oligonucleotide Challenge of Preformed Splicing Complexes A standard splicing reaction was carried out with the globin pm-mRNA, incubated 60 min at WC, then divided into four aliquots and incubated for a further IO min at 3oOC with either: (1) dH2Cl (lane ctrl) or (2-4) 2’-OMe RNA oligonucleotides a-c, (lanes a-c). Splicing complexes were analyzed on nondenaturing polyacrylamide-agarose composite gels.

added pre-mRNA, and is also not seen in the presence of a mutant pre-mRNA that is defective in presplicing complex formation (Figures 8A, 88, and 8C). We therefore conclude that the U2 snRNP particle is still able to interact stably with pre-mRNA when the a oligonucleotide is bound to the 5’ terminus of U2 snRNA. As the U2 snRNP-oligonucleotide a hybrid shows ATP dependence for pre-

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Each lane shows HeLa cell nuclear extract incubated with 5’ end-labeled oligonucleotide a and analyzed on a nondenaturing polyacrylamide-agarose composite gel. After preincubation with oligonucleotide a, each sample was then further incubated, either plus or minus ATR with: (A) HeLa cell nuclear extract alone; (B) HeLa cell nuclear extract plus unlabeled, wild-type globin pre-mRNA; (C) HeLa cell nuclear extract plus unlabeled, mutant globin pre-mRNA. This mutant premRNA has two separate point mutations: at the 5’SS the 2nd nucleotide of the intron is changed from U to A; and at the 3’SS, the last nucleotide of the intron is changed from G to U (Lamond et al., 1967). The positions of both the major U2 snRNP bands and the presplicing “A” complex are indicated by arrows.

mRNA binding and sensitivity to specific point mutations on the pre-mRNA, it is likely that it interacts with premRNA in a qualitatively similar fashion to “wild-type” U2 snRNP Oligonucleotide b Induces a Highly Specific Structural Change in U2 snRNA The mobility of U2 snRNP particles in a native gel shows no apparent change when they are bound to either the a or b oligonucleotides (Figure 2A). An identical result is obtained when the U2 snRNP complexes are bound to unlabeled oligonucleotides and detected by hybridization on a Northern blot (data not shown). This indicates that binding of oligonucleotide probes to U2 snRNA does not radically change the structure or composition of the snRNP particle. This is supported by the immunoprecipitation experiments that demonstrated that oligonucleotide binding does not displace either the Sm or AYE” antigens from U2 snRNP (Figure 28). However, although no apparent change in the composition of U2 snRNP takes place, we have detected a highly specific change affecting the structure of the 5’terminus of U2 snRNA (i.e., the region complementary to oligonucleotide a), which occurs upon the binding of oligonucleotide b. This effect is revealed when the binding of 5’ end-labeled oligonucleotide a to U2 snRNP is compared either with or without prebinding of unlabeled oligonucleotide b (Figure 7). A large increase in the binding of oligonucleotide a is reproducibly observed when oligonucleotide b is prebound to U2 snRNP (Figure 7, compare lanes A and B). This stimulatory effect appears specific to oligonucleotide b, as preincubation of a HeLa cell nuclear extract with six other oligonucleotides of different sequence

Figure 7. Oligonucleotide U2 snRNP

b Stimulates

Binding of Oligonucleotide

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Each lane shows HeLa cell nuclear extract incubated with 5’ end-labeled oligonucleotide a and analyzed on a nondenaturing polyacrylamide-agarose composite gel. Unlabeled oligonucleotides were present in each sample as follows: lane A, no oligonucleotide; lane B, 2’-OMe RNA oligonucleotide b; lane C, non-UBspecific DNA oligonucleotide; lane D. 2’-OMe RNA oligonucleotide a; lanes E-H, non-UPspecific 2’-OMe RNA oligonucleotides.

failed to cause a similar stimulation (Figure 7, lanes C-H). Thus, while the binding of oligonucleotide b does not apparently change the composition or gel mobility of U2 snRNP it does facilitate binding of oligonucleotide a to the adjacent sequence of U2 snRNA. This could result from either an increase in the stability of the hybrid formed between the a oligonucleotide and U2 snRNA and/or through an increase in the accessibility of the U2 5 region.

To examine this effect further, an RNAaseH cleavage experiment was performed with a DNA oligonucleotide of the same sequence as the 2’-OMe RNA oligonucleotide a (Figure 8). Neither the a nor the b 2’-OMe RNA oligonucleotides cause RNAaseH to cleave U2 snRNA (Figure 8, lanes 6 and 7) but a DNA oligonucleotide of the same sequence as 2’-OMe RNA a does (lane 3). Addition of this DNA oligonucleotide together with the b 2’-OMe RNA oligonucleotide resulted in a stimulation of U2 cleavage by RNAaseH (lane 5). Thus, the binding of the 2’-OMe RNA b oligonucleotide to U2 snRNP also causes an increase in cleavage of the adjacent 5’ terminus of U2 snRNA by RNAaseH, demonstrating that either the DNA oligonucleotide is able to bind more effectively or else that this region is more accessible to RNAaseH when oligonucleotide b is present. A control experiment shows that addition of the RNAaseH-resistant 2’-OMe RNA a oligonucleotide together with the DNA a oligonucleotide reduces the fraction of U2 cleaved by RNAaseH (lane 4). Additional experiments showed that the effect of oligonucleotide b binding appears confined to changing the 5’ terminal region of U2 snRNA. The c oligonucleotide remains unable to bind to U2 snRNP in the presence of oligonucleotide b, as do two additional 2’-OMe RNA oligonu-

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cleotides complementary to each of the two internal stem regions flanking the Sm binding site (data not shown). Thus, the structural change responsible for the increased accessibility of the 5’terminus of U2 snRNA is likely to be specific and localized within the U2 particle. We also note that it is very unlikely that the structural change in the 5 terminus of U2 snR.NA is directly responsible for the inhibition of presplicing complex formation caused by oligonucleotide b, as formation of the presplicing complex is not inhibited by the binding of oligonucleotide a to the same 5’ terminal region (Figures 4 and 6). Discussion We have shown that 2’-OMe RNA oligonucleotide probes complementary to specific regions of human U2 snRNA bind to the corresponding complementary sequences of the U2 snRNP particles present in HeLa cell nuclear extracts. The 2’-OMe RNA-snRNA hybrids form rapidly and are sufficiently stable so as to survive gel electrophoresis. This has allowed convenient detection of snRNP particles by virtue of their specific binding to 5’end-labeled 2’-OMe RNA oligonucleotides. We note that the oligonucleotide-snRNA hybrids apparently are not affected by any irreversible unwinding activities (Bass and Weintraub, 1988). As ATP stimulates rather than inhibits oligonucleotide binding to U2 snRNP (Figure 3) this also argues against any helicase activity acting to destabilize hybrid formation. It is possible that the stability of the RNA-2’OMe RNA hybrids derives from the presence of inosine residues and methyl groups on the oligonucleotides which may effectively block unwinding enzymes. The masking of separate regions of U2 snRNA by antisense 2’-OMe RNA oligonucleotides inhibits pre-mRNA splicing in vitro. Oligonucleotide b prevents U2 snRNP from binding pre-mRNA to form the presplicing A complex, while oligonucleotide a allows A complex to form but halts subsequent assembly of a functional spliceosome (Figure 4). These data demonstrate that separable domains of function exist within the U2 snRNP particle that

Figure 6. Comparison of Targeted RNAaseH Cleavage of the C’Terminus of U2 snRNA in the Presence of 2’-OMe RNA and DNA Oligonucleotides (A) shows RNAs extracted from HeLa cell nuclear extracts analyzed on a 10% denaturing polyacrylamide-urea gel. The RNAs were detected by staining with ethidium bromide. (B) shows the same RNA samples analyzed by Northern hybridization with a UP-specific riboprobe. In both (A) and (6) the assays were done with the following oligonucleotides: lane 1, no oligonucleotide control; lane 2, no oligonucleotide plus RNAaseH; lane 3, DNA oligonucleotide a plus RNAaseH; lane 4, both DNA a and 2’-OMe RNA a oligonucleotides plus RNAaseH; lane 5, both DNA a and 2’-OMe RNA b oligonucleotides plus RNAaseH; lane 6, 2’-OMe RNA a oligonucleotide plus RNAaseH; lane 7, 2’-OMe RNA b oligonucleotide plus RNAaseH.

act at different stages of the spliceosome assembly pathway. The phenotype of the b oligonucleotide, which masks the region on U2 snRNA complementary to the pre-mRNA branch site, is consistent with an essential role for RNA-RNA base pairing in the initial binding of mammalian U2 snRNP to pre-mRNA. This is also supported by the oligonucleotide challenge experiment, which shows that the binding of U2 snRNP to pre-mRNA in the presplicing complex can be disrupted by subsequent addition of oligonucleotide b (Figure 5). As the region of complementarity between U2 snRNA and oligonucleotide b is much longer than that between U2 snRNA and pre-mRNA, disruption is likely to result because the greater stability of the snRNA-oligonucleotide hybrid will displace the U2 snRNA-pre-mRNA base pairing. These data support the view that mammalian U2 snRNP interacts with the branch site region in a similar fashion to yeast U2 snRNP, which is shown to require snRNA-pre-mRNA base pairing (Parker et al., 1987). The phenotype of the a oligonucleotide suggests that the 5’ terminal sequences of U2 snRNA may be involved in interactions with other components of the splicing apparatus which occur after U2 snRNP binds to pre-mRNA. It is possible that the change in the accessibility of the 5’ terminus of U2 snRNP, which was detected upon the binding of oligonucleotide b (Figures 7 and 8), may represent a transition that facilitates such interactions. This could involve either direct interaction of the 5’ terminal region of U2 snRNA with other snRNPs or splicing factors or else act indirectly by facilitating interactions in other regions of the U2 particle. We suggest that oligonucleotide b may trigger this structural transition in U2 snRNP because it base pairs with the same U2 snRNA sequences that base pair with sequences at the intron branch site. Base pairing cannot be the sole determinant of spliceosome assembly, however, as the binding of oligonucleotide b alone is insufficient to cause U2 snRNP to associate with the other snRNP components of the spliceosome (see Figure 3). Presumably, additional factors that bind to pre-mRNAs are required, possibly including IBP (Tazi et al., 1986;

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Gerke and Steitz, 1988) and UPAF or SF3 (Ruskin et al., 1988; Kramer, 1988) which are factors shown to bind independently of U2 snRNP at 3’ splice sites. On the basis of the data presented here, we have reached a different conclusion regarding the structure/ function relationship of elements in U2 snRNP to that previously derived from RNAaseH cleavage studies (Frendeweyet al., 1987; Chabot and Steitz, 1987; Zillmann et al., 1988). These authors used DNA oligonucleotides with sequences similar to the 2’-OMe RNA a and b oligonucleotides to direct RNAaseH cleavage of U2 snRNA in HeLa cell nuclear extracts. They reported that cleavage of the 5’terminus of U2 prevented formation of a presplicing complex while cleavage in the region complementary to the branch site did not prevent presplicing complex formation but instead blocked assembly of a functional spliceosome. This is the exact opposite of what we observe in the antisense inhibition experiments. It is not clear how these contrasting results can be reconciled, and further studies are obviously needed to clarify this apparent discrepancy. It must be stressed, however, that RNAaseH cleavage studies are quite distinct experiments from the antisense inhibition experiments reported here. For example, the consequence of RNAaseH cleavage on U2 snRNP structure is very likely to differ from the perturbation induced by the binding of an antisense 2’-OMe RNA oligonucleotide, and such differences may explain the seemingly contradictory data. One possibility is that cleavages produced by RNAaseH on a population of U2 snRNP particles are not homogeneous. Therefore, a significant fraction of U2 snRNA cleaved in the presence of the DNA oligonucleotide which binds to the “b” region (see Figure 1) may in practice retain the sequences complementary to the intron branch site and thus still be able to form a presplicing complex. It also remains to be established whether or not the separate pieces of U2 snRNA generated by RNAaseH cleavage actually stay associated in the U2 snRNP particle. We suggest that the use of antisense oligonucleotides to study snRNP structure and function offers certain advantages over the RNAaseH approach. First, it does not cause destruction of the targeted snRNP and therefore cannot lead to physical dissociation of different regions within a particle. This makes it possible to assess the effect of simultaneously blocking two or more domains within the same particle. Second, the 2’-OMe RNA oligonucleotides are able to inhibit snRNP function at relatively low concentrations compared with the concentration of DNA oligonucleotide needed for efficient snRNP cleavage. The possibility of high oligonucleotide concentrations causing nonspecific interactions is, therefore, minimized. Third, it allows tagging of snRNP particles using 5’ end-labeled oligonucleotide probes. In this way, certain antisense inhibition experiments can be carried out under conditions that do not block general splicing activity in the nuclear extract (see Figure 6). Fourth, the rapid binding kinetics of the oligonucleotide probes avoids the extended incubation times necessary for RNAaseH to produce quantitative digestion and thus facilitates “challenge” experiments that assay the requirement for specific func-

tional domains at different stages of spliceosome assembly. The future application of antisense 2’-OMe RNA oligonucleotides should therefore provide a useful tool for the mapping of other snRNP and RNP particles. Experimental

Procedures

Materials Radiochemicals ([@zP]ATP and [aJzP]UTP) were purchased from Amersham. T4 DNA ligase and all restriction enzymes were purchased from New England Biolabs. T7 RNA polymerase and RNASIN were purchased from Promega Biotec and T3 RNA polymerase was from Stratagene. RNAaseH was purchased from BRL.

in Vitro

Splicing Asseys

HeLa ceil nuclear extract was prepared as described by Dignam et al. (1983). Transcription of uniformly labeled, capped pm-mRNAs and in vitro splicing assays were done as previously described (Lamond et al., 1987). The structures of the pm-mRNAs have been previously described. Adeno pm-mRNA was made from plasmid pBSAd1 cut with SauM (Konarska and Sharp, 1987). Wild-type and mutant gfobin premRNAs were made from plasmids pBSAL4 and pBSAL14, respectively, both digested with Pvu2 (Lamond et al., 1987). Oligonucleotides were 5’ end-labeled using T4 polynucleotide kinase as previously described (Sproat et al., 1969) and used to detect U2 snRNP particles in HeLa cell nuclear extracts at a final concentration of approximately 1 pmollul. Oligonucleotide binding assays were performed using the same conditions as for splicing assays, but incubated for 30-45 min. For antisense inhibition assays, the 2’-CMe RNA oligonucleotides (2-5 pmollyl) were preincubated with nuclear extract for 10 min at 3ooc prior to addition of pm-mRNA and then incubations were continued for a further 60 min. RNAaseH assays were performed for 60 min at 30X at a final concentration of 0.1 U of RNAaseHIuI of assay. lmmunoprecipitations were performed essentially as described by Hamm et al. (1987).

Synthesis

of Oligonucieotides

Both DNA and RNA oligonucleotides were synthesized using an Ap plied Biosystems DNA synthesizer as described by Sproat et al. (1969). In each 2’-OMe RNA oligonucleotide, 2’-OMe inosine and not guanosine was used to base pair with cytidine. The sequences of the 2’-CMe RNA oligonucleotides used were: anti-U2 a, 5’-CCAAAAIICCIAIAAICIAU-3’ anti-U2 b, S-AUAAIAACAIAUACUACACUUIA-3’ anti-U2 c, 5’-AIiUACUICAAUACCAIIU-3’ DNA oligonucleotides were used with the identical sequences but with thymidine in place of uridine and guanosine in place of inosine. Gel System All nondenaturing gels were composed of 3.5% polyacrylamide (1:60 bisacrylamide:acrylamide ratio)/0.5% agarosa. The running buffer used was 75 mM his-glycine. Gels were run at 4OC at a constant voltage of 35 V/cm for 1.5-5 hr according to the experiment. Normally, for analysis of U2 snRNP particles, gels were run for 1.5-2.5 hr, and for analysis of splicing complexes, the time was extended to 5 hr. For Northern analysis, the gels were electroblotted onto GeneScreenPlus membrane (NEN); transfer buffer was 6 mM citrate and 8 mM NasHPO,. Transfers were performed at 250 mA of constant current for either 2 hr (from denaturing poiyacrylamide-urea gels) or 14 hr (from nondenaturing polyacrylamide-agarose composite gels). Prehybridizations were done in 10% dextran sulphate, 1% SDS, 1 M NaCI, and 150 mglml of denatured herring sperm carrier DNA, and incubated for l-2 hr at 65%. Hybridizations were done using the same conditions but with denatured herring sperm carrier DNA at 250 mg/ml and 5 x 104 cpm of uniformly labeled riboprobe. Blots were washed twice in 250 ml of 2x SSC (5 min washes at room temperature), twice in 250 ml of 2x SSC and 1% SDS (30 min washes at SOOC), and twice in 250 ml of 0.1x SSC (30 min washes at room temperature). Blots were exposed wet at 4OC using preflashed film with one intensifying screen.

The authors

thank

Philippe

Neuner

for expert

technical

assistance

in

Cell 396

the preparation of oligonucleotides. We are also especially grateful lo Drs. lain Mattaj, Henk Stunnenberg. Ben Blencowe, Silvia Barabino, Angela Kramer, and to Professors Lennart Philipson and Walter Keller for carefully reading the manuscript and making helpful suggestions. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked bdvertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received

March

30, 1989; revised

April 28, 1989.

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