The EMBO Journal vol.8 no.13 pp.4171 -4178, 1989

Mapping U2 snRNP pre-mRNA interactions usingbiotinylated oligonucleotides made of 2'-OMe RNA

Silvia M.L.Barabino, Brian S.Sproat, UrsulaRyder, Benjamin J.Blencowe and AngusILamondEuropean Molecular Biology Laboratory. Meyerhofstrasse 1, Postfach102209, D6900 Heidelberg 1, FRG

Communicated by L.Philipson

Biotinylated 2'-OMe RNA oligonucleotides comple-mentary to two separate regions of human U2 snRNAhave been used as affinity probes to study U2 snRNP-pre-mRNA interactions. Both oligonucleotides bindspecifically and allow highly selective removal of U2snRNP from HeLa cell nuclear extracts. Pre-mRNAsubstrates can also be specifically affinity selected througholigonucleotides binding to U2 snRNP particles in splicingcomplexes. Stable binding of U2 snRNP to pre-mRNAis blocked by the pre-binding of an oligonucleotide to thebranch site complementary region ofU2 snRNA, but notby an oligonucleotide binding to the 5' terminus of U2.Both oligonucleotides affinity select the intron product,but not the intron intermediate, when added afterspliceosome assembly has taken place. The effect of2'-OMe RNA oligonucleotides on splicing complexformation has been used to demonstrate that complexescontaining U2 snRNP and unspliced pre-mRNA areprecursors to functional spliceosomes.Key words: oligonucleotides/spliceosomes/U2 snRNA

IntroductionAnalyses in both mammalian and yeast in vitro splicingextracts have shown that U1, U2, U4/U6 and U5 smallnuclear ribonucleoprotein particles (snRNPs) are majorsubunits of functional spliceosomes (Grabowski and Sharp,1986; Pikielny et al., 1986; Bindereif and Green, 1987;Cheng and Abelson, 1987; Konarska and Sharp, 1987;Lamond et al., 1988). The assembly of spliceosomes on pre-mRNA substrates involves an ordered pathway of snRNPbinding. Formation of an active spliceosome is preceded byassembly of pre-splicing complexes containing unspliced pre-mRNA together with snRNPs and additional protein factors(for recent reviews see Green, 1986; Maniatis and Reed,1987; Sharp, 1987; Steitz et al., 1988). The products of thesplicing reaction, i.e. fully excised intron and joined exons,are also found associated with RNP complexes (post-splicingcomplexes) which are most probably derived from thespliceosome.An important, early step in spliceosome assembly is the

binding of U2 snRNP to the branch site of unspliced pre-mRNA to form a stable pre-splicing complex (Black et al.,1985; Ruskin and Green, 1985; Konarska and Sharp, 1986;Kramer, 1987). In this complex the branch site anddownstream sequences up to the 3' intron-exon junctionare protected from nuclease digestion. In the case of budding

(©) IRL Press

yeast the U2 snRNP-pre-mRNA interaction has been shownto involve base pairing between a region near the 5' end ofU2 snRNA and a complementary sequence at the branch site(Parker et al., 1987). An essential role for base pairing wasdemonstrated in vivo by suppression of inhibitory branch sitepoint mutations with corresponding changes in thecomplementary U2 snRNA sequences that restored the basepairing potential. Although the degree of sequence con-servation at the branch site of mammalian pre-mRNAs islower than that for Saccharomyces cerevisiae, the potentialto form four or more base pairs with the analogous regionof human U2 snRNA is conserved (Frendewey et al., 1987;Parker et al., 1987). The sequence of the mammalian branchsite and its degree of complementarity to U2 snRNAsignificantly affects splicing efficiency (Reed and Maniatis,1988; Zhuang et al., 1989). Base pairing alone cannot bethe sole determinant of U2 snRNP-pre-mRNA binding,however, as purified U2 snRNP cannot bind to a pre-mRNAsubstrate without additional protein factor(s). Several groupshave reported the purification of activities required for thestable association of purified U2 snRNP with pre-mRNAin the pre-splicing complex (Ruskin et al., 1988; Kramer,1988).We have recently reported the chemical synthesis of

modified RNA oligonucleotides that are highly resistant tonuclease degradation (Sproat et al., 1989). These 2'-OMeRNA oligonucleotides can be used as antisense probes toform stable hybrids with targeted snRNAs in HeLa cellnuclear extracts (Blencowe et al., 1989; Lamond et al.,1989). Here we show that biotinylated antisense 2'-OMeRNA oligonucleotides complementary to human U2 snRNAallow highly specific affinity selection of U2 snRNP. This

UALCC

u c

C GU A

GC G U U

G-C G CU AC A A U

A A G C

U2 A U UCA-U G-CA_u AU A-U AC C A

sA GAUUUUUGUAUCAG AC UA U U GC ACGCAU CACCUGG C

AU ,, AU CGUGGCGGACC Ga c> UA. AA U C U

C30,d G,u*-;C-G

s

22'G GACbpp AU .

p C

AUCGCUU A

Fig. 1. Sequence of the human U2 snRNA. drawn according to thesecondary structure model of Keller and Noon (1985). The nucleotidesin U2 snRNA that show complementarity to the pre-mRNA branch siteare marked with black dots. The binding sites for the U2a and U2b2'-OMe RNA oligonucleotides are indicated with black lines. On eacholigonucleotide the 3' biotinylation site is indicated with a circle andthe 5' biotinylation site with a diamond.

41 71

A B

2

L

-...

Fig. 3. Detection of U2 snRNP particles using a non-denaturing gelassay. (A) HeLa nuclear extract was separated by electrophoresis on anon-denaturing polyacrylamide/agarose composite gel, electroblottedonto a nylon membrane and U2 snRNP particles detected by Northernhybridization with an anti-U2 riboprobe. (B) HeLa nuclear extract wasincubated with 5' end-labelled biotinylated oligonucleotides understandard splicing conditions and then analysed by non-denaturing gelelectrophoresis as in (A). In this case U2 snRNP was detected directlythrough its binding to the 32P-labelled oligonucleotides. Oligonucleotidelabelling and gel assay conditions were as described by Lamond et al.(1989). Samples correspond to: (A) lane 1, HeLa nuclear extractincubated without oligonucleotide; lane 2, HeLa nuclear extractincubated with unlabelled 5'-biotinylated U2a; lane 3, HeLa cellnuclear extract incubated with unlabelled 5'-biotinylated U2b. (B)Lane 1, HeLa nuclear extract incubated with 5' end-labelledU2a-5'bio; lane 2, HeLa nuclear extract incubated with 5' end-labelledU2b-5'bio.

Fig. 2. Affinity selection of U2 snRNP using tetrabiotinylated 2'-OMeRNA oligonucleotides. (A) Diagram of the affinity selection assay (seeMaterials and methods). (B, C) Affinity selection experimentscomparing the RNA recovered from streptavidin-agarose beads withRNA remaining in the supernatant fractions after incubation withdifferent biotinylated oligonucleotides. The RNAs were analysed on10% denaturing polyacrylamide/urea gels and detected by (B) ethidiumbromide staining or (C) by Northern hybridization with UsnRNA-specific riboprobes. Both the 'Intb' and 'Tag4' lanes correspond tosamples affinity selected with two separate control oligonucleotides thatare not complementary to any of the snRNAs.

has facilitated the study of U2 snRNP -pre-mRNA inter-actions at different stages of spliceosome assembly.

Results

Affinity selection of U2 snRNAIn order to generate U2-specific affinity probes, biotinylatedoligonucleotides made of 2'-OMe RNA have beensynthesized complementary to two separate regions of U2snRNA: nucleotides 1-20 (U2a) and nucleotides 27-49(U2b) (Figure 1). Both oligonucleotides have in addition,four modified deoxycytidines at their 5' termini, each link-ed through an amino alkyl spacer arm to biotin (Sproat et al.,1989). The binding of these biotinylated 2'-OMe RNAoligonucleotides to complementary sequences on U2 snRNAwas used to affinity select U2 snRNP from HeLa nuclearextract using streptavidin-agarose beads (Figure 2).Analysis of RNA eluted from streptavidin-agarose beadsafter affinity selection with the two, 5'-biotinylated anti-U2oligonucleotides shows a highly specific recovery of U2snRNA (Figure 2B and C, lanes U2a and U2b). A parallelanalysis of supernatant fractions also shows that U2 snRNAis selectively depleted. Co-addition of the U2a and U2b

oligonucleotides slightly increases the amount of U2 snRNAaffinity selected (Figure 2B, lane U2a + U2b). Quantitativeanalysis of long exposures of the Northern blot shown inFigure 2C indicates that both anti-U2 oligonucleotides af-finity select 102-103 more U2 snRNA than controls doneeither in the absence of oligonucleotide (Figure 2C, lane nooligo) or with a biotinylated 2'-OMe RNA oligonucleotidethat is not complementary to any of the snRNAs (Figure 2C,lane TAG4).The data presented in Figure 2 confirm that both anti-U2

2'-OMe RNA oligonucleotides are highly specific affinityprobes. The data also show that the U2a-5'bio oligo-nucleotide affinity selects U2 snRNA more efficiently thanU2b-5'bio. This was surprising, as a previous analysis (usingnon-biotinylated 2'-OMe RNA oligonucleotides) showed thatthe U2b oligonucleotide bound more strongly to U2 snRNPthan U2a (Lamond et al., 1989). In that studyoligonucleotide binding to U2 snRNP was assayed by non-denaturing polyacrylamide gel electrophoresis. We thereforeused the gel assay to test whether the presence of biotinaffected the relative binding efficiencies of theoligonucleotides (Figure 3). This shows the 5' end-labelledU2b-5'bio binds more strongly to U2 snRNP than 5' end-labelled U2a-5'bio (Figure 3B). This corresponds to the sameresult reported for the non-biotinylated oligonucleotides.Control experiments in which U2 snRNP was detected byNorthern hybridization demonstrate that the incubation ofHeLa nuclear extract with biotinylated oligonucleotides doesnot cause disruption of the U2 particle (Figure 3A). Thepresence of biotin does not, therefore, change the relativebinding properties of the oligonucleotides as judged by thegel assay. We note that the affinity selection and gel assaysare likely to differ in their sensitivity to aspects of the

S.Barabino et al.

4172

v.

Mapping U2 snRNP with 2'-OMe RNA oligonucleotides

A..- :3t---

- ..._

....

N at

B. .n I. J

-' 8

a. _

n * -

U. _ _D D -| || |

- -_ __ __ _

Fig. 4. Affinity selection of U2 snRNP with 3'-biotinylated 2'-OMeRNA oligonucleotides. (A) RNA recovered from bothstreptavidin-agarose beads and supernatant fractions, following affinityselection with 3'-biotinylated anti-U2 oligonucleotides, was separatedon 10% denaturing polyacrylamide/urea gels and detected by Northemhybridization with anti-snRNA riboprobes. Samples are: lane 1, nooligonucleotide control; lane 2, U2a-3'bio; lane 3, U2b-3'bio; lane 4,U2a-3'bio + U2b-3'bio. (B) Histogram showing the relative depletionof U2 snRNA from HeLa nuclear extract by the 5'- and3-biotinylated forms of the U2a and U2b oligonucleotides. The levelof depletion for each oligonucleotide is expressed as the ratio ofU2/U4 snRNA in supematant fractions relative to the U2/U4 snRNAratio in the 'no oligonucleotide' control lane. The data were obtainedby densitometric scanning of autoradiographs from the Northernhybridization experiments shown in Figures 2 and 4.

oligonucleotide -U2 snRNP binding interaction. Thus, thegel assay is likely to be primarily influenced by the stabilityof oligonucleotide binding. The low signal from the U2aoligonucleotide could be due to instability of the U2snRNA -U2a hybrid under the conditions of polyacrylamidegel electrophoresis. In the affinity selection assay, bindingto streptavidin - agarose can be influenced not only byoligonucleotide - snRNP stability, but also through stericeffects, e.g. steric hindrance from proteins in the U2 snRNPparticle may limit access of the streptavidin - agarose beadsto biotin residues. Thus biotin located at the 5' terminus ofthe U2b-5'bio oligonucleotide may be relatively inaccess-ible when bound within the snRNP particle (cf. Figure 1).

In order to test directly for steric effects in the affinityselection assay, 2'-OMe RNA U2a and U2b oligonucleotideswere synthesized with biotin residues linked to their 3' ratherthan 5' termini. Attachment of 3'-terminal biotin was doneby incorporating four additional biotin-linked uridinederivatives at the 3' end of each oligonucleotide. Theefficiency of U2 snRNA affinity selection by the U2a-3'bioand U2b-3'bio oligonucleotides is similar (Figure 4).Quantitative analysis of the data presented in Figures 2 and4 shows an increase in the relative depletion of U2 snRNA

Fig. 5. Affinity selection of purified total HeLa nuclear RNA by 5'-and 3'-biotinylated anti-U2 oligonucleotides. After affinity selection ofpurified total HeLa nuclear RNA with either (A) 5'-biotinylated or (B)3'-biotinylated anti-U2 oligonucleotides, RNA remaining in supernatantfractions was separated on 10% denaturing polyacrylamide/urea gelsand detected by Northem hybridization with anti-UsnRNA riboprobes.In both panels samples are: lane 1, no oligonucleotide control; lane 2,U2a; lane 3, U2b; lane 4, U2a + U2b.

by the U2b oligonucleotide when biotin is at the 3' ratherthan 5' terminus (Figure 4B). In contrast, the level of U2snRNA depletion by the 3' and 5' biotinylated forms of theU2a oligonucleotide is similar. These data demonstrate thatthe site of biotinylation on an oligonucleotide can influencethe affinity selection efficiency of a targeted RNP.The binding of both 5' and 3' biotinylated forms of the

anti-U2 oligonucleotides to purified total HeLa nuclear RNAwas also assayed by streptavidin - agarose affinity selection(Figure 5). The results show that the oligonucleotides re-tain their specificity for U2 snRNA in the absence of snRNPproteins, consistent with their binding to U2 snRNP throughsnRNA -2'-OMe RNA base pairing. As predicted by a sterichindrance model, the U2b oligonucleotide affinity selectednaked U2 snRNA equally well with biotin at the 5' or 3'terminus. This strongly supports the idea that proteins in theU2 snRNP particle block access of streptavidin-agarosebeads to biotin located at the 5' end of the U2boligonucleotide. Also, both biotinylated forms of the U2aoligonucleotide are extremely inefficient at selecting U2snRNA from purified HeLa nuclear RNA in comparisonwith their selection efficiency from HeLa nuclear extract(compare Figures 2 and 4 with Figure 5). This could be dueto a requirement for a protein factor to stabilize the U2snRNA - U2a oligonucleotide interaction. If such a factorwere unstable under the conditions of gel electrophoresis,this could explain why the U2a oligonucleotide gives a lowsignal for U2 binding in the gel assay (Figure 3). Analternative explanation for inefficient affinity selection ofnaked U2 snRNA by oligonucleotide U2a is that removalof snRNP proteins allows the U2 snRNA to adopt analternative secondary structure that precludes U2a binding.

Affinity selection reveals U2 snRNP-pre-mRNAinteractionsHaving established that the oligonucleotide probes interactspecifically with U2 snRNP, we have used them to analyseU2 snRNP-pre-mRNA interactions using a 'double-indirect' affinity selection assay (Figure 6A). The recoveryof 32P-labelled pre-mRNA from streptavidin-agarosebeads was compared in splicing assays containing differentbiotinylated 2'-OMe RNA oligonucleotides (Figure 6B). TheU2a oligonucleotide affinity selected 30% of the pre-mRNA in this assay (Figure 6B, lane U2a). Little or no pre-mRNA was affinity selected either in the absence of

4173

n-:1;

za:.111

.i:

-t_-

4-,

3- J..-Adm.

..& -mm..:

2- MO ow-

S.Barabino et al.

........

.

,!.. i . -' r '_

*i. _Wk, .w;: .j.i_Fig. 6. 'Double indirect' affinity selection of pre-mRNA. (A) Diagramillustrating the 'double indirect' affinity selection assay. (B, C) Affinityselected 32P-labelled pre-mRNA recovered from streptavidin-agarosebeads is shown analysed on denaturing polyacrylamide/urea gels. Thepre-mRNAs used were (B) AdenoPV'uI and (C) wild-type and mutantrabbit ,B-globin. The mutated nucleotides in the AL14 substrate are

indicated with arrows. RNAs were analysed either on 7% (globin) or

10% (adeno) denaturing polyacrylamide/urea gels. Size markers are an

end-labelled MspI digest of pBR322 (lane MRKS) and unsplicedsubstrate (lane pre-mRNA).

4174

oligonucleotide (Figure 6B, lane no oligo), or when the splic-ing assay with the biotinylated U2a oligonucleotide was per-formed in the absence of ATP, i.e. under conditions knownto block U2 snRNP from binding to pre-mRNA. The U2boligonucleotide, which masks the branch site complementaryregion of U2 snRNA, did not affinity select pre-mRNA(Figure 6B, lane U2b). Co-addition of both U2a and U2bresulted in no affinity selection of pre-mRNA (Figure 6B,lane U2a + U2b). The U2b oligonucleotide is thereforedominant over U2a in this assay. Addition of U2a togetherwith a control oligonucleotide that cannot bind U2 snRNP(U2c; Lamond et al., 1989) did result in affinity selectionof pre-mRNA (Figure 6B, lane U2a + U2c). Anoligonucleotide complementary to the pre-mRNA was usedas a positive control and affinity selected approximately threetimes more pre-mRNA than U2a (Figure 6B, lane TAG4).The lower level of substrate selected by U2a compared tothe pre-mRNA complementary oligonucleotide is consistentwith the observation that < 50% of pre-mRNA is complex-ed with U2 snRNP under these assay conditions, as judgedby native gel analyses (our unpublished observations).The data presented in Figure 6B show that pre-mRNA can

be affinity selected from HeLa splicing extracts through thebinding of U2 snRNP particles that are in turn bound to abiotinylated 2'-OMe RNA oligonucleotide, i.e. double-indirect affinity selection (cf. Figure 6A). As an additionalcontrol to demonstrate that the ATP-dependent selection ofpre-mRNA substrates by the U2a oligonucleotide resultsfrom binding of the oligonucleotide-U2 snRNP hybrid topre-mRNA, affinity selection of a mutant substrate wasassayed (Figure 6C). Affinity selection of wild-type (ALA)and mutant (AL14) f-globin pre-mRNAs was compared.The AL14 pre-mRNA has point mutations at both 5' and3' splice sites that inhibit U2 snRNP binding (Lamond et al.,1987, 1989). The affinity selection efficiency was internallycontrolled by incubating both wild-type and mutant substratesin the same splicing extract. The data show that the wild-type pre-mRNA is preferentially affinity selected over themutant (Figure 6C, lane U2a). Neither wild-type nor mutantpre-mRNAs are affinity selected in the absence ofoligonucleotide or when the splicing extract is incubatedwithout ATP. We therefore conclude that affinity selectionof substrate RNA results from the specific interaction of aU2 snRNP-oligonucleotide complex with a pre-mRNAsplice site.

Affinity selection of splicing productsPre-incubation of HeLa nuclear extract with either the U2aor U2b 2'-OMe RNA oligonucleotides blocks splicing andinhibits assembly of functional spliceosomes (Lamond et al.,1989). Therefore, to test whether complexes containing theintermediates and products of the splicing reaction can beaffinity selected through the binding of oligonucleotides toU2 snRNP in the spliceosome, anti-U2 oligonucleotides wereadded after spliceosome assembly (Figure 7). As in theexperiments where oligonucleotides were added to HeLaextract before the substrate RNA (Figure 6), affinity selectionof unspliced pre-mRNA was observed with the U2aoligonucleotide but not with U2b. Both the U2a and U2boligonucleotides, however, selected approximately equivalentamounts of the fully excised intron lariat but none of theintron lariat-3' exon splicing intermediate (Figure 7, lanesU2a and U2b). Control experiments done with RNA purifiedfrom a splicing reaction showed that neither of the anti-U2

'--

2

Mapping U2 snRNP with 2'-OMe RNA oligonucleotides

*:--

Fig. 7. Affinity selection of pre-mRNA splicing products. 32P-LabelledRNAs recovered from a splicing reaction before (lane total) and afteraffinity selection with the anti-U2 oligonucleotides (lanes U2a andU2b) or with an oligonucleotide complementary to the pre-mRNA(lane TAG4) is shown analysed on a 7% denaturingpolyacrylamide/urea gel. The unspliced pre-mRNA and splicingintermediates and products are marked with arrowheads. Size markersare an end-labelled MspI digest of pBR322 (lane MRKS) andunspliced substrate (lane pre-mRNA).

oligonucleotides could affinity select either unspliced pre-

mRNA or the splicing intermediates and products (data notshown). This excludes the possibility that selection of theexcised intron involves direct binding of oligonucleotides tointron RNA released from the post-splicing complex. As a

positive control, an oligonucleotide complementary to thepre-mRNA substrate was used. This probe affinity selectedunspliced pre-mRNA and all species of splicing intermediatesand products (Figure 7, lane TAG4). The failure of anti-U2oligonucleotides to select splicing intermediates musttherefore reflect the specificity of the oligonucleotides andnot represent an inherent limitation of the affinity assay.These results indicate that there is a differential accessibili-ty of U2 snRNP to oligonucleotide probes when U2 is incomplexes containing splicing intermediates as opposed to

splicing products.

A U2 snRNP-pre-mRNA complex is a kineticprecursor of a functional spliceosomePrevious studies of spliceosome assembly have indicated that

formation of a functional spliceosome (here termed B

complex) is preceded by the formation of a pre-splicingcomplex (here termed A complex) containing unspliced pre-mRNA and U2 snRNP (Grabowski and Sharp, 1986;

Pikielny et al., 1986; Bindereif and Green, 1987; Cheng andAbelson, 1987; Konarska and Sharp, 1987; Lamond et al.,1988). Although the pre-splicing A complex has the kineticproperties of an authentic intermediate in spliceosomeassembly it has proven difficult to show that the A complexis converted to a functional spliceosome. Therefore, toaddress whether the A complex is a spliceosome precursoror a dead-end product of the in vitro reaction we haveexploited the effect of both the U2a 2'-OMe RNAoligonucleotide and MgC12 concentration on spliceosomeassembly (Figure 8). The binding of U2a to the U2 snRNPblocks spliceosome assembly but not A complex formation(Lamond et al., 1989) . Incubation of pre-mRNA in HeLanuclear extract at low MgCl2 concentration also results inA complex formation but not assembly of functionalspliceosomes (Figure 8C, lanes 1 and 2; unpublishedobservations). Detectable spliceosome assembly can,however, be restored by increasing the MgCI2 concen-tration. The precise concentrations of MgCl2 required forthese effects was determined empirically and varies slightlybetween different preparations of HeLa nuclear extract.

In Figure 8B the appearance of splicing products iscompared in parallel splicing assays containing pre-mRNAand the U2a oligonucleotide. In both assays identicalcomponents are present, the only difference being that onesample was pre-incubated at 1 mM MgCl2 with pre-mRNAand the other was pre-incubated without pre-mRNA (cf.Figure 8A). The data show that intermediates and productsof the splicing reaction are only observed when pre-mRNAwas present during the pre-incubation. We interpret this asthe result of oligonucleotide U2a inhibiting all spliceosomeassembly except for that stemming from the A complexaccumulated during pre-incubation of pre-mRNA at lowMgCl2 concentration. In support of this view a parallelassay of splicing complex assembly shows that B (i.e.spliceosome) complexes do not form after increasing theMgCl2 concentration when oligonucleotide U2a is presentduring the pre-incubation (Figure 8C, lane 3). B complexesdo form if oligonucleotide U2a is added together with theextra MgCl2 (lanes 4 and 5). Little or no B complex isformed if the MgCl2 concentration is not increased from 1to 2-3 mM (lane 1). The level of complex B formed afterincreasing the concentration of MgCl2 is substantiallyhigher when no U2a is added than in the presence of U2a,even after prolonged incubation of the U2a-containing sample(Figure 8C, compare lane 2 with lanes 4 and 5). This isconsistent with spliceosome formation in the presence of theU2a oligonucleotide only involving A complexes pre-assembled during the pre-incubation.To eliminate the possibility that the presence of the U2a

oligonucleotide indirectly affected spliceosome formation,a simple experiment was done to compare the rate of splicingwith or without pre-incubation of pre-mRNA at low MgCl2concentration (Figure 8D). If the A complex accumulatedduring the pre-incubation is a spliceosome precursor thenthe rate of splicing should be faster than for the non pre-incubated control. If instead the A complex is a dead endproduct then the pre-incubated sample should splice lessefficiently than the control. The data show that splicedproducts appear some 10-20 min faster in the pre-incubatedsample than in the non pre-incubated control. No differencein the rate of splicing is observed, however, if the pre-incubation is done without ATP (data not shown). Weconclude that an ATP-dependent complex that is a

41 75

IoLa EX r::bna'13ecd wiK ep-' NA

at iow Mg :cneraioJ))r3 jin 9g Anomoca 0Qfri

*cs lJ2a o!go' sc ji..*1 ....~~~~~~~~~~~~~~~~~~~....

2.'3,2_

::.

p :r __j,:a__r

_ 0 C _3_0 6_0'106C

Fig. 8. A U2 snRNP-pre-mRNA complex is a spliceosome precursor. (A) Schematic description of the experimental approach. (B) Kinetics of

splicing assayed in the presence of the U2a oligonucleotide either with (lane 8-12) or without (lanes 3-7) preincubation at 1 mMMgCI2. A

splicing reaction using AdenoSau3A pre-mRNA was pre-incubated at 1 mM MgCl2 for 30 min at 30°C. Subsequently, the MgCl2 concentration was

increased to 2 mM and the U2a oligonucleotide added simultaneously. Incubation was continued and samples taken at the time points indicated (lanes8-12). Splicing products were analysed on a 10% denaturing polyacrylamide/urea gel. Lanes 3-7 are equivalent time points from a control reactionin which the pre-mRNA was not included in the pre-incubation at low MgCl2 concentration, but added together with the U2a oligonucleotide and

MgCI2. Lane 1 shows size markers (end-labelled MspIl digest of plasmid pBR322). Lane 2 shows unspliced AdenoSau3A pre-mRNA. (C) Non-

denaturing gel analysis of splicing complex assembly. Each lane shows 32P-labelled pre-mRNA pre-incubated with HeLa cell nuclear extract in 1 mMMgCl2 for 30 min at 30'C. Lanes 1 and 2 are controls. In lane 1 incubation was continued for a further 30 min at 30°C without increasing theMgCl2 concentration. In lane 2 the MgCl2 concentration was increased to 2 mM after pre-incubation and incubated for a further 30 min at 30°C.Lane 3; same as lane 2, but U2a oligonucleotide added at start of pre-incubation. Lane 4; same as lane 2, but U2a oligonucleotide added after pre-incubation and then incubated for a further 30 min at 30°C. Lane 5; same as lane 4, but incubation continued for 60 min after the pre-incubation.(D) Effect of pre-incubation at 1 mM MgCI2 on splicing kinetics. Lanes 8-12 show samples taken at successive time points from a splicing reactionthat had been pre-incubated in 1 mM MgCl2 for 30 min at 30°C. Splicing was assayed at the indicated times after the MgCl2 concentration was

raised to 2 mM. As a control (lanes 3-7), HeLa nuclear extract was pre-incubated in the absence of substrate and then pre-mRNA added at thesame time as the MgCl2 concentration was increased to 2 mM. Splicing was assayed at the indicated time points as for the pre-incubated sample.

spliceosome precursor assembles during pre-incubation. Thiscomplex is sensitive to MgCl2 concentration and to anti-U2snRNP oligonucleotides and most probably corresponds tothe previously described A complex.

DiscussionAntisense 2'-OMe RNA oligonucleotides coupled to biotinare shown here to be highly specific probes for affinityselecting snRNP particles and analysing snRNP -pre-mRNA

4176

S.Barabino et al.

A

* t l Ow;_oi- or,e SO !i.

Mapping U2 snRNP with 2'-OMe RNA oligonucleotides

Table I. Sequences of the 2'-OMe RNA oligonucleotides

Oligonucleotide Length Sequence Complementary Biotinylationnucleotides in U2

U2a 20 CCAAAAIICCIAIAAICIAU 1-20 5' or 3'U2b 20 AIAACAIAUACUACACUUIA 27-46 5' or 3'aTAG4 25 ICAAIICIAUUAAIUUIIIUAACIC 5'

bIntB 21 AUUAUCAUIAIIIUCCAUIIU 5'

aComplementary to 3' terrminal region of pre-mRNAbComplementary to intron internal region of rabbit globin pre-mRNA

interactions. Biotinylated oligonucleotides complementary totwo separate regions of U2 snRNA allowed the selectiveremoval of U2 snRNP from HeLa cell nuclear extract.However, it was observed that the affinity selection efficiencycould be affected by the position of biotin residues on theprobe, probably due to snRNP proteins interfering with thebinding of biotin to streptavidin -agarose. This indicates thatfor analysing RNP structures the optimization of affinityselection efficiency depends on having the ability to controlthe site of biotinylation on the probe. We note that the systemdescribed here, involving chemical synthesis of modifiedRNA oligonucleotides, not only allows the position of biotinresidues to be controlled but also generates probes that arerendered highly resistant to nuclease degradation by virtueof the ribose methylation (Sproat et al., 1989). These probescan therefore be used in crude extracts that have contam-inating nuclease activity.

U2 snRNP -pre-mRNA interactionsThe affinity selection of splicing substrates by the biotinylatedU2a oligonucleotide demonstrates that its binding does notprevent U2 snRNP forming a stable complex with pre-mRNA. The failure of the U2b oligonucleotide to select pre-mRNA shows that no stable U2 snRNP-pre-mRNAcomplex forms in the presence of this probe. These resultsconfirm the conclusions drawn in a previous studyconcerning the roles of separate U2 snRNA domains inspliceosome assembly (Lamond et al., 1989). The bindingof the U2b oligonucleotide masks U2 snRNA sequencescomplementary to the pre-mRNA banch site. The datasupport an essential role for snRNA - pre-mRNA basepairing in determining the stable interaction of mammalianU2 snRNP with the branch site. A similar base pairinginteraction has been shown to be essential for the bindingof yeast (S. cerevisiae) U2 snRNP to the pre-mRNA branchsite in vivo (Parker et al., 1987). The ability of U2 snRNPto form a stable complex with pre-mRNA (A complex) whenthe U2a oligonucleotide is bound indicates that the 5'terminus of U2 snRNA is either not directly involved in Acomplex assembly or else can still function when U2a ispresent. The failure of the A complex to proceed to forma functional spliceosome in the presence of U2a, however,suggests an essential role for the 5' terminal region of U2snRNP in a later step in the assembly pathway.The conclusions presented above differ from previous

proposals concerning the roles of separate U2 snRNPdomains (Chabot and Steitz, 1987; Frendewey et al., 1987;Zillmann et al., 1988). Using DNA oligonucleotides to directquantitative RNase H cleavage of U2 snRNA at positionsequivalent to those masked by the binding of the 2'-OMeRNA U2a and U2b oligonucleotides, these authors reportedthat the 5' terminus of U2 snRNA was required for A

complex formation while the branch site complementaryregion (i.e. 'U2b' region) was not essential. The cleavageof targeted regions of U2 snRNP by RNase H may affectits structure in a different way from masking the equivalentsequences with antisense oligonucleotides, thus leading todifferent effects on U2 snRNP function. Alternatively, it ispossible that the high levels of DNA oligonucleotidesrequired for quantitative RNase H cleavage of U2 snRNAin HeLa nuclear extracts indirectly affects spliceosomeformation. In the present studies 2'-OMe RNAoligonucleotides were used at concentrations more than10-fold lower than the DNA oligonucleotides in the RNaseH experiments. The ability to use 2'-OMe RNAoligonucleotides at relatively low concentrations should helpto minimize the possibility of non-specific effects. We alsonote that a recent study in Xenopus oocytes has reported thattargeted RNase H cleavage of U2 snRNP with a DNAoligonucleotide complementary to the U2b region bothinhibits splicing and prevents pre-splicing A complexformation (Hamm et al., 1989). The Xenopus data thereforeagree with the antisense results presented here. Furtherstudies in HeLa nuclear extracts are required to clarify howspecifically the cleavage of U2 snRNA by RNase H affectsthe function of the U2 snRNP particle.The ability of both biotinylated anti-U2 oligonucleotides

to specifically affinity select the fully excised intron lariat,but not the pre-mRNA splicing intermediates, indicates adifference in the structure and/or accessibility of thespliceosome compared to the post-splicing complex. Thisconclusion is supported by additional studies which showthat biotinylated 2'-OMe RNA oligonucleotides comple-mentary to specific regions of U4 and U6 snRNAs alsoaffinity select the products but not intermediates of splicing(Blencowe et al., 1989). As previous 'oligonucleotidechallenge' experiments demonstrated that neither the U2anor U2b oligonucleotides disrupt pre-assembled spliceosomes(Lamond et al., 1989), their failure to affinity select splicingintermediates cannot be due to specific destabilization ofspliceosomes as opposed to post-splicing complexes. Wesuggest that the U2 binding sites in functional spliceosomesare relatively inaccessible, either due to conformationalchanges in the U2 snRNP or because of steric hindranceresulting from the presence of additional spliceosome fac-tors. In the case of the U2b oligonucleotide it is especiallystriking that neither unspliced pre-mRNA nor the intronlariat -3' exon intermediate can be selected but only the fullyexcised intron lariat (Figure 7). This implies that the post-splicing complex not only contains U2 snRNP in a confor-mation accessible to oligonucleotide U2b binding but thatin this complex the U2 snRNA-pre-mRNA branch site basepairing interaction is no longer essential for stability. Incontrast, the base pairing interaction does appear to be

4177

S.Barabino et al.

essential for maintaining the stability of the pre-splicingcomplex as the U2b oligonucleotide can disrupt pre-assembled A complex (Lamond et al., 1989). These resultsare therefore consistent with a change in the mode of U2snRNP binding to pre-mRNA at different stages of thesplicing reaction.

Materials and methodsOligonucleotides2'-OMe RNA oligonucleotides were synthesized as described by Sproat et al.(1989). In each 2'-OMe RNA oligonucleotide inosine and not guanosinewas used to base pair with cytosine. The sequences of the oligonucleotidesare shown in Table I. The 5'-biotinylated oligonucleotides have at their 5'terminus four 2'-deoxycytidine residues that have been modified to carrybiotin groups linked via an amino alkyl spacer arm on the exocyclic aminogroup of the cytidine ring. In the 3-biotinylated oligonucleotides four biotinmolecules were linked through a similar spacer arm to four modified uridineresidues.

In vitro splicing assaysHeLa cell nuclear tract was prepared as described by Dignam et al. (1983).Adeno pre-mRNA was made from plasmid pBSAdl (Konarska and Sharp,1987) cut with Sau3A or PvuI. Wild-type and mutant rabbit f-globin pre-mRNAs were made from plasmids pBSAL4 and pBSAL14 respectively,both digested with Pvull (Lamond et al., 1987). Transcription of uniformlylabelled, capped pre-mRNAs and in vitro splicing assays were carried outas previously described (Lamond et al., 1987).

Streptavidin - agarose affinity selection assayBefore use, 150-200 jtl of streptavidin-agarose beads suspension (BRL)per 100 1l reaction volume were rotated for 15 min at 4°C in a pre-blockingmix (100 pg/ml tRNA, 100 ag/ml glycogen, 1 mg/ml BSA) to minimizenon-specific binding. Subsequently the beads were pelleted and washed threetimes with 500 1d 50 mM washing buffer (WB50: 20 mM Tris-HCI, pH7.6, 0.01% Nonidet P40, 1.5 mM NaN3, 50 mM NaCl). After the finalwash the beads were resuspended in 1/3 of the initial bead volume ofWB250 (same as WB50 but 250 mM NaCI). For U2 snRNP affinityselection experiments 100 ul reactions were used containing: 35% (v/v) HeLacell nuclear extract (-5 mg/ml final concentration), 1 mM MgCl2,1.5 mM ATP, 5 mM creatine-phosphate and 2-5 pmol/Al 2'-OMe RNAoligonucleotides. The samples were incubated for 30 min at 30°C then2-3 1l of pre-blocked streptavidin-agarose beads per td of reaction mixtureand 500 1l of WB250 were added and the mixture rotated for 1 h at 4°C.The samples were then spun for I min at 5000 r.p.m. in a microfuge. Thesupernatants were transferred to new Eppendorf tubes and incubated withProteinase K (2 mg/ml) and 0.1 S% SDS for 45 min at 65°C. RNAs wereprecipitated with 2.5 vol absolute ethanol using glycogen (20 jtg/ml) ascarrier. The pelleted beads were washed three times (10 min/wash inWB250 at 4°C) and then digested with Proteinase K (1 mg/ml) for 45 minat 65°C. RNA was released from the beads by heating for 15 min at 85°Cand precipitated with 2.5 vol absolute ethanol using glycogen (20 yg/ml)as carrier. All samples were analysed on 10% denaturing polyacryla-mide/urea gels and RNA detected by staining with ethidium bromide(2 ag/mni). For Northern hybridization analysis, gels were electroblotted ontoHybond N membrane (Amersham) and hybridized to snRNA riboprobesas described by Blencowe et al. (1989). Quantitative analysis was done using'Image' software (v. 1.10) on a digitized image of autoradiographs obtain-ed with a Macintosh II video scanning system.

AcknowledgementsThe authors are especially grateful to Phillipe Neuner for assistance withpreparation of the oligonucleotides used in this study. We also thank JorgHamm, Matthias Hentze, lain Mattaj, Cathy Mitchelmore and LennartPhilipson for critical review of the manuscript. S.B. acknowledges supportfrom an EEC Junior Training Grant (Biotechnology Division).

ReferencesBindereif,A. and Green,M.R. (1987) EMBO J., 6, 2415-2424.Black,D.L., Chabot,B. and Steitz,J.A. (1985) Cell, 42, 737-750.Blencowe,B.J., Sproat,B.S., Ryder,U., Barabino,S. and Lamond,A.I. (1989)

Cell, 59, in press.Chabot,B. and Steitz,J.A. (1987) Mol. Cell. Biol., 7, 281-293.Cheng,S.-C. and Abelsen,J. (1987) Genes Dev., 1, 1014-1027.Dignam,J.D., Lebowitz,R.M. and Roeder,R.G. (1983) Nucleic Acids Res.,

11, 1475-1489.Frendewey,D., Kramer,A. and Keller,W. (1987) Cold Spring Harbor Symp.

Quant. Biol.,53, 287-298.Grabowski,P.J. and Sharp,P.A. (1986) Science, 233, 1294-1299.Green,M.R. (1986) Annu. Rev. Genet., 20, 671-708.Hamm.J., Dathan,N.A. and Mattaj,I.W. (1989) Cell, in press.Keller,E. and Noon,W. (1985) Nucleic Acids Res., 13, 4971-4981.Konarska,M.M. and Sharp,P.A.(1986) Cell, 46, 845-855.Konarska,M.M. and Sharp,P.A. (1987) Cell, 49, 763-774.Kramer,A. (1987) J. Mol. Biol., 196, 559-573.Kramer,A. (1988) Genes Dev., 2, 1155-1167.Lamond,A.I., Konarska,M.M. and Sharp,P.A. (1987) Genes Dev., 1,532-543.

Lamond,A.I., Konarska,M.M., Grabowski,P.J. and Sharp,P.A. (1988)Proc. Natl. Acad. Sci. USA, 85, 411-415.

Lamond,A.I., Sproat,B.S., Ryder,U. and Hamm,J. (1989) Cell, 58,383 -390.

Maniatis,T. and Reed,R. (1987) Nature, 325, 673-678.Parker,R., Siliciano,P. and Guthrie,C. (1987) Cell, 49, 220-239.Pikielny,C.W., Rymond,B.C. and Rosbash,M. (1986) Nature, 324,

341 -345.Reed,R. and Maniatis,T. (1988) Genes Dev., 2, 1268-1276.Ruskin,B. and Green,M.R. (1985) Cell, 43, 131-142.Ruskin,B., Zamore,P.D. and Green,M.R. (1988) Cell, 52, 207-219.Sharp,P.A. (1987) Science, 235, 766-771.Sproat,B.S., Lamond,A.I., Beijer,B., Neuner,P. and Ryder,U. (1989)

Nucleic Acids Res., 17, 3373-3386.Steitz,J.A., Black,D.A., Gerke,V., Parker,K.A., Kramer,A., Frendewey,D.

and Keller,W. (1988) In Birnstiel,M.L. (ed.), Structure and FunctionofMajor and Minor Small Nuclear Ribonucleoprotein Particles. Springer-Verlag, Berlin, pp. 71-99.

Zhuang,Y., Goldstein,A.M. and Weiner,A.M. (1989) Proc. Natl. Acad.Sci. USA, 86, 2752-2756.

Zillmann,M., Zapp,M.L. and Berget,S.M. (1988) Mol. Cell. Biol., 8,814-821.

Received on 1989, Accepted 1989

Affinity selection of splicing complexesSplicing complex selection was performed on standard 20 pI splicingreactions containing 2-5 pmol/ul of 2'-OMe RNA oligonucleotides. Thesamples were incubated for 30 min at 30°C before addition of the pre-blockedstreptavidin-agarose beads and then processed as described above. Theeluted 32P-labelled pre-mRNA was analysed on 7% (globin) or 10%(adeno) denaturing polyacrylamide/urea gels. In the case of the affinityselection of pre-assembled splicing complexes (Figure 7), a standard splicingreaction was incubated for 1 h 45 min at 30°C and then divided into 20 ,ulaliquots and 2-5 pmol//l of 2'-OMe RNA oligonucleotides added. Thesamples were incubated for a further 10 min at 30°C before the additionof the streptavidin -agarose beads and then processed as described above.

4178