Molecular Cell, Vol. 6, 317–328, August, 2000, Copyright 2000 by Cell Press

Functional Recognition of the 59 Splice Site byU4/U6.U5 tri-snRNP Defines a Novel ATP-DependentStep in Early Spliceosome Assembly

spliceosome assembly. The concurrent association ofU1 and U2 snRNPs with the pre-mRNA defines the “pre-spliceosome” (complex A) (Reed and Palandjian, 1997).Following formation of complex A, U4, U5, and U6snRNPs join the spliceosome as a preformed tri-snRNP

Patricia A. Maroney,† Charles M. Romfo,†and Timothy W. Nilsen*Center for RNA Molecular BiologyDepartment of Molecular Biology and MicrobiologyCase Western Reserve University School of Medicine

to produce complex B. Upon tri-snRNP addition, an10900 Euclid Avenueintricate series of RNA-RNA rearrangements ensues, re-Cleveland, Ohio 44106sulting in the formation of the catalytically competentcomplex C. Key steps in formation of the catalyticallyactive spliceosome include pairing of the 59 splice site

Summary with U6 snRNA (an interaction that is mutually exclusivewith the U1/59 splice site pairing) and formation of U2/

A sensitive assay based on competition between cis- U6 base-pairing interactions at the expense of U4/U6pairing (Nilsen, 1998; Staley and Guthrie, 1998).and trans-splicing suggested that factors in addition

While this canonical view of spliceosome assemblyto U1 snRNP were important for early 59 splice sitehas proven to be reliable and predictive, there existrecognition. Cross-linking and physical protection ex-notable exceptions to the obligatory order of additionperiments revealed a functionally important interac-of some components. For example, U2 snRNP can spe-tion between U4/U6.U5 tri-snRNP and the 59 splicecifically bind to the branch point region in certain mole-site, which unexpectedly was not dependent uponcules that lack a 59 splice site (e.g., Ruskin and Green,prior binding of U2 snRNP to the branch point. The1985; Chiara and Reed, 1995). In one well-establishedearly 59 splice site/tri-snRNP interaction requires ATP,pathway, 59 splice site-independent recruitment of U2occurs in both nematode and HeLa cell extracts, andsnRNP is mediated by exonic enhancer elements lo-

involves sequence-specific interactions between the cated in the 39 exon (e.g., Lavigueur et al., 1993; Wanghighly conserved splicing factor Prp8 and the 59 splice et al., 1995; Zuo and Maniatis, 1996). There are similarsite. We propose that U1 and U5 snRNPs functionally exceptions to the order of assembly described abovecollaborate to recognize and define the 59 splice site that involve 59 splice site recognition. In a comprehen-prior to establishment of communication with the 39 sive series of experiments, Konarska and colleaguessplice site. have shown that oligoribonucleotides mimicking 59

splice sites can engage U6 snRNP directly, effectivelybypassing a role for U1 snRNP (Konforti et al., 1993;IntroductionKonforti and Konarska, 1994, 1995). Indeed, productiveuse of such oligonucleotides in splicing is observed onlySplicing of nuclear pre-mRNAs occurs in a large ribo-when the 59 end of U1 snRNP is sequestered (Konfortinucleoprotein complex known as the spliceosome,and Konarska, 1995). A similar bypass of U1 snRNPwhich is comprised of five snRNPs and a large numberfunction, thought to be mediated by direct engagement(.50) of non-snRNP-associated proteins (reviewed inof U6 snRNA, is observed when certain pre-mRNAs areBurge et al., 1999; Staley and Guthrie, 1998). Unlike thespliced in U1 snRNP inactivated or depleted extractsribosome, a ribonucleoprotein machine of comparable(Tarn and Steitz, 1994, 1995; Crispino and Sharp, 1995;size and complexity, the spliceosome must be assem-Crispino et al., 1996). Processing of some of these mole-bled de novo on each intron. Essential to initiation ofcules requires high concentrations of SR proteins. Nota-assembly is recognition of intronic boundaries, the 59bly, both of these examples of U1-independent splicingand 39 splice sites. Despite intensive investigation, nei-have been viewed as extraordinary reactions, not neces-ther the precise determinants of splice site recognitionsarily reflective of “normal” spliceosome assembly.nor the molecular details of splice site communication

In addition to the exceptions noted above, the consen-are fully understood.sus model of spliceosome assembly fails to adequatelyNevertheless, a vast amount of data has led to a con-explain a number of phenomena associated with 59sensus view of an ordered pathway of spliceosome as-splice site choice. Specifically, a deterministic role for

sembly. In this model, U1 snRNP and associated factors U1 snRNA base pairing (or U6 pairing) is unlikely torecognize the 59 splice site, resulting in the formation of account for activation of some cryptic donor sitescommitment (or E) complex; it is thought that functional (Nilsen, 1994). Similarly, in certain unusual circum-association of the 59 and 39 splice sites is established stances, U5 snRNP unambiguously dictates the positionin this complex (Abovich and Rosbash, 1997; Reed and of the 59 splice site (Newman and Norman, 1991, 1992;Palandjian, 1997). The U1 snRNP-59 splice site interac- Cortes et al., 1993). In this regard, Newman and Normantion proceeds at 08C and is ATP independent. Subse- (1992) demonstrated that, in the presence of a mutantquently, U1 snRNP promotes U2 snRNP binding to the 59 splice site, base pairing between U5 snRNP and 59branch point sequence; association of U2 snRNP is de- exonic nucleotides activated a cryptic splice site severalpendent upon incubation at higher temperature and is nucleotides upstream from the site of U1 interaction.widely viewed as the first energy requiring step in Here, we have used a sensitive assay to probe func-

tional recognition of the 59 splice site. This novel assayis based upon competition between cis- and trans-splic-* To whom correspondence should be addressed (e-mail: twn@po.ing in an in vitro system derived from nematode em-cwru.edu).

† These authors contributed equally to this work. bryos. In nematodes, most pre-mRNAs are processed

Molecular Cell318

by both cis- and trans-splicing. In trans-splicing, the helps to recruit U2 snRNP to the branchpoint (Figure1A; Experimental Procedures).59 most exon of the mature mRNA is acquired from

a specialized Sm snRNP, the spliced leader (SL) RNP As an initial test of the competition assay, we inacti-vated U1 snRNP via annealing of a 29Ome oligonucleo-(Blumenthal, 1995; Nilsen, 1997). Accurate processing

requires that SL addition be prevented at internal cis-39 tide complementary to its 59 end. Given the primary roleof U1 snRNP in 59 splice site recognition, we anticipatedsplice sites. Several studies have demonstrated that

cis- and trans-39 splice sites appear to be functionally that cis-splicing would be inhibited and trans-splicingwould be activated; trans-splicing does not depend theequivalent, since cis-39 acceptors uncoupled from their

upstream 59 splice site are efficiently recognized and 59 end of U1 snRNP (Nilsen, 1997). While debilitation ofused by the trans-splicing apparatus (Conrad et al., U1 snRNP resulted in almost complete inhibition of cis-1991; C. M. R., P. A. M., and T. W. N., unpublished data.). splicing, activation of trans-splicing was surprisinglyConversely, both in vivo and in vitro, a functional 59 modest under these conditions (Figure 1C, lane 2). Bysplice site in cis-splicing is necessary and sufficient contrast, occlusion of the 59 splice site with a comple-to prevent inappropriate trans-splicing at downstream mentary 29Ome oligonucleotide (nucleotides 24 to 110)splice sites and inactivation of a cis-59 splice site allows led to a more significant increase in the level of trans-trans-splicing (Conrad et al., 1993; Figure 1). Accord- splicing even though cis-splicing was somewhat lessingly, SL addition to an otherwise nonpermissive cis-39 inhibited (Figure 1C, lane 3). These results suggestedsplice site provides a powerful reporter both for initial either that oligonucleotide-blocked U1 snRNP could stillrecognition of cis-59 splice sites and for functional com- associate at some level with the 59 splice site or thatmunication between 59 and 39 splice sites. Using an factors in addition to U1 snRNP might be involved inappropriate cis-splicing substrate, we generated a panel early 59 splice site recognition.of mutations in the 59 splice site region and monitoredactivation of trans-splicing. The observed pattern of ac- Activation of trans-Splicing in the Presencetivation was not fully consistent with disruption of the of Mutant 59 Splice SitesU1 snRNP/59 splice site base-pairing interaction, and As shown in Figure 1B, lane 3, and 1C, lane 4, mutationan unexpected dependence on exonic nucleotides 21 of intronic nucleotides 11 to 16 led to full activation ofand 22 was revealed. trans-splicing. To define more precisely the contribution

Site-specific cross-linking indicated that the impor- of 59 splice site nucleotides to suppression of trans-tance of these nucleotides is correlated with a specific splicing, we introduced a series of dinucleotide muta-interaction between the highly conserved U5 snRNP- tions [(11,12)(13,14)(15,16)] in the intronic compo-associated protein, Prp8, and the 59 splice site. This nent of the 59 splice site; in addition, the 39 terminalinteraction requires ATP and incubation at 308C and two nucleotides of the 59 exon (-1,-2) were altered. Asunambiguously occurs independent of stable U2 snRNP discussed below, the latter nucleotides are highly con-association with the branch point. Several lines of evi- served and have previously been implicated as determi-dence indicate that the early Prp8/59 splice site inter- nants of 59 splice site recognition. When tested in splic-action occurs only if Prp8 is a constituent of U4/U6.U5 ing reactions, the series of mutant substrates yielded antri-snRNP. These results are likely to be of general signif- intriguing range of phenotypes. Alteration of the nearlyicance because identical interactions were observed in invariant GU (11,12) resulted in full activation of trans-HeLa cell extracts. The data define a previously unrec- splicing, whereas alteration of nucleotides 13 and 14ognized ATP-requiring step that proceeds in the ab- yielded essentially no activation (compare Figure 2A,sence of U2 snRNP association with the branch point lanes 3 and 4). Substantial activation of trans-splicingand reveal an unexpected role for U4/U6.U5 tri-snRNP was observed with mutant 15,16 (Figure 2A, lane 5)both in early 59 splice site recognition and in early func- and, remarkably, with mutant 21,22 (Figure 2A, lane 2).tional communication between splice sites. These re- These results clearly indicated that different sequencesults help to explain a variety of heretofore puzzling elements within the 59 splice site play distinct roles inbiochemical and genetic observations in the splicing initial recruitment of and/or stable binding of 59 spliceliterature and have substantive implications for under- recognition factors. Furthermore, 59 exonic sequencestanding the mechanisms of 59 splice site identification elements must be considered important for this processand the mechanism of recruitment of U4/U6.U5 tri- (see below and Discussion).snRNP to the maturing spliceosome. Significantly, the data obtained with mutant 59 splice

sites did not reveal a strict correlation between the ex-Results tent to which U1 pairing was disrupted and activation

of trans-splicing. For example, mutant 13,14 is pre-dicted to eliminate two internal base pairs of the U1/59A Sensitive Competition Assay for 59 Splice

Site Function splice site helix, while mutant 15,16 affects only oneterminal base pair; however, the activation of trans-The principle of the cis-/trans-splicing competition

assay is shown schematically in Figure 1A. The wild- splicing was much greater with mutant 15,16 (Figure2A, compare lanes 4 and 5). Despite the fact that thetype pre-mRNA is efficiently cis-spliced, and internal SL

addition is barely detectable (Figure 1B, lane 1). Upon pattern of activation of trans-splicing by different 59splice site mutants did not correlate with destabilizationremoval (Figure 1B, lane 2) or mutational inactivation

(Figure 1B, lane 3) of the cis-59 splice site, efficient trans- of U1 snRNP binding, these results could not excludethe possibility that the extent of trans-splicing was nev-splicing is observed. Because trans-splicing depends

upon 59 splice site independent recruitment of U2 snRNP ertheless determined by U1 snRNP occupancy of the59 splice site.(C. M. R., P. A. M., and T. W. N. unpublished data),

the pre-mRNA was designed to contain a 39 exon with To address this possibility, we used a variety of tech-niques, including affinity selection, in attempts to quanti-sequence elements that mediate SR protein-dependent

recruitment of U2AF to the 39 splice site. U2AF in turn tate U1 snRNP binding to 59 splice site variants. Unfortu-

59 Splice Site Recognition by tri-snRNP319

Figure 1. Trans-Splicing as a Reporter for cis59 Splice Site Function

(A) The basis of the competition assay be-tween cis- and trans-splicing is shown sche-matically. The presence of a functional 59 do-nor site in cis prevents use of the spliceacceptor as a site of SL addition; inactivationof the donor site (represented by the X overthe 59 splice site) permits trans-splicing. Forthe pre-mRNA splicing substrates used, U2snRNP recruitment is 59 splice site indepen-dent (see Results).(B) In vitro splicing of the indicated 39 end-labeled substrates was carried out as de-scribed in the Experimental Procedures, andidentities of splicing intermediates and prod-ucts are indicated schematically. In lane 3,the 59 splice site was mutant in nucleotides11 to 16 (indicated by the X). The RNAmigrating between the substrate and trans-spliced product in lane 3 is a cis-spliced prod-uct resulting from the inefficient use of a cryp-tic 59 splice site activated by the mutation.(C) Splicing in the presence of no oligonucle-otide, lane 1; a 29Ome oligonucleotide com-plementary to nucleotides 1–14 of the 59 endof U1 snRNA, lane 2; a 29Ome oligonucleotidecomplementary to nucleotides 24 to 110 ofthe 59 splice site region of the substrate, lane3. Lane 4 is a reaction containing the samesubstrate as B, lane 3. Substrate designa-tions are as in (B).

nately, these experiments were not informative because was identical at the two temperatures. Remarkably, cis-splicing was essentially abolished at 378C in mutant 21,we could only detect U1 snRNP association with the

wild-type 59 splice site; no measurable binding was ob- 22, but significantly there was no concomitant increasein trans-splicing (Figure 2B, lanes 3 and 4, and seeserved with any of the mutants (data not shown). As a

final approach to address the U1 snRNP occupancy below).The foregoing results, considered in their entirety,question, splicing of the entire panel of substrates was

assayed at 308 and 378C (Figure 2B). We reasoned that strongly suggested that U1 snRNP was not the soledeterminant of suppression of trans-splicing and, asdestabilization of U1 pairing might be exacerbated at

high temperature; similar strategies have been used by a corollary, was not the only factor involved in earlyrecognition of the 59 splice site. Both the pattern ofothers to assess U1 snRNP/59 splice site interactions

(Staley and Guthrie, 1999). As shown in Figure 2B (lanes splicing and the in vitro “temperature sensitivity” ob-served with mutant 21,22 suggested that these exonic1 and 2 and 11 and 12), both cis- and trans-splicing

proceed efficiently at 378C. However, with the exception nucleotides were important recognition elements for ad-ditional factor(s).of the 21,22 mutant, the ratio of cis- to trans-splicing

Molecular Cell320

Figure 2. Differential Activation of trans-Splicing by Specific Mutations in the cis-59

Splice Site

(A) In vitro splicing of 39 end-labeled sub-strates containing either wild-type (lane 1) ortwo nucleotide 59 splice site mutations as in-dicated (lanes 2 through 5) were carried outas described in the Experimental Procedures;the substrate in lane 6 was mutant in nucleo-tides 11 to 16. Cis- and trans-spliced prod-ucts are indicated. The potential for basepairing between the wild-type 59 splice siteand the 59 end of Ascaris U1 snRNA (Sham-baugh et al., 1994) is shown on the right.(B) The same substrates used in (A) werespliced in vitro at either 308C or 378C. TheRNA resulting from use of the cryptic 59 splicesite described in the legend to Figure 1 isevident in lanes 3, 4, 5, and 11.

Prp8 Contacts the 59 Splice Site Independent of U2 bands of lower molecular weight; transfer of label to theproteins was completely dependent upon UV irradiationsnRNP Binding to the Branch Point

In an attempt to identify factors associated with the 59 and incubation with extract (Figure 3A). If the high mo-lecular weight cross-linked protein was relevant to 59splice site, we performed short wavelength UV cross-

linking experiments with RNAs containing a single la- splice site recognition, it would be expected to showsequence specificity. Therefore, identical cross-linkingbeled phosphate within the nearly invariant GU dinu-

cleotide (Figure 3). To accumulate initial 59 splice site experiments were performed with the panel of 59 splicesite mutants used above (Figure 2A). Cross-linking to therecognition factors and to avoid detection of factors

that interact with the 59 splice site during spliceosome high molecular weight protein was essentially abolishedwith the 21,22 and 11,12 mutants and greatly reducedmaturation, spliceosome assembly was arrested prior

to the stable binding of U2 snRNP. U2 snRNP associa- in the 15,16 mutant; strong cross-linking was still ob-served with the 13,14 mutant (Figure 3B). These resultstion was prevented via the use of a 29Ome oligonucleo-

tide (U2b) complementary to the branch point binding were consistent with the notion that the cross-link re-flected a functionally important interaction and revealedregion. In the presence of this oligonucleotide, splicing

(both cis- and trans-) is completely inhibited, as is the an intriguing correlation between activation of trans-splicing and the efficiency of cross-linking to the z220formation of A complex, the first U2 snRNP containing

splicing complex (data not shown; C. M. R., P. A. M., kDa protein.The size of the cross-linked band, as well as the appar-and T. W. N., unpublished data).

Uniquely labeled substrate was incubated under ent sequence specificity of cross-linking, suggested thatit might be the Ascaris ortholog of the highly conservedthese conditions and subjected to irradiation with 254

nm UV light. Following digestion with ribonuclease, pro- splicing factor Prp8. Prp8 is an intrinsic protein compo-nent of the U5 snRNP, which has been implicated in ateins were fractionated by PAGE (Figure 3A). This proto-

col yielded a remarkably simple pattern of labeled pro- bewildering array of interactions throughout spliceo-some assembly and catalysis (see Discussion). Indeed,teins; a prominent band at z220 kDa and a few fainter

59 Splice Site Recognition by tri-snRNP321

by two independent criteria, oligonucleotide inhibitionand 39 splice site mutation, interaction of the z220 kDaprotein with the 59 splice site occurs independent of U2snRNP binding.

To determine if the nematode results could be repro-duced in an unrelated system, we performed identicalcross-linking experiments in HeLa cell nuclear extracts.The cross-linking results obtained with these extractswere essentially identical to those obtained in Ascarisextracts; a single protein of z220 kDa molecular weightwas labeled (Figure 4A, lane 1). The extent of cross-linking in HeLa cell extracts was not affected by either39 splice site mutation (Figure 4A, lane 2) or by inhibitionof U2 snRNP binding with the U2b oligonucleotide (Fig-ure 4A, lane 1).

Although we suspected that the z220 kDa proteinmight be Prp8, it was necessary to test this notion di-rectly. Figure 4B shows that the cross-linked protein,labeled either in HeLa cell or Ascaris extracts, was im-

Figure 3. Sequence-Specific Interaction of a High Molecular Weight munoprecipitated under denaturing conditions (Luo etProtein with the 59 Splice Site al., 1999) by polyclonal antisera raised against human(A) Wild-type cis-splicing substrate (Figure 1, lane 1) was labeled Prp8 (kindly provided by Melissa Moore, Brandeis Uni-with a single radioactive phosphate between the invariant GU of the versity). In aggregate, these results indicated that Prp859 splice site as described in the Experimental Procedures. This specifically interacts with the 59 splice site independentRNA was then incubated under splicing conditions in the presence of stable binding of U2 snRNP to the branchpoint.of a 29Ome oligonucleotide complementary to the branch point rec-ognition sequence of U2 snRNA (see the Experimental Procedures).Following incubation, reactions were either mock irradiated (lane 1)

The Prp8/59 Splice Site Interaction Occursor irradiated (lane 2) with 254 nm UV light. Following digestion ofthe RNA with ribonuclease, labeled proteins were fractionated on when Prp8 Is a Constituent of U4/U6.U510% SDS polyacrylamide gels and visualized by autoradiography tri-snRNP and Is ATP Dependentas described in Experimental Procedures. The positions of protein As noted above, Prp8 is known to be an intrinsic proteinmolecular weight standards are indicated. of the U5 snRNP. Because U5 snRNP can exist as a(B) Wild-type or 59 splice site mutant RNAs were site specifically

mono-particle or associated with the U4/U6 di-snRNPlabeled as in (A). Incubation, UV irradiation, and labeled protein(Black and Pinto, 1989; reviewed by Will and Luhrmann,visualization were exactly as in (A) lane 2.1997), it was necessary to determine the context inwhich Prp8 interacted with the 59 splice site and todefine the requirements for this interaction. All subse-

numerous cross-linking studies have placed Prp8 at or quent experiments were carried out in parallel in bothnear the 59 splice site (see Newman, 1997, for review). HeLa cell and Ascaris extracts, with equivalent resultsHowever, with the exception of certain experiments con-

in both systems. To avoid redundancy, only the HeLa cellducted with 59 splice site oligoribonucleotides (Reyes

data are presented. To address the context question, aet al., 1996), these previous analyses have been interpre-number of oligonucleotide inhibition (29Ome sequestra-ted in the context of the canonical view of spliceosometion or targeted RNase H ablation) experiments wereassembly; i.e., U5 snRNP only associates with the matur-performed. First, we tested the effect of a 29O allyI-2-ing spliceosome post complex A (i.e., after stable U2amino adenosine-containing oligonucleotide comple-snRNP binding to the branchpoint).mentary to the phylogenetically invariant loop of U5Because our results appeared to contravene the pre-snRNA (Lamm et al., 1991). Although the loop of U5vailing view of prespliceosome assembly, it seemed im-snRNA is not absolutely required for splicing (O’Keefeperative to ensure that the cross-link occurred in theet al., 1996; Segault et al., 1999), it is known to be inabsence of stable U2 snRNP binding. Furthermore, ifclose proximity to the 59 splice site (Newman, 1997).observations in the nematode system were relevant to 59Thus, annealing of an oligonucleotide would be pre-splice site recognition in general, the same experimentaldicted to interfere stericly with any U5 snRNP/59 spliceapproaches would be expected to yield similar results insite interaction. The anti-U5 oligonucleotide preventedother systems. To address the first point, we performedthe cross-link to Prp8 (data not shown), suggesting thatcross-linking experiments with a substrate in which thethe 59 splice site interaction required functional U5invariant AG at the 39 splice site was changed to UC.snRNP. Next, we determined whether oligonucleotidesWe have previously shown that stable U2 snRNP bindingcomplementary to U4 or U6 snRNAs had any effect.to our cis-splicing substrate is absolutely dependentTwo 29Ome anti-U6 oligonucleotides (complementary toupon the integrity of the AG dinucleotide. AG depen-bases 42–60, U6a or 77–95, U6b) did not affect the extentdence results from the fact that binding of the essentialof cross-linking (Figure 5, lanes 3 and 5); however, asplicing factor U2AF to this substrate requires the AG29Ome oligonucleotide complementary to U6 basesdinucleotide; failure to bind U2AF results in failure to33–47 (U6c) completely prevented the appearance ofbind U2 snRNP (Wu et al., 1999; our unpublished data).cross-linked Prp8 (Figure 5, lane 4). All three anti-U6As shown in Figure 4A, mutation of the 39 splice site didoligonucleotides inhibited splicing (data not shown;not affect the cross-linking of the z220 kDa protein in

Ascaris extracts (Figure 4A, lanes 3 and 4). Therefore, Black and Steitz, 1986) but probably at different steps

Molecular Cell322

Figure 4. Prp8 Interacts with the 59 SpliceSite in the Absence of U2 snRNP Binding tothe Branch Point Region

(A) Wild-type or 39 splice site mutant(AG→UC) RNAs were site specifically labeledat the 59 splice site as in Figure 3. The twoRNAs were then incubated as described inthe legend to Figure 3 in either HeLa cell orAscaris extract. Cross-linking and processingof labeled proteins was exactly as describedin the legend to Figure 3.(B) Cross-linking reactions of 39 splice sitemutant RNAs identical to those in A (lanes 2and 4) were performed in HeLa cell or Ascarisextracts. Following ribonuclease digestion,aliquots were analyzed directly on SDS gels(lanes 1 and 4), after binding to protein A aga-rose (lanes 2 and 5), or after binding to anti-Prp8 polyclonal antisera prebound to proteinA agarose (lanes 3 and 6). Denaturing immu-noprecipitations were as described (Luo etal., 1999).

during spliceosome assembly and catalysis (see Discus- site-specific labeling and nuclease digestion (Wu et al.,1999; P. A. M., C. M. R., and T. W. N., unpublishedsion). Finally, degradation of U4 snRNA by oligodeoxy-

nucleotide-directed RNase H digestion dramatically in- data). In brief, in this technique (basically a modernizedvariation of “bind and chew” methodologies) (e.g.,hibited cross-linking to Prp8 (Figure 5, lane 8). The fact

that either inactivation of U4 snRNA or sequestration of Mount et al., 1983; Chabot et al., 1985), RNAs carryingsite-specific labels are incubated under the desired con-U6 snRNA inhibited the interaction of Prp8 with the 59

splice site strongly suggested that the interaction occurs ditions and subsequently digested with ribonuclease;protected fragments are then visualized after electro-only when Prp8 is associated with U5 snRNP as part of

the U4/U6.U5 tri-snRNP. phoresis. Because of the specific label, the exact sitesof protection can be determined easily. Since this meth-It is well established that the U1 snRNP-59 splice site

interaction occurs on ice and does not require ATP. To odology measures bulk occupancy, it is not subject tosome of the caveats associated with cross-linking ap-determine the requirements for the tri-snRNP/59 splice

site interaction, cross-linking was measured following proaches, and it seemed well suited to addressing thetri-snRNP/59 splice site interaction question.incubation either on ice or at 308C. As shown in Figure

6, cross-linking was undetectable at low temperature Figure 7 shows the pattern of protected fragmentsgenerated when 59 splice site labeled RNA (mutated at(Figure 6, lane 4); under these conditions a prominent

cross-link is observed to an unidentified protein of the 39 splice site to prevent U2 snRNP association) wasincubated with HeLa cell extract under a variety of condi-z100 kDa.

To determine if the Prp8/59 splice site interaction re- tions. No protection was observed without incubation(Figure 7, lane 1); however, two distinct clusters ofquired energy, ATP was depleted via preincubation (Mi-

chaud and Reed, 1991). In ATP-depleted extract, no nuclease resistant fragments (A and B, Figure 7, lane 2)were evident after incubation under conditions in whichcross-linking was detected (Figure 6, lane 2); however,

robust cross-linking was restored when depleted ex- the Prp8/59 splice site interaction was observed. Protec-tion of these clusters was largely dependent upon thetracts were supplemented with ATP, but not with nonhy-

drolyzable analogs (Figure 6, lane 3; data not shown). 59 end of U1 snRNP, since masking of U1 nucleotides1–14 with a 29Ome oligonucleotide caused a markedreduction in both sets of fragments (Figure 7, lane 3).Early Association of the U4/U6.U5 tri-snRNPTwo anti-U6 snRNA oligonucleotides, U6a and U6b, didwith the 59 Splice Site Results in Physicalnot alter the pattern of protection, just as they did notProtection of the 59 Splice Site Regionaffect cross-linking (Figure 7, lanes 4 and 5). Strikingly,To this point, we have used UV-induced cross-linkinghowever, the anti-U6 snRNA oligonucleotide (U6c) thatto Prp8 as the sole assay to monitor the interaction ofhad prevented cross-linking also completely abolishedU4/U6.U5 tri-snRNP with the 59 splice site. Interpretationprotection of fragment set B (Figure 7, lane 6); underof such results can be complicated by the limitationsthese conditions, protection of fragment set A appearedinherent to any cross-linking technique. For example, itto be enhanced.is difficult to assess the fraction of molecules actually

The direct correlation between the nuclease resis-engaged in a particular interaction because efficiencytance of fragment set B and cross-linking of Prp8 to thecannot be quantitated with precision. Furthermore, it is59 splice site prompted us to examine the temperatureimpossible to distinguish between transitory interac-and ATP dependence of protection. Appearance of frag-tions (i.e., those trapped by cross-linking) and more sta-ment set B required incubation at 308C; protection ofble complexes.fragment set A occurred on ice (Figure 7, lanes 11 andAccordingly, we sought to demonstrate the tri-snRNP/12). In the absence of ATP, fragment set B was not59 splice site interaction using an independent method-observed (Figure 7, lane 8); under these conditions, frag-ology. We have recently described a rapid method for

assessing association of factors with RNAs based upon ment set A appeared to accumulate. The protection of

59 Splice Site Recognition by tri-snRNP323

Figure 5. Inactivation of Either U6 or U4 snRNPs Prevents Association of Prp8 with the 59 Splice Site

Cross-linking analysis of 59 splice site labeled pre-mRNAs bearing a 39 splice site mutation were carried out in HeLa cell extract as describedin the legend to Figure 4. The extract was preincubated with the indicated 29Ome oligonucleotides (lanes 1–5) or DNA oligonucleotides andRNase H (lanes 6–8) as described in the Experimental Procedures. The regions of complementarity between the 29Ome oligonucleotides andhuman U6 or U4 snRNAs are depicted schematically. The U6c and U6a oligonucleotides partially overlap, as indicated by the magenta coloring.

fragment set B was fully restored upon the addition of and the temperature and energy requirements, we con-clude that fragment set A results from protection of theATP to depleted extract (Figure 7, lane 9).

To map the boundaries of the protected fragments, 59 splice site by U1 snRNP and associated factors. Theadditional protection observed in fragment set B resultsthey were excised from gels such as those shown in

Figure 7 and subjected to site-specific cleavage with from tri-snRNP association with the 59 splice site. Fur-thermore, the data suggest that U1 snRNP binding pre-RNase H (Lapham and Crothers, 1996; Yu et al., 1997).

The results (data not shown) indicated that protected cedes (and probably is a prerequisite for) tri-snRNP as-sociation (see Discussion). While it seems likely thatfragment set A spanned nucleotides 25 to 114, while

protected fragment set B spanned nucleotides 215 to protection of fragment set B results from concurrentoccupancy of the 59 splice site by U1 snRNP and the114 (see schematic, Figure 7). Based on the pattern of

protection, the sensitivity to oligonucleotide inhibition, U4/U6.U5 tri-snRNP, we cannot formally exclude the

Molecular Cell324

cross-linking was not the result of trapping a fleetinginteraction.

Discussion

A thorough understanding of the mechanism of pre-mRNA splicing depends critically upon knowing the pre-cise order of addition of the constituents of the spliceo-some. To date, the pathway of spliceosome assemblyhas been largely defined via biochemical approachessuch as glycerol gradient sedimentation and native gelelectrophoresis (reviewed in Moore et al., 1993). Impor-tantly, these techniques can only reveal the existence ofmacromolecular complexes that are stable to particularisolation conditions. Indeed, the development of gentleapproaches such as gel filtration and modified nativegels (e.g., Reed and Palandjian, 1997; Abovich and Ros-bash, 1997; Raghunathan and Guthrie, 1998) has led tothe identification of a variety of previously unrecognizedinteractions. Nevertheless, it remains an accepted factin the spliceosome literature that U4/U6.U5 tri-snRNPassociates with the maturing spliceosome only after sta-ble binding of U2 snRNP.

Figure 6. The Prp8/59 Splice Site Interaction Requires ATP and Incu-Using cross-linking and physical protection, we havebation at 308C

shown that U4/U6.U5 tri-snRNP can engage the 59 spliceCross-linking reactions using site-specifically labeled pre-mRNA

site in the absence of U2 snRNP binding; U2 snRNPwith a mutant 39 splice site (AG→UC) were carried out in untreatedassociation was prevented either by mutation of the 39HeLa cell extract (lane 1); extract that had been preincubated tosplice site in an AG-dependent intron or by physicallydeplete ATP as described in the Experimental Procedures (lane 2);blocking the U2 snRNP branch point recognition se-or in depleted extract that was supplemented with ATP (lane 3). In

lane 4, the sample contained ATP but was kept on ice. Lane 5 quence. The interactions we observe are highly se-was identical to lane 1. Incubation conditions are described in the quence specific, occur in both nematode and HeLa cellExperimental Procedures. extracts, and require ATP. The stringent requirements

for the association of tri-snRNP with the 59 splice sitestrongly argue that this interaction reflects the physio-logically relevant mode of recruiting the tri-snRNP topossibility that protection of fragment set B is mediated

by tri-snRNP alone following U1 snRNP displacement the spliceosome. In this view, formation of complex Bdoes not reflect “recruitment” but rather a stabilization(see Discussion). In any case, the nuclease protection

assay indicates that the tri-snRNP/59 splice site interac- that occurs as a consequence of the intraspliceosomalRNA—RNA rearrangements (i.e., replacement of U1 bytion is not transitory; accordingly, we conclude that the

Figure 7. U4/U6.U5 tri-snRNP-Dependent Pro-tection of the 59 Splice Site Region

Pre-mRNA (39 SS AG→UC), with a specificlabel at the 59 splice site, was incubated inHeLa cell extract under splicing conditionsfor 10 min at 308C. Following incubation, re-actions were placed on ice and digested withmicroccocal nuclease prior to deproteiniza-tion (see the Experimental Procedures).Nuclease resistant RNA fragments were frac-tionated on denaturing gels and visualized byautoradiography. In lane 1, incubation wasterminated by nuclease digestion immedi-ately following addition of labeled RNA. Inlanes 2 through 6, the extract was preincu-bated with the indicated 29Ome oligonucleo-tides. In lanes 7 through 9, incubations wereexactly as described for lanes 1 through 3 inFigure 6. Lane 10 was treated the same aslane 1, and lane 12 was treated the same aslane 2; lane 11 was incubated on ice. Theboundaries of protected fragment sets A andB (indicated) are shown schematically belowthe autoradiogram.

59 Splice Site Recognition by tri-snRNP325

U6 and formation of U2/U6 helices) that are prerequisites in S. cerevisiae has demonstrated an interaction be-for catalytic activation. tween exon positions 21 and 22 and U1 snRNA (Ser-

We have yet to physically isolate the predicted U1- aphin and Kandels-Lewis, 1993). Similarly, genetic andU4/U6.U5 pre-mRNA complex, and such a complex has biochemical approaches have shown that these samenot been reported in the extensive investigations of early nucleotides interact with both the RNA and proteinevents in mammalian or yeast spliceosome formation (Prp8) constituents of U5 snRNP (Newman, 1997). How-(reviewed in Reed and Palandjian, 1997). However, the ever, as discussed by Seraphin and Kandels-Lewismajority of these analyses have been conducted at low (1993), it has not been established whether recognitiontemperature and in the absence of ATP, conditions un- is sequential or simultaneous. Formally, the splicingder which the interactions we observe would not occur. phenotypes we observe upon alteration of nucleotidesAlthough direct evidence for early (i.e., U2 snRNP-inde- 21 and 22 could result from disruption of U1 and/or U5-pendent) recognition of the 59 splice site by U4/U6.U5 specific contacts. However, the sensitivity to elevatedhas not previously been reported, many experimental temperature conferred by this mutation (which is spe-observations have hinted at such recognition. In this cific to cis-splicing) argues strongly that the robust acti-regard, numerous site-specific cross-linking studies vation of trans-splicing seen with the 21, 22 mutanthave shown that the U5 snRNP (both protein [Prp8] and cannot be due to disruption of a U1/59 splice site con-RNA constituents) makes intimate contact with the 59 tact. Specifically, the fact that inactivation of cis-splicingsplice site prior to the first catalytic step of splicing at 378C with the 21, 22 mutant does not lead to further(reviewed in Newman, 1997). Of most relevance to the enhancement of trans-splicing indicates that the 59work described here, these studies revealed two tempo- splice site is still occupied (presumably by U1 snRNP).rally and physically distinct “modes” of interaction of Given these considerations, we interpret the activationU5 snRNP with 59 exon sequences. In Hela cell extracts, of trans-splicing in the 21, 22 mutant to indicate aone U5 snRNP/pre-mRNA interaction, detected with a weakened early engagement of the 59 splice site by tri-cross-linking reagent placed at the 22 position, was snRNP.shown to occur very early in splicing reactions (Wyattet al., 1993); a similar interaction was detected in yeast

Implications for Early 59 Splice Site Recognitionextracts with a cross-linking reagent at the 28 positionThe fact that the 39 boundaries of protected fragment(Newman et al., 1995). In both systems, a second distinctsets A and B are coincident strongly suggests simulta-U5 snRNP/pre-mRNA interaction was detected whenneous occupancy of the 59 splice site by U1 snRNP andpre-mRNAs contained a cross-linking reagent at the 21tri-snRNP. Furthermore, the studies cited above provideposition. This interaction occurred with much slowerprecedent for such simultaneous recognition (Hall andkinetics than the first (Sontheimer and Steitz, 1993; New-Konarska, 1992; Kuhn et al., 1999; Staley and Guthrie,man et al., 1995). Importantly, the early interaction was1999). Based on our data and these precedents, weshown to be ATP dependent, and in the HeLa systemsuggest that recognition of authentic 59 splice sites nor-cross-linking was observed both to Prp8 and U5 snRNAmally requires concurrent interactions with both U1(Wyatt et al., 1993). We think it highly likely that thissnRNP and the U4/U6.U5 tri-snRNP. Such corecognitionearly interaction is the same interaction that we observe.would provide a straightforward explanation for a varietyThe late interaction would then reflect more stable con-of poorly understood phenomena relating to 59 splicetacts established upon destabilization of U1 snRNPsite selection.binding.

Neither we nor others can define the context in whichAdditional suggestive evidence for the involvement ofa given sequence will function as a 59 splice site (Horo-U4/U6.U5 in early spliceosome assembly derives fromwitz and Krainer, 1994; Black, 1995). In this regard, it isdocumented cross-links between U1 and U5 snRNAswell established that U1 snRNP binds to sequences that(Ast and Weiner, 1996, 1997), as well as two-hybrid inter-are not used as 59 splice sites (e.g., Eperon et al., 1993).actions between the protein constituents of U1 and U5Accordingly, U1 snRNP binding in and of itself cannotsnRNPs (Abovich and Rosbash, 1997). Furthermore, as-be sufficient to nucleate spliceosome assembly. Wesociation of tri-snRNP with the prespliceosome hasspeculate that U1 snRNP, which is present in vast ex-been observed when either the U1/59 splice site or U4/cess over tri-snRNP, binds to both authentic andU6 base-pairing interactions are hyperstabilized (Kuhn“pseudo” 59 splice sites; U4/U6.U5 tri-snRNP then asso-et al., 1999; Staley and Guthrie, 1999; and see below).ciates only with authentic sites. In this scenario, onlyFinally, an oligoribonucleotide mimicking a 59 splice sitethose sites that are compatible with cooccupancy arespecifically induced the formation of a U1, U2, U4/U6.U5used. This model accommodates the rather loose re-complex, although the relevance of this pseudospliceo-quirements for U1 snRNP binding in higher eukaryoticsome to splicing was not established (Hall and Konar-cells and provides for the appealing possibility that 59ska, 1992).splice sites are inspected twice prior to being recog-We have demonstrated that tri-snRNP establishes annized as authentic. It is intriguing that association of tri-early contact with the 59 splice site, but is this interactionsnRNP requires ATP hydrolysis; perhaps this require-required for splicing? This question is difficult to answerment reflects a “proofreading” function (Burgess andunambiguously because the components of the U4/Guthrie, 1993; see below).U6.U5 tri-snRNP are essential for multiple steps in the

Corecognition of the 59 splice site by U1 snRNP andsplicing process; accordingly, inactivation of any com-tri-snRNP also provides appealing explanations for twoponent inhibits the process as a whole without revealingother phenomena, activation of some cryptic 59 splicethe inhibited step. Nevertheless, we believe that oursites and U1-independent splicing. For cryptic sites, weresults with the 21, 22 mutant provide strong evidencesuggest that certain mutations in authentic sites preventfor the functional relevance of the early tri-snRNP/59their use in splicing but still permit some level of U1splice site interaction.

In this regard, compensatory base mutational analysis snRNP binding; U1 snRNP in turn recruits tri-snRNP.

Molecular Cell326

Unable to bind to the authentic splice junction, tri- it seems particularly relevant that one phenotypic con-snRNP via the protein (Prp8) and RNA components of sequence of a mutation that debilitates the ATPase ac-U5 snRNP binds to and activates nearby sequences. It tivity of Brr2 is failure of tri-snRNP to associate withwill be interesting to test these predictions in docu- spliceosomes (Raghunathan and Guthrie, 1998). As dis-mented examples of cryptic 59 splice site use (e.g., Co- cussed by Raghunathan and Guthrie (1998), this pheno-hen et al., 1994; Hwang and Cohen, 1996). Finally, the type would not be expected based on the canonicalfact that both U1-dependent and U1-independent splic- view of spliceosome assembly.ing share the same 59 splice site sequence requirements Taking all of these observations into consideration,led Crispino and Sharp (1995) to propose the existence we suggest that U4/U6.U5 interacts with the 59 spliceof a non-U1 recognition factor they designated “Factor site only if the U4 and U6 snRNAs are in a specificX.” Given our results, it seems highly probable that Fac- conformation (perhaps partially dissociated via the ac-tor X is U4/U6.U5 tri-snRNP (as speculated earlier Cris- tion of Brr2). Although speculative, such a scenariopino and Sharp, 1995). would explain both the remarkable specificity of inhibi-

tion of the tri-snRNP/59 splice site interaction by distinctATP Requirement for U4/U6.U5 Interaction anti-U6 oligonucleotides (Figures 5 and 7) and the ATPwith the 59 Splice Site requirement for this interaction.The sequence specificity of the Prp8 contact with the 59exon/intron boundary was initially demonstrated using Experimental Proceduresoligoribonucletides that mimic 59 splice sites (Reyes et

Pre-mRNA Template Constructional., 1996). Our data regarding specificity are in agree-DNA fragments corresponding to a nematode cis-59 exon (Hannonment with this prior study and reinforce the significanceet al., 1991) and its internally shortened downstream intron wereof that work; nevertheless, there exist intriguing differ-fused precisely at the 39 splice site AG to an exon that serves asences between the experimental results obtained byan SL acceptor (Hannon et al., 1990) using conventional techniques.Reyes et al. (1996) and the results we have presented.The resultant construct contained a 108 nucleotide 59 exon, a 93First, in the oligonucleotide studies, cross-linking tonucleotide intron, and a 202 nucleotide 39 exon. The trans-39 exon

Prp8 was observed only when the 59 end of U1 snRNA was chosen because it contains sequence elements that promotewas sequestered (Reyes et al., 1996). In contrast, we the SR protein-dependent recruitment of U2 snRNP in the absenceobserve a diminution of Prp8 cross-linking when the of a 59 splice site (Figure 1A; C. M. R., P. A. M., and T. W. N.,same sequestering oligonucleotide is used, indicating unpublished data). For some experiments, the 59 exon and its ac-

companying 59 splice site were removed, resulting in a “39 half-that the interaction we detect is dependent on U1 snRNPmolecule” containing 79 nucleotides of residual intron and the 202function (Figure 5). Second, cross-linking to the oligori-nucleotide 39 exon. Mutations in the 59 splice site region and the 39bonucleotide occurred on ice and did not require ATPsplice site were introduced by site-directed mutagenesis as de-(Reyes et al., 1996). We observe no cross-linking to Prp8scribed (Kunkel et al., 1987). In all cases, mutations were designedon ice in the absence of ATP. One appealing interpreta-to change wild-type bases to their complement. In 39 splice sitetion of these differences is that the results obtained inmutant constructs (see Results), an AG dinucleotide eleven nucleo-

the oligoribonucleotide experiments reflect a bypass of tides downstream from the authentic 39 splice site was also changedan otherwise required step in 59 splice site recognition; to its complement.i.e., U1 snRNP recognition. This interpretation is consis-tent with the observation that the oligoribonucleotides Substrate Labelingengage in splicing only when U1 snRNP is inactivated For all in vitro splicing reactions, substrates, as indicated in the(Konforti and Konarska, 1995). Furthermore, the differ- Figures, were 39 end-labeled (England and Uhlenbeck, 1978) withent requirements for interaction of U4/U6.U5 with the [32P]pCp to a specific activity of z100,000 cpm/ng. Site-specific

labeling at the 59 splice site of wild-type and mutant pre-mRNAsoligoribonucleotide and our splicing substrate suggestwas carried out essentially as described (Wu et al., 1999). In brief,that the ATP requirement we observe is in some wayfull-length RNAs were cleaved at the desired position using site-related to U1/U6 transactions at the 59 splice site.directed RNase H cleavage (Lapham and Crothers, 1996; Yu et al.,The displacement of U1 by U6 at the 59 splice site1997). Following purification of the half-molecules by denaturinghas received intensive scrutiny of late (Kuhn et al., 1999;PAGE, the 39 half-molecule was treated with phosphatase and sub-Staley and Guthrie, 1999), and this single step in thesequently labeled to high specific activity with [g32P]ATP and poly-

splicing pathway is remarkably complex. As noted nucleotide kinase. After labeling, the half-molecules were rejoinedabove, hyperstabilization of either the U1/59 splice site using splinted ligation (Moore and Sharp, 1992) and repurified.interaction or U4/U6 base pairing inhibits the exchange(Kuhn et al., 1999; Staley and Guthrie, 1999), and in both In Vitro Splicing, Cross-Linking, and Nucleaseof these unusual situations, the tri-snRNP is apparently Protection Assayspresent in pre-spliceosomes. Intriguingly, certain mu- Splicing reactions were performed as described (Yu et al., 1993) fortant alleles of Prp8 suppress the splicing defects that 20 min at the indicated temperatures. For cross-linking, substrates

were incubated in Ascaris or HeLa nuclear extracts (Dignam et al.,result from hyperstabilization of the U4/U6 duplex (Kuhn1983) under splicing conditions for 10 min at 308C. Subsequently,et al., 1999). Perhaps most interestingly, RNA-depen-aliquots of the incubations were spotted on an ice cold metal blockdent ATPases have been clearly implicated in modulat-and irradiated with 254 nm UV light for 10 min in a Stratalinkering the 59 splice site/U1 interaction (Prp28) and the U4/(Stratagene). Cross-linked samples were diluted with ice cold TEU6 interaction (Brr2/Snu246) (Raghunathan and Guthrie,(10 mM Tris [pH 8.0] and 1 mM EDTA) and digested with 2 mg of1998; Staley and Guthrie, 1999). In higher eukaryotes,RNase A at 378C for 30 min. Labeled proteins were visualized on

both proteins are intrinsic components of the U5 snRNP 10% polyacrylamide SDS gels.(Lauber et al., 1996); in S. cerevisiae, however, Prp28 is For nuclease protection, substrates were incubated in HeLa nu-apparently not snRNP associated. An attractive possibil- clear extracts under splicing conditions for 10 min at 308C. Samplesity suggested by our data is that ATP hydrolysis by one were then diluted with splicing mix minus PEG (Yu et al., 1993),(or both) of these proteins is required for early associa- supplemented with 4 mM CaCl2, and treated with 300 units of micro-

coccal nuclease (Worthington) on ice for 15 min; digestion wastion of tri-snRNP with the 59 splice site. In this regard,

59 Splice Site Recognition by tri-snRNP327

stopped by addition of 8 mM EGTA. Protected RNAs were recovered Chiara, M.D., and Reed, R. (1995). A two-step mechanism for 59 and39 splice-site pairing. Nature 375, 510–513.by phenol-chloroform extraction and ethanol precipitation. Aliquots

of each reaction were analyzed on an 8% denaturing polyacrylamide Cohen, J.B., Snow, J.E., Spencer, S.D., and Levinson, A.D. (1994).gel. Suppression of mammalian 59 splice-site defects by U1 small nu-

clear RNAs from a distance. Proc. Natl. Acad. Sci. USA 91, 10470–Oligonucleotide Inhibition and ATP Depletion 10474.29O methyl oligonucleotides used for snRNP occlusion experiments Conrad, R., Thomas, J., Spieth, J., and Blumenthal, T. (1991). Inser-were complementary to bases 24 to 110 of the cis-59 splice site tion of part of an intron into the 59 untranslated region of a Caeno-(59SS, 50 ng), bases 1 to 14 of U1 snRNA (U1, 50 ng), bases 29 to rhabditis elegans gene converts it into a trans-spliced gene. Mol.44 of U2 snRNA (U2b, 200 ng), bases 42 to 60 (U6a, 250 ng), 77 to Cell. Biol. 11, 1921–1926.95 (U6b, 250 ng), and 33 to 47 (U6c, 250 ng) of U6 snRNA. Extracts

Conrad, R., Liou, R.F., and Blumenthal, T. (1993). Conversion of awere preincubated with the indicated amounts of 29O methyl oligo-trans-spliced C. elegans gene into a conventional gene by introduc-nucleotides under splicing conditions for 5 min at room temperature.tion of a splice donor site. EMBO J. 12, 1249–1255.For targeted RNase H degradation, extracts were pretreated withCortes, J.J., Sontheimer, E.J., Seiwert, S.D., and Steitz, J.A. (1993).400 ng of U4b oligonucleotide (complementary to nts. 64–83 ofMutations in the conserved loop of human U5 snRNA generate thehuman U4) or 400 ng of a control oligonucleotide complementaryuse of novel cryptic 59 splice sites in vivo. EMBO J. 12, 5181–5189.to 5S rRNA for 20 min at 308C in the presence of 2 mM ATP, 5 mM

MgCl2 and 2 Units of RNase H (BRL). After occlusion or targeted Crispino, J.D., and Sharp, P.A. (1995). A U6 snRNA:pre-mRNA inter-RNase H degradation, extracts were used directly in splicing, cross- action can be rate-limiting for U1-independent splicing. Genes Dev.linking, or nuclease protections assays. 9, 2314–2323.

ATP depletion was similar to that described by Michaud and ReedCrispino, J.D., Mermoud, J.E., Lamond, A.I., and Sharp, P.A. (1996).

(1991). Extracts were preincubated under splicing conditions minusCis-acting elements distinct from the 59 splice site promote U1-

added ATP and creatine phosphate for 10 min at room temperature.independent pre-mRNA splicing. RNA 2, 664–673.

Following depletion, substrates were added with or without 2 mMDignam, J.D., Lebovitz, R.M., and Roeder, R.G. (1983). AccurateATP and 20 mM creatine phosphate and assayed for cross-linkingtranscription initiation by RNA polymerase II in a soluble extractor nuclease protection.from isolated mammalian nuclei. Nucleic Acids Res. 11, 1475–1489.

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Hall, K.B., and Konarska, M.M. (1992). The 59 splice site consensushelp with the figures and preparing the manuscript. C. M. R. was

RNA oligonucleotide induces assembly of U2/U4/U5/U6 small nu-supported by the postdoctoral training grant AG00105. This re-

clear ribonucleoprotein complexes. Proc. Natl. Acad. Sci. 89, 10969–search was supported by National Institutes of Health grants GM-

10973.31528 and AI-28799 (T. W. N.).

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