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DynamicExchangesofRNAInteractionsLeadingtoCatalyticCoreFormationintheU12-DependentSpliceosomeARTICLEinMOLECULARCELLFEBRUARY2001ImpactFactor:14.46DOI:10.1016/S1097-2765(01)00169-1Source:PubMed

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Molecular Cell, Vol. 7, 217226, January, 2001, Copyright 2001 by Cell Press

Dynamic Exchanges of RNA InteractionsLeading to Catalytic Core Formationin the U12-Dependent Spliceosome

and snRNA molecules that participate in base pairingand have established that these critical RNARNA inter-actions occur in both the U2- and U12-dependentspliceosomes in mammalian cells, as well as in the U2-dependent spliceosome of yeast (Tarn and Steitz, 1997;

Mikko J. Frilander and Joan A. Steitz*Department of Molecular Biophysics

and BiochemistryYale University School of MedicineHoward Hughes Medical Institute

Nilsen, 1998). Most significantly, studies of the U12-New Haven, Connecticut 06536-0812dependent spliceosome have illuminated models forcatalytic core interactions in the U2-dependent spliceo-some, since quite divergent snRNA sequences assumeSummaryequivalent architecture to juxtapose the reactants forthe first step of the splicing reaction (Hall and Padgett,Important general insights into the mechanism of pre-1996; Tarn and Steitz, 1996a, 1996b; Incorvaia and Pad-mRNA splicing have emerged from studies of the U12-gett, 1998; Shukla and Padgett, 1999; Figure 1B).dependent spliceosome. Here, photochemical cross-

In the U2-dependent spliceosome, the order of RNAlinking analyses during U12-dependent spliceosomeRNA rearrangements that build the catalytic core hasassembly have surprisingly revealed that an upstreambeen deduced from experiments using yeast or HeLa59 exon region is required for establishing two essen-nuclear extracts (Sawa and Abelson, 1992; Wassarmantial catalytic core interactions, U12/U6atac helix Ib andand Steitz, 1992; Konforti et al., 1993; Sontheimer andU6atac/59 splice site contacts, but not for U5/59 exonSteitz, 1993; Konforti and Konarska, 1994) and frominteractions or partial unwinding of U4atac/U6atac. Astudies of yeast mutants in vivo and in vitro (Li andnovel intermediate, representing an alternative path-Brow, 1996; Kuhn et al., 1999; Staley and Guthrie, 1999).way for catalytic core formation, is a ternary snRNABriefly, in the earliest stages of spliceosome assembly,complex containing U4atac/U6atac stem II and U12/U2-type introns are recognized by U1 and U2 snRNPs,U6atac helix Ia that forms even without U6atac replac-which form base-pairing interactions with the 59ss anding U11 at the 59 splice site. A powerful oligonucleotideBPS, respectively. Subsequent entry of the U4/U6.U5displacement method suggests that the blocked com-tri-snRNP into the nascent spliceosome triggers a largeplexes analyzed to deduce the interdependence ofnumber of rearrangements (for reviews, see Nilsen,these multiple RNA exchanges are authentic interme-1998; Staley and Guthrie, 1998). Most significant are thediates in U12-dependent spliceosome assembly.switch from U1/59ss to U6/59ss interactions (Staley andGuthrie, 1999) and the unwinding of the U4/U6 duplex,

Introduction which enable formation of U6/U2 helices Ia and Ib, be-lieved to be at the active site of the spliceosome (Mad-

Removal of noncoding intron sequences from pre- hani and Guthrie, 1992). Since many of the RNARNAmRNA molecules is carried out by a large RNAprotein interactions that occur are mutually exclusive, precisecomplex termed the spliceosome. Two parallel spliceo- ordering of annealing and unwinding steps is criticalsomes, called U2 and U12 dependent, have been de- for spliceosome assembly (Staley and Guthrie, 1998).scribed from the cells of higher eukaryotes. Most introns Previous experiments have suggested that recognitionare removed by the U2-dependent spliceosome, while of the 59ss by U6 takes place prior to the unwinding ofa subset of introns (,1%) contain divergent but highly the U4/U6 duplex and therefore precedes the formationconserved 59 splice site (59ss) and branchpoint signals of critical U2/U6 catalytic core interactions. In this(BPS) and are removed by the U12-dependent spliceo- model, recognition of the 59ss twice has been viewedsome (Hall and Padgett, 1994; Tarn and Steitz, 1997; as a proofreading mechanism that enhances the fidelityBurge et al., 1998). Both spliceosomes are composed of of spliceosome action (Kandels-Lewis and Seraphin,five small nuclear ribonucleoprotein (snRNP) particles. 1993; Lesser and Guthrie, 1993; Staley and Guthrie,Only U5 is shared between the two spliceosomes, while 1998; Kuhn et al., 1999).U1, U2, U4, and U6 are found exclusively in the U2- Here, we have used the U12-dependent spliceosomedependent spliceosome and the corresponding U11, to investigate the timing of RNARNA interactions es-U12, U4atac and U6atac snRNPs in the less abundant sential for building the core of an active spliceosome.U12-dependent spliceosome (Tarn and Steitz, 1997; The U12-dependent spliceosome offers special techni-Burge et al., 1999). cal advantages, since psoralen cross-links have been

The pathways for the formation of the two spliceo- mapped within both stem II of the U4atac/U6atac duplexsomes are highly similar, with a network of dynamic and within U12/U6atac helices Ia and Ib (Tarn and Steitz,

1996b; our unpublished results; see Figures 1A and 1B).base-pairing interactions guiding the assembly process.Thus, the formation and dissolution of these interactionsPhylogenetics, genetics, and biochemical cross-linkingcan be followed by psoralen cross-linking (Tarn andexperiments have identified the regions in the pre-mRNASteitz, 1996b) and ordered relative to other RNARNAinteractions, such as the U12/branchpoint sequence* To whom correspondence should be addressed (e-mail: joan.steitz@pairing (Tarn and Steitz, 1996a). Contrary to the idea thatyale.edu).a reiterative process of 59ss recognition must precede Present address: Institute of Biotechnology, PL 56 (Viikinkaari 9)

00014, University of Helsinki, Finland. catalytic core formation, we find that functional U6atac/

Molecular Cell218

We investigated the effect of mutations within theP120 splicing substrate and discovered that progressivetruncations of the 59 exon led to decreased formationof U12/U6atac helix Ib (Figure 2A, lanes 310). In con-trast, two other major spliceosomal cross-links, U12/BPS and U12/U6atac helix Ia, were unaffected. The ap-pearance of helix Ib was typically 5- to 10-fold weakerwith a substrate containing an 8 nucleotide (nt) 59 exon(P120-8, lane 10) than with a standard 158 nt 59 exon(P120-158, lane 3) and was not influenced by whetherthe substrate was G or A capped (data not shown; seeOMullane and Eperon [1998]). Use of the same trun-cated substrates in splicing assays revealed that intronexcision from the P120-8 substrate was correspondinglyimpaired (Figure 2B, compare lane 15 with lanes 6, 9,and 12).

An unanticipated conclusion from the results in Figure2A is that a ternary complex containing both U4atac/U6atac stem II and U12/U6atac helix Ia apparently forms

Figure 1. RNARNA Cross-Links Diagnostic of the Assembly of the during the assembly of a functional U12-dependentU12-Dependent Spliceosome spliceosome (see Figures 1A and 1B). This doubly cross-(A) Location of the psoralen U4atac/U6atac cross-link in the di- linked U4atac/U6atac/U12 species (Frilander and Steitz,snRNA secondary structure. 1999) can be visualized by comparing the profiles with(B) A model for the spliceosomal catalytic core in which U12 and

probes for U12 snRNA (Figure 2A, top panel), for U4atacU6atac form base-pairing interactions with each other and with the(middle panel), and for U6atac (bottom panel). The low59ss and BPS regions of the intron, respectively. The solid lightningabundance of this ternary cross-link did not allow pre-bolts indicate the positions of psoralen cross-links mapped on

snRNA or pre-mRNA molecules (Tarn and Steitz, 1996b; our unpub- cise mapping, but it seems most likely that U6atac islished results), while the open lightning bolt depicts a cross-link cross-linked to U12 via U12/U6atac helix Ia and U4atacbetween U6atac snRNA and a 4SU residue at the 12 position of the via stem II for two reasons. First, the component singleintron (Yu and Steitz, 1997b). Thick black lines represent 29-O-methyl cross-links are the most prominent in our gels; second,RNA oligonucleotides used in this study. The boxes in the U6atac

the ternary cross-link is present even in the absence ofsnRNA indicate regions proposed to interact with 59ss (light gray),the U12/U6atac helix Ib cross-link (Figure 2A, lane 10).or with U12 to form helices Ia (dark gray) and Ib (black), as shown

in (B). The filled circles are 59 cap structures. Accordingly, progressive exon truncation not only repro-ducibly increased the levels of the ternary complex(about 2-fold) but also the intensity of the singly cross-59ss and U12/U6atac helix Ia interactions can take placelinked U4atac/U6atac band (Figure 2A, lanes 310).

in either order. Recognition of a 59 exon region is re-Since the psoralen cross-link in the U4atac/U6atac du-

quired to complete catalytic core assembly, with a likelyplex occurs in stem II (see Figure 1A), we interpret the

intermediate containing U6atac base-paired to bothinhibition of U12/U6atac helix Ib formation and in-

U4atac (via stem II) and U12 (via helix Ia). creased detection of a ternary U12/U4atac/U6ataccross-link to result from a defect in U4atac/U6atac stem

Results II unwinding for substrates containing short 59 exons.To ensure that the apparent dependence on 59 exon

A Region in the 59 Exon Required for U12/U6atac sequences for U12/U6atac helix Ib formation is not re-Helix Ib Formation stricted to the P120 splicing substrate, we constructedInitially we employed psoralen cross-linking to assess similar truncations for an unrelated substrate containingthe unwinding of U4atac/U6atac base pairing relative to a U12-type intron. Even though the SCN4AENH1 sub-the formation of the U12/U6atac helices Ia and Ib during strate (Wu and Krainer, 1998) exhibited lower psoralenassembly of the U12-dependent spliceosome. These cross-linking activity, inhibition of helix Ib formation andtwo interactions are mutually exclusive since they in- increased levels of the ternary U4atac/U6atac/U12volve the same U6atac nucleotides (indicated in Figure cross-link are clearly visible when its 59 exon is reduced1A). The well-studied P120 splicing substrate (here to 8 nucleotides (Figure 2A, lanes 1115). We concludecalled P120-158) was used under standard conditions that dependence on 59 exon sequences is a generalin HeLa nuclear extract (Figure 2A, lane 3). Cross-linked property of U12-type introns, even though no sequencespecies were identified by Northern analysis with probes conservation in this region is apparent in known U12-specific for U12 (top panel), U4atac (middle panel), or type introns (Burge et al., 1998) and our extensive site-U6atac snRNA (bottom panel) (Tarn and Steitz, 1996b). directed mutagenesis did not reveal a specific sequenceAfter 45 min (well before the appearance of splicing requirement (data not shown).intermediates or products), cross-links diagnostic of To test whether sequence-independent recognitionU12/BPS pairing and U12/U6atac helices Ia and Ib of the 59 exon region is required, we asked whetherformed, but only in the presence of substrate (compare a 29-O-methyl RNA oligonucleotide complementary tolane 3 to 1). A small (1.5- to 2-fold) but reproducible nucleotides 26 to 222 relative to the 59ss (59 exon oligo;substrate-dependent decrease in the level of U6atac/ Figure 1B) would likewise inhibit U12/U6atac helix Ib

formation. Figure 2C, lane 4, shows that in the presenceU4atac cross-links is observed in the same two lanes.

Pathways for U12-Dependent Spliceosome Assembly219

Figure 2. Truncation of the 59 Exon Inhibits the Formation U12/U6atac Helix Ib and Splicing

(A) Unlabeled P120 (lanes 210) or SCN4AENH1 (lanes 1115) pre-mRNA substrates and their deletion derivatives, all containing a GpppGcap, were incubated under standard splicing conditions (Frilander and Steitz, 1999) for 45 min at 308C, followed by psoralen cross-linking onice. Recovered RNAs were separated in 7% urea-polyacrylamide gels, transferred to nylon filters, and detected using 32P-labeled probescomplementary to U12 (top panel), U4atac (middle panel), or U6atac (bottom panel, P120 substrate only) snRNAs. The lengths of the 59 exonsare indicated above each lane and the probe used for each panel on the left. The sizes of DNA molecular weight markers (M), a 32P-labeledpBR322 MspI digest, are indicated on left. A nonspecific band generated without UV irradiation is indicated with an asterisk.(B) The effect of 59 exon truncation of the P120 substrate on splicing activity. 32P-labeled splicing substrates containing truncated 59 exonswere incubated under standard splicing conditions for 0, 2, or 3 hr. The recovered RNAs were analyzed in a 5% urea-polyacrylamide gelfollowed by autoradiography. The lengths of the 59 exons are shown at the top. Splicing intermediates and products are indicated on theright. The asterisk represents a degraded substrate fragment.(C) The effect of 29-O-methyl RNA oligonucleotides on helix Ib formation. Psoralen cross-linking was performed as described in panel A, usingthe P120-158 substrate in the absence (lane 3) or presence of 1 mM 59 exon (lane 4) or 0.5 mM U12-Helix I (lane 6) oligo. The control reactioncontained the P120-8 substrate (lane 5). The differences in the levels of U12 internal cross-links were not reproducible. The identities of cross-linked products are indicated on the right and the asterisk and markers are as in panel A.

of the P120 59 exon oligo there is a significant decrease would also perturb U6atac/59ss, U11/59ss, or U5/59exoninteractions. In yeast, U2/U6 helix Ib has been suggestedin the cross-linked band diagnostic of helix Ib, while

helix Ia and the U12/BPS cross-links are only slightly to play a role in 59ss choice (Luukkonen and Seraphin,1998), and a genetic interaction between helix Ib andaffected (compare to lane 3). In repeated experiments,

the effects of blockage with the 59 exon oligo proved to the invariant loop I of U5 snRNA has been described(Xu et al., 1998). We constructed two unlabeled splicingbe somewhat more variable than exon truncation; the

level of helix Ib formation with the P120-8 substrate was substrates, each containing two site-specific photo-activatable 4SU residues, at positions 22 and 12 relativeconsistently about 10-fold lower than with the P120-158

substrate, while with oligonucleotide blockage helix Ib to the 59ss, for use in cross-linking reactions to detectinteractions with snRNAs. Splicing substrate P120-4SU-formation was approximately 5-fold diminished, and oc-

casionally a faint doublet appeared in place of the U12/ 47 has a 47 nt 59 exon and displays normal helix Ibformation in psoralen cross-linking experiments (dataU6atac helix Ib band (Figure 2C, lane 4).

Together, the results from 59 exon truncations (Figure not shown), while P120-4SU-8 has a 8 nt 59 exon anddisplays a defect in helix Ib formation as assayed by2A) and oligonucleotide blockage (Figure 2C) indicate

that recognition of the 59 exon in a sequence-indepen- psoralen cross-linking (data not shown). Based on docu-mented U5/59 exon interactions in the U2-dependentdent manner is required for the proper formation of U12/

U6atac helix Ib. It is possible that interactions with the spliceosome (Newman and Norman, 1991, 1992; Sont-heimer and Steitz, 1993; Newman et al., 1995), we antici-RNA backbone may be involved; these might be ex-

pected to be impaired by introduction of a rigid double pated that the 4SU residue at the 22 position wouldreveal similar U5 interactions in the U12-dependenthelical conformation upon the binding of the 59 exon

oligo. spliceosome. The 4SU at the 12 position of the intron isknown to capture U11/59ss and U6atac/59ss interactions(Yu and Steitz, 1997b).Formation of U12/U6atac Helix Ia in the Absence

of U6atac/59ss Interactions The results in Figure 3A provide direct evidence for afunctional interaction between the 59 exon of a U12-Next we investigated whether the same substrate ma-

nipulations that inhibit U12/U6atac helix Ib formation dependent intron and the U5 snRNP as well as the dem-

Molecular Cell220

the singly cross-linked U6atac/substrate band. This dif-ference suggests that equal numbers of U5 and U6atacsnRNAs may contact substrate molecules simultane-ously but that formation of the U5/substrate cross-linkis more efficient than that of the U6atac/substrate cross-link. Alternatively, the majority of U5/substrate interac-tions may take place in the absence of U6atac/substrateinteractions, with the cross-linking efficiencies beingcomparable. We cannot distinguish between these twopossibilities.

As with the formation of U12/U6atac helix Ib (Figure2A), the U6atac/59ss interaction was inhibited by trun-cating the 59 exon or by binding the 59 exon oligo up-stream of the 59ss (Figure 3A, top panel). Inhibition ofboth the singly and doubly U6atac-containing cross-linked species (labeled U6atac/sub and U5/U6atac/sub,respectively, in Figure 3A) was approximately 6-fold withthe P120 59 exon oligo (lane 5) and more than 30-foldwith the P120-4SU-8 substrate (lane 6) compared to theP120-4SU-47 substrate (lane 4). In contrast, the U5/59exon interaction (labeled U5/sub) displayed only modestinhibition with the 59 exon oligo (1.53) or 59 exon trunca-tion (33). Likewise, U11 cross-linking to the 59ss of thesubstrate (Figure 3A, bottom panel) was unaffected(,1.53) by manipulation of the 59 exon. One might havepredicted that blocking U6atac binding would increaseU11/59ss cross-links, but the lack of effect in lanes 5Figure 3. Analysis of U5/59 Exon, U6atac/59ss, and U11/59ss Inter-

actions by 4SU Cross-Linking and 6 can be explained by our native gel analyses, which(A) Spliceosome assembly and cross-linking reactions were per- indicated that only a subset (10%) of prespliceosomalformed as in Figure 2A, except that the substrate was P120-4SU-47 complexes containing U11/U12 bound to pre-mRNA as-(lanes 25 and 7) or P120-4SU-8 (lane 6) and no psoralen was added. semble into more mature spliceosomes (Frilander andCross-linked snRNAs were detected using the U6atac-specific Steitz, 1999). We conclude that a region in the 59 exonprobe (top panel), followed by probe removal and rehybridization

somehow selectively promotes U6atac binding but notwith U5- (middle panel) or U11-specific probes (bottom panel).U11 or U5 interactions in the vicinity of the 59ss.(B) RNase H identification of U6atac- and U5-specific cross-linked

species. 4SU cross-linking was performed as in panel A using the Finally, we tested the effect of including the U12-P120-4SU-47 substrate. The isolated RNA species from the cross- Helix I oligo (Figure 1B), which completely blocks thelinking reaction were incubated with RNase H alone (lane 1) or RNase appearance of both U12/U6atac helix Ia and Ib psoralenH plus 100 pmol of DNA oligonucleotides complementary to U5 cross-links (see Figure 2C, lane 6). Strikingly, it did not(lane 2), U6atac (lane 3), the P120 intron (lane 4), or the P120 39 exon

significantly alter either the U6atac/59ss or U5/59exon(lane 5). Subsequently, the reactions were analyzed using U6atac-interaction, as detected by 4SU cross-linking (Figure 3A,(upper panel) or U5 snRNA-specific probes (lower panel) as de-

scribed for panel A. lane 7 compared to lane 4). This result contrasts theobservation that U6atac/59ss interactions are inhibitedby 59 exon manipulation (Figure 3A, top panel, lanes 5and 6), while the helix Ia formation is only slightly af-onstration that U5 and U6atac interact simultaneouslyfected (Figure 2C, lanes 4 and 5). Apparently, U12/U6a-with a pre-mRNA undergoing splicing. With the P120-tac helix Ia and U6atac/59ss interactions can occur in4SU-47 substrate, two cross-linked species were de-either order, since each can be observed in the absencetected in lane 4 using either U5- (Figure 3A, middle panel)of the other.or U6atac-specific probes (Figure 3A, top panel). The

faster migrating bands, which differ in mobility in gelsprobed for U5 and U6atac snRNAs, represent cross- Reversal of the 29-O-Methyl RNA Oligonucleotide

Block Restores Normal RNARNA Interactionslinks between the pre-mRNA and U5 or U6atac snRNAalone. The upper bands display identical mobility in both and Splicing Activity

It might be argued that cross-linking analyses of splicingpanels and were confirmed by RNase H targeting experi-ments (Figure 3B) to be doubly cross-linked, containing complexes arrested by blocking oligonucleotides (Fig-

ures 2 and 3) are not relevant to normal spliceosomeboth U5 (lane 2) and U6atac (lane 3) snRNAs linked tothe pre-mRNA substrate (lanes 4 and 5). The intensity assembly. We therefore devised a method to reverse

the oligonucleotide block, which was modeled after anof the U5/U6atac/substrate cross-link in gels probed forU5 snRNA (Figure 3A, middle panel) was consistently oligonucleotide displacement strategy used to purify the

telomerase RNP (Lingner and Cech, 1996). Each blockingapproximately 20-fold lower than the U5/substratecross-link (and therefore difficult to visualize in lanes 4 oligonucleotide was extended with an unpaired 39 tail

(Figure 4A), and reversal of the oligonucleotide blockand 7; however, see Figure 4C), whereas in gels probedfor U6atac snRNA (Figure 3A, top panel) the double was accomplished by addition of a second complemen-

tary 29-O-methyl RNA oligonucleotide. This release oli-cross-link was typically only 1.5- to 2-fold weaker than

Pathways for U12-Dependent Spliceosome Assembly221

Figure 4. Reversal of 29-O-Methyl RNA Oligonucleotide-Mediated Arrest of U12-Dependent Spliceosome Assembly

(A) Schematic illustration of 29-O-methyl RNA oligonucleotide blockage and its subsequent release using a second, complementary 29-O-methyl RNA oligonucleotide.(B) Restoration of psoralen cross-link formation. Spliceosome assembly was carried out on the P120-47 substrate as described in Figure 2,either without (lanes 16) or with 1 mM 59 exon (lanes 712), 0.5 mM U12-Helix I (lanes 1318), or 0.5 mM U12-BPS (lanes 1924) oligonucleotidecontaining an unpaired 39 tail (see Experimental Procedures). Release oligonucleotides were added 45 min after the start of incubation to thereactions indicated (1). After the total reaction time shown above each lane, psoralen was added to the reactions, which were then cross-linked and processed as described in Figure 2.(C) Restoration of 4SU cross-link formation. Reactions were performed as in panel B, except that the substrate was P120-4SU-47 and psoralenwas not added prior to irradiation. The probes were the same as in Figure 3. The identities of cross-linked species are indicated on the right.(D) Restoration of splicing activity. Splicing of the 32P-labeled P120-158 substrate was carried out under standard conditions using the sameoligonucleotides as in panel B. The reactions were incubated for the times indicated, and the recovered RNAs were analyzed as describedin Figure 2B. The release oligonucleotides were added as indicated 2 hr after the start of incubation. Immediately prior to addition of therelease oligonucleotide, a U12-Helix I 29-O-methyl RNA oligonucleotide was added to reactions 811 to prevent early splicing complexes notalready containing U12/U6atac helix Ia from continuing to assemble. Similarly, a 29-O-methyl RNA oligonucleotide U6atac120 was added toreactions 1417 immediately prior to the addition of the release oligonucleotide to prevent intermediates not yet containing the U6atac/59ssinteraction from continuing spliceosome assembly. Splicing intermediates and products are indicated on the right. Nonspecific bands (inpanel B) or the degradation products of the P120 splicing substrate (in panel D) are indicated with an asterisk. The markers (M) are as inFigure 2A.

gonucleotide first pairs with the free tail and then with Each release oligonucleotide was able to restore theinteractions inhibited by its corresponding tailed blockingthe full length of the blocking oligonucleotide, thus dis-

sociating the blocking oligonucleotide from its target oligonucleotide, whereas no rescue was observed withblocking oligonucleotides not containing unpaired tails(Figure 4A).

We tested three separate block/release 29-O-methyl (not shown). For 59 exon blockage, the release oligonu-cleotide restored both the U12/U6atac helix Ib and theRNA oligonucleotide pairs (Figures 4B and 4C). The

blocking oligonucleotides were tailed versions of the 59 U6atac/59ss interactions (Figures 4B and 4C, top panel,lanes 911); with the U12-Helix I pair, blockage of U12/exon and U12-Helix I oligos used above and a U12-BPS

oligo (see Figure 1B), which blocks U12/BPS recognition U6atac helices Ia and Ib was reversed (Figure 4B, toppanel, lanes 1517). Upon release of the U12-BPS block,and therefore causes an early assembly defect (Tarn

and Steitz, 1996a; Frilander and Steitz, 1999). As ex- which affects an early step in spliceosome assembly,the normal time course of RNARNA interactions ensuedpected, the U12-Helix I and U12-BPS oligos functioned

comparably to their tailless counterparts (Figure 4B, top (compare lanes 25 and 2123 in Figures 4B and 4C).Note in particular the difference in the kinetics of reap-panel, lanes 18 and 24; Figure 4C, top and bottom pan-

els, lanes 18 and 24). Also, the 59 exon oligo inhibited pearance of the U12/U6atac helix Ib and the disappear-ance of U4atac/U6atac upon release of the U12-Helix IU12/U6atac helix Ib formation (Figure 4B, top panel,

compare lanes 6 and 12) and the U6atac/59ss interaction oligo block versus that of the U12-BPS oligo (lanes1517 versus 2123). A direct demonstration of displace-(Figure 4C, top panel, lane 12), while the U5/59 exon

interaction was only slightly affected (Figure 4C, bottom ment is presented in lanes 1318 and 1924 of the toppanel of Figure 4B, where strong cross-links betweenpanel, lane 12).

Molecular Cell222

the blocking oligonucleotides and U12 (labeled U12/ unwinding of the U4atac/U6atac duplex may be detect-able only in the U12-dependent system. However, theoligo) are observed. Lanes 1517 and 2123 show thattwo systems have important differences: in the U2-these cross-links vanish upon addition of the releasedependent system, the regions that form U2/U6 helicesoligonucleotide, consistent with quantitative release ofIa and Ib are both contained within stem I of the U4/U6the block.di-snRNP, while in the U12-dependent system they areFinally, we examined splicing activity to ask whetherdivided between stems I and II (see Figure 1). Moreover,trapped complexes, once released, can proceed toan additional U2/U6 helix (helix II) exists in the majorsplicing intermediates and products. After 2 hr incuba-spliceosome that in yeast has been suggested to betion with one of the three blocking oligonucleotides inredundant with U2/U6 helix Ib (Field and Friesen, 1996).a standard splicing reaction, the corresponding releaseTherefore, it is possible that the two spliceosomes couldoligonucleotide was added and each reaction followedfollow slightly different assembly pathways.for an additional 4 hr. The results presented in Figure

In both pathways depicted in Figure 5, the base-pair-4D show that the 59 exon and the U12-BPS oligos areing interactions identified in assembly intermediatesvery efficient in preventing splicing, even for 6 hr of(U6atac/59ss interaction in pathway I or U6atac/U12 he-incubation (lanes 12 and 24), while with U12-Helix I oligo,lix Ia interaction in pathway II) are quite short and seemsome background is observed (lane 18). The releaseunlikely to mediate stable association of the tri-snRNPoligonucleotide for 59 exon blockage is able to restorewith the prespliceosome. However, native gel analysissplicing not only efficiently but also rapidly: intermedi-of arrested complexes specific for either one or the otherates and products are maximal already 1 hr after releasepathway revealed weak but consistent signals corre-(lanes 811). Similarly, with U12-Helix I blockage, releasesponding to spliceosomal B complex (our unpublishedrestores splicing activity (lanes 1417), albeit not as rap-results), suggesting that some intermediate complexesidly. In contrast, upon release of the U12-BPS block,are able to survive the conditions of native gel electro-the splicing reaction follows a normal time course (lanesphoresis. Because U5/59 exon contacts (at position 22)2023), consistent with a block at an early stage ofare not dependent on prior U6atac/59ss or U12/U6atacspliceosome assembly. Together these results suggestinteractions, they probably coexist with both assemblythat authentic reaction intermediates are captured byintermediates (see Figure 5), further suggesting that U5both the 59 exon and the U12-Helix I oligonucleotidewith its associated proteins (possibly in conjunction withblock.other RNAprotein or proteinprotein interactions) couldstabilize the initial tri-snRNP association. Early entry ofDiscussionthe U4atac/U6atac.U5 tri-snRNP, with initial U5 interac-tions influencing 59ss choice, is consistent with the re-Our results have documented a novel requirement forcent conclusions of Maroney et al. (2000), as well asthe 59 exon of the substrate and provide insights intothose of others (Newman and Norman, 1991; Wyatt etthe order of RNARNA interactions leading to catalytical., 1992; Cortes et al., 1993; Ast and Weiner, 1996, 1997;core formation in the U12-dependent spliceosome.Kuhn et al., 1999; Kuhn and Brow, 2000) on assembly

Truncation or sequestration of an upstream region inof the U2-dependent spliceosome. Joining of the tri-

the 59 exon inhibited U6atac/59ss interactions (and pre-snRNP has been suggested to be guided by U5 snRNA

vented splicing) but allowed normal U5/59 exon and U12/and its associated Prp8/220 protein through dual inter-

U6atac helix Ia contacts, requiring that at least stem I action with the pre-mRNA (59 exon and 59ss) and U1of the U4atac/U6atac duplex (see Figure 1A) become snRNP (Abovich and Rosbash, 1997; Ast and Weiner,unwound. We further observed that the U6atac/59ss in- 1997; Reyes et al., 1999; Maroney et al., 2000). Later, interaction does not depend on prior formation of the U12/ the active spliceosome, U5 remains in close proximityU6atac helix Ia because it is unaffected by the presence to the 59ss, as previously deduced for the major spliceo-of the U12-Helix I blocking oligonucleotide. We conclude some (Sontheimer and Steitz, 1993; Newman et al., 1995;that U6atac/59ss and the U6atac/U12 helix Ia interac- Teigelkamp et al., 1995). Our observation of a ternarytions can occur independently and that U6atac base- U5/U6atac/substrate cross-link (Figure 3) constitutes di-paired to both U4atac and U12 is a potential intermedi- rect evidence for either spliceosome that the pre-mRNAate in assembly of the U12-dependent spliceosome. A is contacted simultaneously by both U5- and U6-likemodel showing this novel intermediate and its role in one snRNAs at this subsequent stage of the splicingof the two pathways leading to the ultimate assembly of process.the complete catalytic core is presented in Figure 5. Remarkably, obstruction of a region centered around

Our detection of the U12/U6atac/U4atac ternary position 28 in the 59 exon blocks U6atac/59ss contactscross-link (Figure 2A) not only provides direct evidence and formation of U12/U6atac helix Ib. One explanationfor the novel assembly intermediate but also argues that for arrest at this stage of spliceosome assembly is aunwinding of the U4atac/U6atac duplex can be discon- defect in the switch from U11/59ss to U6atac/59ss inter-tinuous with U4atac/U6atac stem I opening first, fol- actions, suggesting that an unwinding activity governinglowed by a separate stem II unwinding event (as in this exchange may be affected. In yeast, Prp28 has beenpathway II, Figure 5). Previous results with the yeast U2- identified as the relevant helicase (Staley and Guthrie,dependent spliceosome were interpreted to mean that 1999), and its human homolog, the U5-100 kDa protein,the U6/59ss interaction is essential to initiate (complete) is an integral part of the U5 snRNP (Teigelkamp etU4/U6 unwinding (Kuhn et al., 1999; Staley and Guthrie, al., 1997). Since we detect nearly normal levels of the1999), but because of the fortuitous arrangement of pso- U5/59 exon 22 cross-link with our 59 exon blocking condi-

tions, it is possible that subsequent contact (by someralen cross-linking sites (see Figures 1A and 1B), partial

Pathways for U12-Dependent Spliceosome Assembly223

Figure 5. A Model for the Formation of the Cat-alytic Core of the U12-Dependent Spliceosome

U11 and U12 prebound to the substrate arejoined by the U4atac/U6atac di-snPNP toform either an intermediate in which U6atacreplaces U11 at the 59ss to form a complexcontaining U4atac/U6atac duplex (pathway I)or a novel intermediate containing a partiallyunwound U4atac/U6atac duplex (pathway II).Both intermediates are resolved by the lossof U4atac and the formation of both U6atac/U12 helices at the catalytic core. In additionto the interactions depicted, U5 makes con-tact with the 59 exon just upstream of the 59ssin the fully assembled catalytic core and mostlikely also in both intermediates. The shadedboxes represent regions in snRNA moleculesand in the intron that base pair to juxtaposethe reactants for the first step of splicing.Other symbols are as in Figures 1A and 1B.

other component of the spliceosome) with the essential its antagonist Prp24 (Raghunathan and Guthrie, 1998b),while the initial activation of the unwindase within theregion farther upstream in the 59 exon is required either

for activating the unwinding activity or for its proper spliceosome is probably regulated by U5/Prp8 bindingupstream of the 59ss (Kuhn et al., 1999). Interestingly,positioning at the 59ss. Accordingly, a recent study of

the U2-dependent spliceosome found that the entry of the activity of yeast Brr2 has been reported to be neces-sary for stable association of the U4/U6.U5 tri-snRNPthe tri-snRNP led to physical protection of a region that

extended to position 215 upstream of the 59ss (Maroney with the spliceosome, suggesting that (partial) unwind-ing may be necessary for early recognition of the 59ss byet al., 2000). A candidate for direct or indirect interaction

with this region is the Prp8/U5-220 kDa protein, which the tri-snRNP (for recent discussions, see Raghunathanand Guthrie [1998a]; Maroney et al. [2000]). Althoughhas been detected in the U12-dependent spliceosome

(Luo et al., 1999). In the U2-dependent spliceosome, this some proteins of the U12-type spliceosome have beencharacterized (Will et al., 1999), it is not known if theprotein is known to make contact with the 59 exon at

least up to position 28 relative to the 59ss (Teigelkamp same or analogous components carry out this task inthe U12-dependent spliceosome.et al., 1995). In a preliminary survey of proteins inter-

acting with this region in the U12-dependent system, we Dependence on 59 exon sequences for establishingU6atac/59ss interactions in the U12-dependent spliceo-detected several additional proteins (our unpublished

results), suggesting that multiple interactions/compo- some may also reflect a difference in the initial recogni-tion of U12- versus U2-type introns: the 59ss and BPSnents may be required for triggering U4atac/U6atac un-

winding. are recognized in a cooperative manner by the U11/U12di-snRNP complex (Frilander and Steitz, 1999), whereasIn the major spliceosome at least some components

of the U4/U6 unwinding machinery have been identified. U1 and U2 binding can proceed independently on themajority of introns (Seraphin and Rosbash, 1991; ChiaraIn vitro studies in the mammalian and yeast systems

have demonstrated that U5-200 kDa protein and its and Reed, 1995). Therefore, the exchange of U11 forU6atac at the 59ss may be significantly more difficult toyeast homolog, Brr2, are capable of dissociating both

stem I and stem II of U4/U6 base-paired snRNAs, even execute than the exchange of U1 for U6 since the U11snRNP must be freed from the U12 snRNP. It has beenin the absence of pre-mRNA (Laggerbauer et al., 1998;

Raghunathan and Guthrie, 1998a). Furthermore, the ex- documented that at least the first step of splicing forthe U2-dependent spliceosome can proceed efficientlytent of U4/U6 unwinding in yeast has been shown to be

governed by the balance between Brr2 helicase and with very short 59 exons (Hertel and Maniatis, 1999). Yet,

Molecular Cell224

template. Templates for the P120 and SCN4AENH1 substrates witha similar requirement for upstream 59 exon sequencestruncated 59 exons were generated by PCR. All splicing substratesmay have escaped previous detection if recognition ofwere purified in denaturing polyacrylamide gels before use.this region is essential for only a subset of introns, per-

The 4SU-containing P120 substrates containing either a 47 or 8haps those designated U1-dependent (Crispino et al., nucleotide 59 exon were constructed using the two-piece RNA liga-1996). tion technique of Moore and Sharp (1992) and RNA ligation condi-

tions described by Frilander and Steitz (1999). The 39 piece for bothThe conclusions in this work rely on the use of 29-O-substrates (starting from intron position 13) was generated frommethyl RNA oligonucleotides, which were used to blockfull-length U-containing P120 substrate by site-specific RNase Hspecific interactions along the spliceosomal assemblycleavage (Yu and Steitz, 1997a). The 59 piece for the P120-4SU-47pathway. A potential problem with their use is that theysubstrate was generated by constructing a plasmid containing a T7

may redirect the spliceosomal assembly pathway and RNA promoter and nucleotides 247 to 12 of the P120 substrate.lead to the formation of dead-end complexes. To ad- The sequence of nts 247 to 12 had been modified to contain U

residues only in positions 22 and 12 relative to the 59ss in thedress this problem, we adapted an oligonucleotide dis-ligated substrate. DNA containing the T7 promoter and the 247 toplacement method previously used in RNP purification12 region of the substrate was amplified from the plasmid and(Lingner and Cech, 1996; see Figure 4) to show thatused for in vitro transcription with T7 RNA polymerase in reactionsthe spliceosomal assembly pathway stalled by specificcontaining 4SUTP (USB) substituted for UTP. The 59 piece for the

oligonucleotides can be restored to a functional state. In P120-4SU-8 substrate was generated using the same strategy as forour analysis, the rapid kinetic appearance of RNARNA P120-4SU-47. The 59 and 39 pieces were annealed using a bridging

DNA oligonucleotide, ligated, and gel purified as described earlierinteractions and splicing products following the release(Frilander and Steitz, 1999). The introduced mutations were con-of blocking oligonucleotide rules out de novo spliceo-firmed by sequencing. The 4SU-containing substrates were splicedsome assembly and strongly suggests that true interme-as efficiently as wild-type substrates of similar length lacking 4SUdiates were captured (Figures 4B4D). We believe thatresidues.

this oligonucleotide block/release method should begenerally applicable to the study of other RNA (or single- Splicing Reactions

In vitro splicing reactions were carried out as described (Frilanderstranded DNA) recognition events.and Steitz, 1999), using 32P-labeled P120 substrate or its 59 exonAs the assembly of a functional spliceosome requiresdeletion mutants. For cross-linking, splicing reactions containedan ordered exchange of a large number of RNARNA20 nM unlabeled P120 or SCN4AENH1 substrate or their deletioninteractions, each presumably catalyzed by a specificderivatives. The reactions were incubated for 45 min at 1308C prior

RNA helicase (Staley and Guthrie, 1998), it is necessary to cross-linking, unless otherwise indicated. The concentrations forto define the number of RNA exchanges in order to the blocking 29-O-methyl RNA oligonucleotides (with or without an

unpaired tail) were 0.5 mM for U12 helix I and U12-BPS oligos andestimate the number of components needed to build an1 mM for the P120 59 exon oligo. A 1.5- to 3-fold molar excess ofactive spliceosome. For example, the partial U4atac/the displacement oligonucleotide compared to the blocking oligonu-U6atac unwinding documented in this work suggestscleotide was used in the block-release experiments.that additional helicases or regulatory components may

be needed. Given the close parallels between the U2- Cross-Linking and Identification of Cross-Linked RNA Speciesand U12-dependent spliceosome, it will be most inter- The conditions for performing and processing psoralen and 4SU

cross-linking reactions have been described previously (Frilanderesting to ask whether partial unwinding of U4/U6 con-and Steitz, 1999). Cross-linked RNAs were detected by Northerncomitant with U2/U6 helix Ia catalytic core interactionsanalysis as described previously (Tarn and Steitz, 1996b), exceptcan be documented during assembly of the majorthat a DNA oligonucleotide probe complementary to nucleotides

spliceosome. An advantage of alternative pathways may 6179 of the U6atac snRNA was used. The identities of the cross-be to confer opportunities for regulation by introducing linked bands were confirmed by RNase H targeting and by reprobingflexibility into the order of RNARNA interactions that the filters successively with probes for different snRNAs. Previously

mapped positions of psoralen cross-links within U12/BPS, U12/eventually attain the same catalytic configuration.U6atac helix Ia and Ib (Tarn and Steitz, 1996b) were confirmed by39 end labeling the U12 snRNA after UV irradiation, followed by gelExperimental Procedurespurification of the cross-linked species. Partial RNase T1 and alkalinehydrolysis analyses of these RNAs were consistent with the previous

29-O-Methyl RNA Oligonucleotidesmapping.

The 29-O-methyl RNA oligonucleotides used for the pretreatmentof the nuclear extracts were as follows: U2b, used to block U2-

Acknowledgmentsdependent splicing (Tarn and Steitz, 1996a); U12-Helix I, comple-mentary to nucleotides 116 of U12 snRNA (Tarn and Steitz, 1996b);

We thank D. Brow, T. Nilsen, E. Sontheimer, W. Y. Tarn, and theU12-BPS, complementary to nucleotides 1128 of U12 snRNA (Tarn

members of the Steitz laboratory for helpful discussions; T. McCon-and Steitz, 1996a); and 59 exon (complementary to nucleotides 26

nell, L. Weinstein, K. Tycowski, and L. Otake for comments on theto 222 upstream of the 59ss in the P120 splicing substrate). In manuscript; and A. Krainer for SCN4AENH12 plasmid. This workoligonucleotide release experiments, a 14 nt 39 tail (59-ACCAGUA was supported by the National Institutes of Health (grant GM26154GUCAGUC-39) was added to each blocking oligonucleotide. This to J. A. S.), the European Molecular Biology Organization (long-termtail was designed not to form extensive base-pairing interactions fellowship ALTF 616-1996 to M. J. F.), and Academy of Finlandwith the target molecule or any spliceosomal snRNA. The corre- (grants 44993 and 48416 to M. J. F.). J. A. S. is an investigator ofsponding release oligonucleotides were 29-O-methyl RNA oligonu- the Howard Hughes Medical Institute.cleotides complementary to the tailed blocking oligonucleotides.

Received September 27, 2000; revised November 14, 2000.Splicing SubstratesCapped full-length P120 and SNC4AENH1 splicing substrates were Referencesproduced by in vitro transcription using either T7 (P120) or SP6(SCN4AENH1) RNA polymerase (Pharmacia) and plasmid pP120 Abovich, N., and Rosbash, M. (1997). Cross-intron bridging interac-linearized with HindIII (Tarn and Steitz, 1996a) or plasmid tions in the yeast commitment complex are conserved in mammals.

Cell 89, 403412.SCN4AENH12 linearized with EcoRI (Wu and Krainer, 1998) as a

Pathways for U12-Dependent Spliceosome Assembly225

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