Mechanical Devices of the Spliceosome: Review Motors, Clocks, Springs, and Things

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Cell, Vol. 92, 315–326, February 6, 1998, Copyright 1998 by Cell Press Mechanical Devices of the Spliceosome: Review Motors, Clocks, Springs, and Things

Jonathan P. Staley and Christine Guthrie* and springs for achieving speed and accuracy (see re- Department of Biochemistry and Biophysics view by Wilson and Noller, 1998). Similar to a motor, the University of California, San Francisco initiation factor eIF-4A, which escorts the 40S ribosome San Francisco, California 94143 from the 5Ј cap to the start codon, is generally thought to use the energy of ATP to disrupt secondary structure along the way. The elongation factor EF-1, which escorts charged tRNA to the ribosomal A site, has been de- Introduction scribed as a clock and is believed to use the energy The discovery of interrupted genes and messenger RNA of GTP hydrolysis to transmit the signal that a correct (mRNA) splicing 20 years ago was greeted with amaze- codon:anticodon match has been made at the decoding ment (Berget et al., 1977; Chow et al., 1977). Although site, setting the stage for peptide bond formation. Fi- introns needed to be removed with single-nucleotide nally, EF-2 appears to use GTP hydrolysis to unlatch a precision to avoid introducing catastrophic errors of tightly coiled spring, which pushes the tRNA:codon unit frame, they contained few conserved sequences, varied from the A site to the P site (Abel and Jurnak, 1996). widely in length, and were found at dozens of locations Since splicing must also be efficient and accurate, we in a single transcript. How did cells achieve this exquisite anticipate that the spliceosome will also utilize motors, precision while splicing such diverse introns? clocks, and springs. In 1985 it was discovered that the splicing apparatus The spliceosome is a surprisingly dynamic machine, was as immense as the ribosome (Brody and Abelson, building anew on each pre-mRNA substrate (Figure 2; 1985). The “parts list” for the spliceosome includes five Moore et al., 1993). Each round of splicing requires the small nuclear RNAs (snRNAs) and more than 50 proteins ordered binding and release of snRNPs. Although the (Will and Lu¨ hrmann, 1997). Thus, like the ribosome, the earliest assembly step is energy independent, all subse- spliceosome is a ribonucleoprotein (RNP) machine. Al- quent assembly steps require NTP hydrolysis. Coinci- though the early years of ribosome research were de- dent with many of these steps, RNA helices are formed voted to the protein components, revolutionary phyloge- and disrupted (Figure 3; Nilsen, 1998). Intriguingly, each netic studies subsequently identified the rRNAs as its ATP-dependent step requires one or more proteins of most highly conserved constituents (see review by Wil- a superfamily of ATPases that share sequence similarity son and Noller, 1998 [this issue of Cell]). Similarly, the with canonical DNA helicases (Gorbalenya and Koonin, splicing field established pivotal roles for the snRNAs 1993), proteins that function as processive motors (Loh- in splicing (Moore et al., 1993). Indeed, both the stunning man and Bjornson, 1996). This similarity has suggested demonstration that peptidyl transferase activity is pri- that the spliceosomal members may consume ATP to marily RNA based and the electrifying discovery of self- unwind RNA (Fuller-Pace, 1994), a popular but unproved splicing RNAs validated an RNA-centered view for both hypothesis. RNP machines and spawned the concept of an ancient This review focuses on recent progress toward ad- “RNA world” (Gesteland and Atkins, 1993). dressing the roles of ATP and protein components of Both the spliceosome and the ribosome are energy- the spliceosome in driving RNA rearrangements. We will consuming machines, but neither requires NTP hydroly- review recent evidence that spliceosomal “DExD/H box” sis for the basic chemical reaction promoted by each ATPases can function as one-step (nonprocessive) mo- machine. No NTP hydrolysis isrequired for the chemistry tors to unwind short RNA duplexes. The precise timing of splicing, because catalysis proceeds by two phos- of the RNA rearrangements suggests that these RNA- phoryl transfer reactions (Figure 1; Moore et al., 1993). dependent ATPases may also function as clocks in In the first reaction, the 2Ј hydroxyl of a conserved, proofreading steps of splicing. In addition, we will de- intronic adenosine attacks the phosphate at the 5Ј splice scribe a spliceosomal analog of EF-2, a ribosomal GTPase site, producing a free 5Ј exon and a branched species, proposed to function like a spring (Abel and Jurnak, termed the lariat intermediate. In the second reaction, 1996). Finally, we will discuss the role of RNA annealing the 3Ј hydroxyl of the 5Ј exon attacks the phosphate at proteins, “things” that promote RNA rearrangements by the 3Ј splice site, yielding ligated mRNA and a lariat energy-independent mechanisms. It is our hope that an intron. Significantly, group II introns self-splice in vitro appreciation of the mechanical aspects of splicing will by an identical two-step transesterification mechanism, enhance efforts to understand how this extraordinary independent of both NTP hydrolysis and protein (Michel machine can accommodate substrate diversity while and Ferat, 1995). constraining errors. For recent, more comprehensive If the basic chemical reaction promoted by the reviews, see Hertel et al., 1997; Newman, 1997; Tarn spliceosome can be catalyzed by RNA alone, then how and Steitz, 1997; Wang and Manley, 1997; Will and Lu¨ hr- can we rationalize the protein parts of this splicing ma- mann, 1997; and Nilsen, 1998. chine and its requirement for energy? A look under the hood of the eukaryotic protein synthesis machine reveals the use of fuel to power molecular motors, clocks, Rearrangements in the Spliceosome Assembling a Catalytically Active Spliceosome The spliceosome assembles or rearranges in a highly * To whom correspondence should be addressed. ordered and stepwise manner (Figure 2; Moore et al., Cell 316

Figure 2. Spliceosome Assembly, Rearrangement, and Disassem- bly Requires ATP, Numerous DExD/H box Proteins, and Prp24 The snRNPs are depicted as circles. The pathway for S. cerevisiae Figure 1. Pre-mRNA Splicing Occurs in Two ATP-Independent is shown. (See Table 1.) Transesterification Reactions Pink, first transesterification reactants; green, second transesterification reactants. The intron is shown as a line with S. cerevisiae highly coordinated and carefully regulated fashion. The consensus sequences. 5Ј splice site is initially recognized through a base-pairing interaction with U1 (Figure 3, [a]). U1 is not required during the transesterification reactions (Yean and Lin, 1993). Assembly begins with the association of the U1 1996), however, and this interaction is switched subse- snRNP with pre-mRNA. Subsequently, the U2 snRNP quently for a mutually exclusive base-pairing interaction and then the U4/U6·U5 triple snRNP bind, U1 and U4 with U6 (Nilsen, 1998). The branch point sequence is are destabilized, and the spliceosome is activated for recognized early by BBP, the branch point binding pro- catalysis. Significantly, this order in assembly is con- tein, in a sequence-specific fashion (Figure 3, [b]; Berg- served from yeast to mammals and even extends to a lund et al., 1997). Thus, subsequent recognition of the newly discovered spliceosome that comprises a unique branch point region by base pairing with U2 snRNA set of snRNAs and splices a novel class of introns in a (Nilsen, 1998) is also mutually exclusive. This switch U12-dependent manner (Tarn and Steitz, 1997). Thus, appears to be the first example for the spliceosome the spliceosomal assembly pathway is likely to reflect in which a protein:RNA interaction is replaced by an fundamental requirements in the construction of a cata- RNA:RNA interaction. Interestingly, the activity of the lytically active machine. U2 snRNP is modulated via a switch between competing At the molecular level, many of the assembly steps U2 conformations (Zavanelli et al., 1994). In the active correspond to changes in RNA:RNA interactions (Figure form, stem/loop IIA is favored; in the inactive form, a 3; Nilsen, 1998). These interactions have been character- mutually exclusive interaction between the loop and ized by three powerful approaches: phylogenetics, ge- a conserved downstream sequence predominates (Fig- netics, and photochemical cross-linking. To the delight ure 3, [c]). of the field, the data from these complementary ap- The addition of the U4/U6·U5 triple snRNP to the proaches have been virtually in complete agreement. spliceosome (Figure 2) heralds a large number of RNA: Thus, functional interactions have been shown to reflect RNA rearrangements (Nilsen, 1998). The triple snRNP is direct interactions and direct interactions have been believed to escort U6 to the spliceosome base paired shown to reflect functional ones. As with the assembly with U4 via stems I and II (Figure 3, [e] and [d], respec- pathway, most of these RNA:RNA interactions have tively). In yeast, this interaction, which involves a total of been validated in yeast and mammals (Nilsen, 1998), as 24 base pairs, is remarkably stable. Despite this stability, well as in the U12-dependent spliceosome (Tarn and both stems of the U4/U6 interaction are disrupted as Steitz, 1997). the spliceosome undergoes extensive remodeling and It is striking that many of these interactions are mutu- becomes activated for catalysis (Figure 3; Nilsen, 1998), ally exclusive; the formation of one requires the disrup- for which U4 is not required (Yean and Lin, 1991). The tion of another (Figure 3). Such a design is likely to stem II region of U6, once freed from U4, folds on itself ensure that the spliceosome can be assembled in a to form an intramolecular stem/loop (Figure 3, [d]). The Review: Spliceosome Mechanics 317

Figure 3. Splicing Requires Numerous Rearrangements A composite summary is shown of six rearrangements required for the first transesterification; the RNA is color coded to indicate the regions that become paired. Thin line, pre-mRNA; thick line, snRNA; BBP, branch point–binding protein. A, the branch point adenosine. The specific rearrangements are shown below the summary. (a) The exchange of U1 for U6. (b) The exchange of BBP for U2. (c) An intramolecular U2 rearrangement. (d) Disruption of U4/U6 stem II; formation of U6 3Ј stem/loop. (e) Disruption of U4/U6 stem I and U2 5Ј stem/loop; formation of U2/U6 helix I. (f) Disruption of the U2 5Ј stem/loop; formation of U2/U6 helix II. S. cerevisiae sequences are shown. For clarity, U5 has been omitted.

stem I region of U6, once freed, base pairs with U2 Rearranging the Catalytically Active Spliceosome snRNA, forming U2/U6 helix I (Figure 3, [e]). Since helix Having executed these RNA rearrangements (Figure 3), I is mutually exclusive with the 3Ј half of the 5Ј stem/ the spliceosome is competent to carry out the first loop of U2 snRNA, the stem/loop mustalso be disrupted; chemical step,a transesterification reaction that cleaves disruption of this stem loop also frees the 5Ј half to the 5Ј exon from the intron and produces a lariat interme- interact with U6, forming U2/U6 helix II (Figure 3, [f]). diate (Figure 1). A rearrangement must follow to permit While abundant evidence supports these conforma- the second transesterification (Figure 4). After the first tional isomerizations (Figure 3), their precise timing transesterification, the leaving group for this reaction, needs to be determined with respect to one another and the 3Ј hydroxyl of the 5Ј exon (Figure 1), is adjacent to to the changing snRNP composition of the assembling the newly formed 2Ј–5Ј phosphodiester bond. For the spliceosome (Figure 2). For example, it is generally second transesterification, however, the 3Ј hydroxyl, thought that the U4/U6 rearrangements coincide with which becomes the attacking group, must be positioned the destabilization of U4 from the spliceosome, but it is near the 3Ј–5Ј phosphodiester bond of the 3Ј splice site. not known whether stem I and stem II are disrupted in In principle, this alignment could be achieved by one of a concerted or sequential manner. These details are two distinct mechanisms. In the first case, the position important for understanding the timing and specificity of the 5Ј exon remains fixed relative to the spliceosome, of the factors that mediate these rearrangements (see while the 2Ј–5Ј phosphodiester bond of the lariat inter- below). mediate is replaced by the 3Ј–5Ј bond of the 3Ј splice Cell 318

Figure 4. Rearrangement of the Reactants between the First and Second Transesterifi- cation (A) Modeling one catalytic site. (B) Modeling two catalytic sites. Left, the configuration just after the first reaction; right, the configuration just before the second. As in Figure 1, the first-step reactants are colored pink; the second-step, green. Interactions important for the second transesterification are indicated by dashes between U5 and the exons and between the first and last nucleotides of the intron. N, any nucleotide. Sequences are conserved from S. cerevisiae to mammals.

site (Figure 4A; Steitz and Steitz, 1993). In the second A combination of genetic and photochemical cross-link- case, the 2Ј–5Ј bond would remain fixed, while the 5Ј ing analyses has revealed interactions between the exon is repositioned near the 3Ј splice site (Figure 4B). highly conserved loop of U5 and both the 5Ј and 3Ј The first case described above is consistent with a exons (Figure 4; Newman, 1997), suggesting a role for U5 single catalytic site; however, the other case necessi- in aligning the exons for the second transesterification tates a second site. Group I self-splicing RNAs utilize a reaction. Genetics have also revealed a non-Watson- single catalytic core; a 3Ј hydroxyl and a 3Ј–5Ј bond Crick interaction between the first and last nucleotides are formed in each step and the second reaction is of the intron, which are almost always guanines (Figure essentially the reverse of the first (Saldanha et al., 1993). 4; Umen and Guthrie, 1995). The G-G interaction can be In group II RNAs and the spliceosome, however, a 3Ј replaced, albeit inefficiently, by an A-C interaction, a hydroxyl and a 2Ј–5Ј phosphodiester bond are produced combination which is found naturally in a minority of in the first reaction, but this 3Ј hydroxyl reacts with a introns in higher eukaryotes (Tarn and Steitz, 1997). 3Ј–5Ј bond in the second (Moore et al., 1993; Michel and Postcatalytic Rearrangements and Recycling Ferat, 1995). Thus, the second transesterification cannot Having performed the task of ligating exons and produc- be a simple reversal of the first. This conclusion is sup- ing mRNA, the spliceosome must liberate the mRNA for ported by stereochemical analyses using chiral phos- export. Furthermore, just as a machine is designed to phorothioates and observations that different chemical perform repetitive tasks, the postcatalytic spliceosomal substituents are required for each step (Moore et al., machinery must be reconfigured to allow a new round 1993; Sontheimer et al., 1997). Still, these data do not of splicing. The snRNP-bound lariat intron must be dis- rule out a single catalytic core (Steitz and Steitz, 1993). assembled, allowing the lariat intron to be degraded and In either case, a rearrangement must occur that proba- the snRNPs to be recycled (Figure 2). Release of mRNA, bly involves two RNA:RNA interactions implicated in disassembly, and recycling all necessarily involve exten- configuring the reactants for the second catalytic step. sive RNA:RNA rearrangements. The base pairing of U6 Review: Spliceosome Mechanics 319

Table 1. The Parts Listsa

S. cerevisiae Mammalsb Motifsc Stepd Putative Function Mud2? U2AF65 3 RRMs, (RS) 1st U2 addition Prp24 ? 3 RRMs recycling annealing U4/U6 Sub2e? UAP56 DECD 1st U2 addition Prp5 ? DEAD 1st U2 addition Prp28 U5-100 kDa DEAD, (RS) 1st tri-snRNP addition Prp2 ? DEAH 1st catalytic activation Prp16 hPRP16 DEAH, (RS) 2nd 2nd step rearrang. Prp22 HRH1 DEAH, S1, (RS) post-2nd mRNA release Prp43 mDEAH9 DEAH post-2nd lariat release Brr2f U5-200 kDa DEIH, DxxH 1st unwinding U4/U6 Snu114 U5-116 kDa EF-2-like ? ? a See text for references. b These factors are required for splicing the major U2-dependent introns; it is not known whether they are also required for U12-dependent introns (Tarn and Steitz, 1997) or whether unique, related proteins are required. c (RS) indicates that this motif is found in the mammalian ortholog only. d Step refers to the transesterification reaction (Figure 1). e ORF ydl084w. f Also known as Rss1 (Lin and Rossi, 1996), Slt22 (Xu et al., 1996), and Snu246 (Lauber et al., 1996).

to the 5Ј splice site, U2 to the branch point, and U5 to 1995), can promote annealing of complementary RNAs. the exons must be severed (Figures 3 and 4). Moreover, Although RRMs are found in all eukaryotes, RS domains the mutually exclusive pairings involving U2, U6, and U4 are more prevalent in higher eukaryotes, where RS phos- (Figure 3) must be restored to their original conforma- phorylation may play a role in regulation. tions. The second and largest class of devices contains members of a superfamily of ATPases (Gorbalenya and Mechanical Devices in the Splicing Machine Koonin, 1993). Members of this superfamily, superfamily An Inventory II, and the related superfamily I possess a number of These RNA rearrangements predict the requirement for conserved motifs, although the consensus sequences mechanical devices that promote base pairing or facili- for these motifs vary considerably between the two (Fig- tate unwinding. Such devices must function in an orderly ure 5B). Splicing members of superfamily II are found and timely fashion and could contribute to the accuracy in the DEAD and DEAH box families (Fuller-Pace, 1994), of splicing. Candidates for such devices can be grouped eponymous for their motif II sequence (Figure 5B), or into three classes distinguished by conserved sequence contain DECH or DECD motif II sequences (Table 1). We motifs (Table 1). Interestingly, a few devices contain will refer to these factors collectively as the “DExD/H multiple types of motifs (Figure 5A). box” proteins. Superfamily I includes well-characterized The first class we will discuss has RNA-annealing ac- DNA helicases such as Rep—oligomeric, directional, tivity. RNA recognition motifs (RRMs), which bind single- and processive enzymes (Lohman and Bjornson, 1996). stranded RNA (Shamoo et al., 1995), and RS domains, Superfamily II includes the extensively studied initia- which contain repeats of arginine/serine dipeptides (Fu, tion factor eIF-4A, which unwinds RNA (Fuller-Pace,

Figure 5. Sequence Characteristics of the Spliceosome’s Mechanical Gadgets (A) Examples of domain structure. DEAD and DEAH, helicase-like domains; C-domain, conserved in the DEAH proteins; S1, a ribosomal motif implicated in RNA binding; RS, rich in serine/arginine dipeptides; RRM, RNA recognition motif; EF-2, elongation factor 2. All factors are from S. cerevisiae except for the mammalian factors U2AF65 and HRH1, the human ortholog of Prp22. (B) Sequence motifs of the DExD/H box domains. DEAD, residues identical between Prp5, Prp28, and U5–100 kDa (Table 1). DEAH, amino acidresidues identical between Prp2, Prp16, Prp22, Prp43, hPRP16, and HRH1 (Table 1). x, any amino acid. The specific sequences for the HCV RNA unwindase and Rep are shown for comparison. Pink, residues common to all compared sequences. Yellow, residues common to all superfamily II sequences. †Note well: from a structural comparison, motif IV of superfamily II does not align with motif IV of superfamily I; rather, motif IV of superfamily II aligns with a novel superfamily I motif designated IVa (Figure 8; Korolev et al., 1997b). Cell 320

Do the RRMs of Prp24 facilitate annealing by bringing strands together or by serving as chaperones to prevent competing conformations (Herschlag, 1995)? Interest- ingly, recent crystal structures of the RRM-containing annealing protein hnRNPA1 reveal that its two RRMs are antiparallel with respect to one another (Shamoo et al., 1997; Xu et al., 1997). This suggests that each RRM binds single-stranded RNA in an antiparallel orientation to facilitate annealing. To fully understand the role of RRMs of Prp24, however, the role of other proteins in Figure 6. A Paradigm for Unwindase Specificity and Timing? the RNP-facilitated mechanism must be determined. For The DExD/H box protein UAP56 (orange) binds U2AF65 (pink) through example, do these additional proteins configure the RNA its linker region (L). U2 binds the branch point. Y’s indicate the to facilitate sequence-specific recognition by the RRMs, polypyrimidine stretch; RS, RRM as in Figure 5A. Sequences are or do they recruit the RRMs directly? from mammals. ATPases and the Unwindase Hypothesis Since the spliceosomal DExD/H box proteins share se- 1994), though not as an oligomeric, directional, or pro- quence similarity with eIF-4A, and eIF-4A unwinds RNA cessive enzyme in the absence of cofactors (Lorsch and in vitro, the spliceosomal DExD/H box factors have been Herschlag, 1998). This class of mechanical devices of suspected to function in RNA unwinding (Fuller-Pace, the spliceosome are consequently excellent candidates 1994). Indeed, several of the spliceosomal DExD/H box to function in RNA unwinding. factors exhibit ssRNA-stimulated ATPase activity (Schwer The third class includes one member of the GTPase and Guthrie, 1991; Kim et al., 1992; O’Day et al., 1996). superfamily (Bourne, 1995). Despite the prevalence of Furthermore, Prp16 was shown to be required for a these mechanical devices in other macromolecular ma- rearrangement that occurs at the 3Ј splice site (Schwer chines (see reviews by Matlack et al., 1998; Ohno et al., and Guthrie, 1992). Yet in the seven years since their 1998; Wilson and Noller, 1998 [all this issue of Cell]), the discovery, attempts to demonstrate in vitro strand dis- first spliceosomal example has been found only recently placement activity for the spliceosomal DExD/H box (Fabrizio et al., 1997). Intriguingly, this GTPase shares factors have failed (Kim et al., 1992; Strauss and Guthrie, extensive sequence similarity with EF-2, which is re- 1994). Four recent reports, however, now document this quired for the translocation step in translation (Abel and activity for splicing proteins. Prp16, Prp22, and U5–200 Jurnak, 1996). kDa all exhibit ATP-dependent unwinding of base- For all three classes of mechanical devices, the funda- paired U4/U6 RNAs as well as of several generic du- mental questions are the same—what are their sub- plexes (C. Gross and B. Schwer, submitted; Lagger- strates and what controls their specificity and timing? bauer et al., in press; J. Wagner et al., submitted; Y. Annealing Critical RNA Pairs Thus far, two spliceosomal RNA annealing reactions are Wang and C. Guthrie, submitted). Since we do not yet known to be facilitated by proteins. To anneal U2 with know if these factors function like DNA helicases, i.e., the pre-mRNA branch point sequence, U2AF65 utilizes as oligomers with directional and processive activity both RRM and RS domains (Valca´ rcel et al., 1996). (Lohman and Bjornson,1996), we suggest the term “RNA Through its RRMs, U2AF65 binds to the polypyrimidine unwindases” to avoid implying mechanistic similarities tract, a mammalian intron consensus sequence down- with the DNA helicases. stream from the branch point (Figure 6). This positions This important progress suggests that these three the RS domain near the branch point and consequently DExD/H box proteins do facilitate RNA rearrangements promotes the annealing of U2 with the branch point and do so by unwinding RNA directly. The next chal- region. Since seven arginines in the RS domain suffice lenge, however, is to assign specific rearrangements to promote annealing, this domain can function much to these proteins in vivo (e.g., Figure 3, [a–f]). If such like the generic polycation spermidine in annealing RNA. assignments can be made, then the interesting question (Note, however, RS domains have other functions, such becomes what dictates the specificity of these proteins as stabilizing protein:protein interactions, that require in splicing, since they can unwind generic RNA sub- the serines in addition to the arginines [Fu, 1995].) The strates in vitro (C. Gross and B. Schwer, submitted; RRM-containing Mud2 protein from yeast may be the Laggerbauer et al., in press; J. Wagner et al., submitted; functional analog of U2AF65 (Abovich et al.,1994), though Y. Wang and C. Guthrie, submitted). It is also quite possi- it contains only one RRM and does not contain an obvi- ble that DExD/H box proteins perform biological func- ous RS domain—perhaps because the U2:branch point tions other than unwinding RNA duplexes. For example, base pairing interaction is much more extensive in yeast. tertiary interactions are likely to play key roles in the In summary, U2AF65 achieves specific annealing by us- assembling spliceosome (Nilsen, 1998)—perhaps to sta- ing a non-sequence-specific mechanism. bilize short helices or to juxtapose distal RNA elements. Extensive genetic and biochemical data have impli- Conceivably, the role of some DExD/H factors could be cated Prp24 in the annealing of U4 and U6 (Figure 2). to disrupt such tertiary interactions. Recombinant Prp24 anneals synthetic U4 and U6 RNAs Alternatively, the direct target of some DExD/H box (Ghetti et al., 1995). Interestingly, Prp24 anneals these proteins might be a protein that binds RNA. Indeed, RNAs much more rapidly in the context of RNPs (Raghu- many of the RNA duplexes in splicing are short (Figure nathan and Guthrie, 1998). Significantly, Prp24 contains 3) and probably require protein binding for stabilization three RRMs but no RS domain. (e.g., Mount et al., 1983). Thus, an RNA rearrangement Review: Spliceosome Mechanics 321

may result indirectly from the disruption of a protein:RNA sensitivity. Intriguingly, four of the yeast spliceosomal interaction. A potential paradigm for such a mechanism DExD/H box proteins, namely Prp16, Prp22, Prp28, and is provided by Mot1, a superfamily II member that down- Brr2, have been identified as cold-sensitive mutants, regulates transcription by displacing TBP from the TATA suggesting that they might be defective in RNA unwind- box in an ATP-dependent fashion (see review by Kado- ing. Consistent with this hypothesis, the mutant prp28–1 naga, 1998 [this issue of Cell]). It is noteworthy that the exacerbates the defect due to hyperstabilization of U1 ATPase activity of Mot1 is strongly stimulated by TBP in vivo and prevents base pairing between U6 and the but not by DNA (Auble et al., 1997). This raises the in- 5Ј splice site in vitro (J. P. S. and C. G., unpublished triguing possibility that DExD/H box proteins that exhibit data). Prp28 may function either to unwind the U1:5Ј weak or no RNA stimulation of ATPase activity will be splice-site helix or to promote formation of the U6:5Ј good candidates for protein-stimulated ATPases. splice-site helix (Figure 3, [a]), perhaps by unwinding a Answers to the foregoing questions demand the iden- helix in the U4/U6·U5 triple snRNP. Indeed, although tification of the specific rearrangements catalyzed by Prp28 does not appear to be an snRNP component in each spliceosomal factor. To date, however, this task yeast, it has been isolated as an integral component of has proven inordinately difficult. Below we consider the U4/U6·U5 triple snRNP in mammals (Teigelkamp et what is known about the functions of individual spliceo- al., 1997); intriguingly, this mammalian counterpart con- somal DExD/H box proteins in relation to specific re- tains an RS domain (Table 1). arrangements. A Question of Timing and Specificity. The DExD/H box Branchpoint Recognition: Roles for UAP56 and Prp5. proteins are required for distinct steps in the assembly The association of U2 snRNP with the pre-mRNA is ATP pathway (Figure 2). What times their action? In addition, dependent (Figure 2), requires a specific conformation given that Prp16, Prp22, and U5–200 kDa unwind ge- of U2 (Figure 3, [c]), and requires the displacement of neric substrates in vitro (C. Gross and B. Schwer, sub- BBP (Figure 3, [b]). The ATPases UAP56 and Prp5 (Table mitted; Laggerbauer et al., in press; J. Wagner et al., 1) are required at this step. UAP56, a DECD box protein, submitted; Y. Wang and C. Guthrie, submitted), how do interacts in vitro with U2AF65 (Fleckner et al., 1997). Sig- they identify their specific targets? The binding of UAP56 nificantly, U2AF65 interacts with the spliceosome tran- to U2AF65 may serve as a paradigm in this respect (Figure siently (Bennett et al., 1992). Thus, U2AF65 could confer 6, see above); that is, the timing of certain DExD/H box temporal specificity to the function of this putative un- proteins may be established by the transient binding windase, in addition to the positional specificity man- of specific cofactors. Most yeast DExD/H box factors dated by targeting UAP56 near the branch point. The are not integral snRNP components. Prp16 and Prp2, substrate for UAP56 is not known but, given its proximity for example, interact transiently with the spliceosome to the branch point, an intriguing possibility is SF1, the (Schwer and Guthrie, 1991; Kim and Lin, 1996). Thus, mammalian ortholog to BBP (Berglund et al., 1997), as suggested for UAP56, the timing of these factors which must be displaced from the branch point region may be controlled by their physical association with to allow U2 binding (Figure 3, [b]). Although both UAP56 and Prp5 (see below) are required for the association the spliceosome via domains unique to each protein. of U2 snRNP with the spliceosome, the best candidate Indeed, in the case of Prp16, its unique N-terminal do- for a yeast ortholog of UAP56—with 61% identity—is main has been shown to be responsible for its transient Sub2 (Table 1), a suppressor of the cold-sensitive binding to the spliceosome (Y. Wang and C. Guthrie, snRNP biogenesis mutant brr1–1 (Noble, 1995). submitted). Specialized binding domains may also serve Interestingly, Prp5 facilitates an ATP-dependent con- to direct DExD/H box proteins to their specific target formational rearrangement in U2 snRNA that results in by delivering them in close proximity. increased sensitivity of the branch point recognition re- Do these domains bind protein or RNA components gion to targeted cleavage by RNase H (O’Day et al., of the spliceosome? Intriguingly, the N terminus of Prp22 1996). Indeed, the RNA-dependent ATPase activity of contains a conserved region similar to the RNA-binding Prp5 is preferentially stimulated by U2 snRNA, consis- domain of the ribosomal protein S1 (Company et al., tent with a model in which Prp5 facilitates the binding 1991; Bycroft et al., 1997). The human ortholog of Prp22, of U2 snRNA to the pre-mRNA by exposing the branch HRH1 (Ono et al., 1994), also contains this RNA-binding point recognition sequence. Since the branch point rec- domain as well as an RS domain (Table 1, Figure 5A) ognition sequence is not predicted to be base paired that may also contribute to binding the spliceosome (Fu, or otherwise structured in the U2 snRNP, however, what 1995; Will and Lu¨ hrmann, 1997). In contrast, the binding is the target? It is notable that mutations in the U2 snRNP of Prp2 to the spliceosome requires prior binding of protein Prp9 and in the stem of U2 stem/loop IIa (Figure another soluble factor, Spp2 (Roy et al., 1995), which 3, [c]) also increase the accessibility of the branch point may control the timing of Prp2 action by binding the recognition sequence (Wiest et al., 1996). Thus, Prp5 spliceosome at a unique step. could displace Prp9, unwind this U2 stem, or both. Antagonizing the Role of a DExD/H Box Protein. The 5Ј Splice Site Recognition: A role for Prp28? The de- DExD/H box protein Brr2 has been implicated in a critical stabilization of U1, which is required to form the U6:5Ј RNA rearrangement—the unwinding of U4/U6 (Raghu- splice-site interaction (Figure 3, [a]), requires ATP (Fig- nathan, 1997). Since Brr2 is an integral snRNP compo- ure 2). Hyperstabilizing the duplex between U1 snRNA nent, however, transient binding will not explain its tim- and the 5Ј splice site through increased base pairing ing or specificity. results in a cold-sensitive, splicing defect (J. P. S. and Although the unwinding of the U4/U6 duplex is a criti- C. G., unpublished data). Indeed, many snRNA muta- cal step in assembly (Moore et al., 1993), surprisingly, tions that hyperstabilize an RNA duplex confer cold the ATP-dependent dissociation of U4 and U6 snRNPs Cell 322

also occurs in the absence of assembly, in the context are distinguished from weak incorrect ones (i.e., “near- of the U4/U6·U5 triple snRNP (Figure 2; Raghunathan cognate”) by an irreversible step that discriminates and Guthrie, 1998). Indeed, a triple snRNP-containing against interactions that rapidly dissociate; these “dis- particle immunopurified with antibodies against Brr2 un- card” pathways are governed by the rate of NTP hydroly- winds triple snRNP-bound U4/U6 in an ATP-dependent sis (Burgess and Guthrie, 1993; cf. Rodnina et al., 1995). fashion (Raghunathan, 1997). Significantly, the brr2–1 The spliceosome may employ a similar fidelity-enhanc- mutation, found in the first of two DExD/H box domains, ing mechanism (Burgess and Guthrie, 1993). Mutations prevents this unwinding. Furthermore, the mammalian in PRP16 broaden the specificity of branch point selec- ortholog U5–200 kDa unwinds a synthetic RNA duplex tion, decreasing fidelity. Invariably,these mutations map (Laggerbauer et al., in press). Still, the direct target for to the DEAH-box domain and result in significantly Brr2 remains to be identified, and proof is lacking that slower rates of RNA-dependent ATP hydrolysis, sug- Brr2 directs unwinding of U4/U6 during assembly of the gesting that a slower Prp16 “timer” allows for splicing of spliceosome. defective substrates, which bypass a discard pathway. Interestingly, other alleles of Brr2 have been isolated Indeed, whereas wild-type cells contain only low levels with distinct phenotypes. For example, the slt22–1 allele of lariat intermediates with mutant branch points, prp16 (Xu et al., 1996), like brr2–1 (Noble and Guthrie, 1996), suppressor alleles increase the levels of the aberrant is defective for the first transesterification reaction but, lariat intermediate presumably by increasing the interval unlike the brr2–1 allele (Raghunathan, 1997), is defective before the timer goes off. Accordingly, the outcome of after the unwinding of U4/U6 and release of U4 during this kinetic proofreading pathway is determined by the assembly (Figure 2). Thus, this protein may act at several relative order of two competing events: if a mutant points in the splicing pathway. Multiple functions may branch point slows a necessary step, then ATP hydroly- reflect the activity of two DExD/H box domains in this sis by PRP16 activates a discard pathway; if the step large protein. Alternatively, the two domains may func- occurs efficiently, then PRP16 hydrolysis facilitates the tion like a dimer to confer processivity, as for the oligo- second transesterification. meric DNA helicases (Lohman and Bjornson, 1996). A strong prediction of this model is that other spliceo- As an integral snRNP protein, how is the function somal DExD/H box proteins will govern similar discard of Brr2 timed—especially considering its activity in the pathways, such that the accuracy of splicing is deter- absence of pre-mRNA? Notably, the U4 and U6 snRNPs mined by the cumulative success of transits along the generated by the Brr2-dependent reactions can be effi- productive branches of the pathways. Given the larger ciently “re-wound” by recombinant Prp24 (Raghunathan number of introns and the lower conservation of intron and Guthrie, 1998). Thus, the release of U4 during sequences in mammals (Moore et al., 1993), error reduc- spliceosome assembly may reflect Brr2 function in the tion may be crucial in mammals. Since a mammalian absence of Prp24 function. Dynamic interconversion be- ortholog of Prp16 has recently been identified (Table 1; tween duplex and free U4/U6 before assembly mayallow Z. Zhou and R. Reed, personal communication) a role for rapidregulation of spliceosome assembly; e.g., splic- for Prp16 in maintaining fidelity in mammalian splicing ing is halted in heat shock via inactivation of a U4/U6·U5 can now be tested. triple snRNP factor (Utans et al., 1992). A Ribosomal-like Translocation in the Spliceosome? It is important to note that Prp24 appears to be essen- Interestingly, the newly identified U5 snRNP component tial for splicing in vitro only under conditions where multi- U5–116 kDa (Fabrizio et al., 1997), or Snu114 in S. cere- ple rounds of assembly and disassembly are required visiae, is highly similar in sequence to the ribosomal (Raghunathan and Guthrie, 1998). By this criterion, the GTPase EF-2, or in prokaryotes EF-G, which affects protein can be considered a recycling factor. Given translocation of the ribosome. U5–116 kDa cross-links the number of rearrangements that must ensue before specifically to GTP in an RNA-stimulated fashion (Fa- the spliceosome is reconfigured for a subsequent round brizio et al., 1997) similar to the RNA-stimulated ATPase (Figures 2 and 3), it seems likely that additional recycling activity of some DExD/H box proteins, suggesting that factors remain to be identified. In this regard, it will be RNA is a target of U5–116 kDa. Surprisingly, the se- of interest to determine the rearrangements promoted quence similarity with EF-2 includes domains recently after the ligation of exons by Prp22 (Company et al., proposed to structurally mimic tRNA bound to EF-1 1991) and Prp43 (Arenas and Abelson, 1997) or their (Abel and Jurnak, 1996). According to the “molecular mammalian orthologs HRH1 (Ohno and Shimura, 1996) mimicry” hypothesis, after GTP hydrolysis releases the and mDEAH9 (Gee et al., 1997), respectively (Table 1; EF-2 spring, the tRNA-like domain displaces the newly Figure 2). formed peptidyl tRNA from the A site to the P site. Proofreading in the Spliceosome? Despite the ab- It is tempting tospeculate that thespliceosomal GTPase sence of quantitative data on the accuracy of splicing, U5–116 kDa may function in an analogous fashion, it is axiomatic that the spliceosome must have evolved namely, in the rearrangement of the substrate with re- mechanisms to constrain the frequency of errors, given spect to the catalytic core. As described above, the the meager intronic consensus sequences. It is thought conserved loop of U5, which interacts with the 5Ј exon, that the protein and DNA synthesis machineries face a undergoes a rearrangement betweenthe two transester- common problem in the need to enhance fidelity beyond ification reactions in the two-catalytic site model (Figure that allowed by the modest differences in binding energy 4B). Intriguingly, this interaction may be analogous to between correct and incorrect interactions. In kinetic the interaction of a tRNA anticodon loop with its codon proofreading pathways, originally proposed on theoreti- (Figure 7). Perhaps U5–116 kDa functions by bumping cal grounds, strong correct interactions (i.e., “cognate”) the U5 loop from one catalytic site to the other and Review: Spliceosome Mechanics 323

work. For example, how is ATP hydrolysis used to unwind RNA? The recent crystal structures for the highly similar superfamily I DNA helicases PcrA (Subramanya et al., 1996) and Rep (Korolev et al., 1997a) and the superfamily II DECH box HCV RNA unwindase from hepatitis C virus (Yao et al., 1997) suggest possible mechanisms for coupling chemical energy and work (Marians, 1997). Despite poor sequence conservation between ATPase superfamily I and superfamily II members (Gorbalenya and Koonin, 1993), the structures of Rep and the HCV RNA unwindase are remarkably similar (Figure 8; Koro- lev et al., 1997b). Both contain an NTP-binding domain, named 1A, and a structurally similar domain, 2A. Inter- Figure 7. A Parallel between the Spliceosome and the Ribosome? estingly, these domains are each strikingly similar to a The binding of a yeast Phe codon by the anticodon loop of the domain in RecA (Subramanya et al., 1996), a key player cognate tRNA is compared with the binding of a 5Ј exon by the in homologous recombination. The conserved motifs for yeast U5 loop in a hypothetical, yet provocative, configuration. N, Rep and the HCV RNA unwindase line the cleft formed any nucleotide. by the interface of domains 1A and 2A. As predicted, motif I binds the phosphates of ADP. The DE of motif II consequently repositioning the 5Ј exon for the second (Figure 5B) likely plays a role analogous to D144 and E96 transesterification. in RecA—namely, binding magnesium and activating water for hydrolysis, respectively. What Makes the Clock Tick and the Wheels Turn? The mechanism for unwinding likely employs differen- Ultimately, to understand the roles for NTP hydrolysis tial affinity for single- and double-stranded RNA modu- in the spliceosomal RNA rearrangements, we will need lated by ATP or ADP, as observed for eIF-4A (Lorsch to determine how this chemical energy is converted to and Herschlag, 1998). The cocrystal structure of Rep

Figure 8. The Conserved Motifs of the ATPase Superfamilies I and II Are Structurally Similar and Form a Network that Links the ATP-Binding Site to a Nucleic Acid–Binding Site (A) HCV RNA unwindase, of superfamily II (Yao et al., 1997). (B) Rep DNA helicase, of superfamily I (Korolev et al., 1997a). The binding of ADP and ssDNA (yellow) in the cleft of Rep is shown. The unwindase motifs are color coded in each structure to highlight their structural similarities (Korolev et al., 1997b). The motifs are designated in the context of the primary sequence below each structure. Motif III is abbreviated in the HCV RNA unwindase; see note in Figure 5 legend and Korolev et al. (1997b) for an explanation of motifs IV and IVa. This figure was generously contributed by S. Korolev, P. Weber, and G. Waksman. Cell 324

reveals that motifs Ia, TxGx, III, and V interact with uncoupled from one another both mechanistically and ssDNA (Figure 8B; Korolev et al., 1997a) and suggests a temporally. mechanism for coupling hydrolysis to unwinding. Since A major goal for the future is identifying whether the motif III, which is implicated in unwinding, is sandwiched ligands and targets of each of the unwinding and “re- between I and II, the binding of the ␥-phosphate can be winding” proteins are RNA, protein, or RNP. This distinc- structurally linked to the binding of nucleic acid by a tion will be critical for understanding the various mecha- network of interactions bridging domains 1A and 2A. nistic strategies used by these molecular gadgets, be Given the greater sequence conservation between the they motors, clocks, or springs. In addition to specific HCV RNA unwindase and the DExD/H box proteins than information about any given rearrangement, these data between the HCV RNA unwindase and Rep (Figure 5B), will inform the exciting long-term goal of understanding the spliceosomal DExD/H box proteins will likely share the design principles underlying the conserved order of structural and mechanistic parallels with the HCV RNA these rearrangements, the mechanisms that ensure this unwindase as well as Rep and PcrA. order, and the role this order plays in achieving accurate A major feature revealed by the Rep structure is that splicing. domain 2B can swivel 130Њ (Figure 8B; Korolev et al., 1997a). It has been speculated that rearrangement of this domain is coupled to translocation and confers the Acknowledgments processivity observed for DNA helicases. The minimal, We thank J. Thorner, J. Lorsch, and D. Herschlag for provocative functional fragment of the HCV RNA unwindase and the discussions; J. Abelson, R. Lu¨ hrmann, T. Nilsen, and R. Reed for small eIF-4A, however, do not contain such a domain, preprints; and G. Chanfreau, C. Collins, A. Frankel, A. Kistler, K. demonstrating that domain 2B cannot be required for Lynch, S. Rader, B. Raumann, D. Ryan, C. Siebel, J. Wagner, Y. unwinding activity. In point of fact, processivity is not a Wang, J. Wilhelm, and members of the Guthrie laboratory for critical function likely to be required for the splicing DExD/H reading of the manuscript. We are indebted to S. Korolev and G. Waksman (Washington University School of Medicine) and P. Weber box proteins; the step size of unwinding for the Rep- (Schering-Plough Research Institute) for contributing Figure 8. We related DNA helicase UvrD is five base pairs (Ali and apologize to our colleagues for work that could not be cited due to Lohman, 1997), which would suffice for most of the a limitation on references. Cited work from this laboratory is sup- spliceosomal helices (Figure 3, [a–f]). Still, swiveling of ported by NIH grant GM21119 to C. G. J. P. S. is supported by a the N- or C-terminal domains of the spliceosomal un- California Division-American Cancer Society Fellowship #1–59–97B. windases could result in a “power stroke” that dis- C. G. is an American Cancer Society Research Professor of Molecu- lar Genetics. places a protein, perhaps the DExD/H box protein itself, from RNA. Taken in the extreme, there is likely to be a unifying References theme in the basic function of all NTP-dependent “mo- Abel, K., and Jurnak, F. (1996). A complex profile of protein elonga- lecular gadgets.” The richness of molecular detail re- tion: translating chemical energy into molecular movement. Struc- vealed by GTPase structures (Sprang, 1997) suggests ture 4, 229–238. that a good mechanical analogy for the mechanism of Abovich, N., Liao, X.C., and Rosbash, M. (1994). The yeast MUD2 these proteins is a spring. 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Notes Added in Proof

The data referred to throughout as Korolev et al., 1997b, is now Korolev et al., 1998.

Long et al. (1997) have identified a weak consensus of adenosines at positions Ϫ2, Ϫ3, and Ϫ4 of the 5Ј exon in S. cerevisiae, support- ing base pairing between the 5Ј exon and the U5 loop: Long, M., de Souza, S.J., and Gilbert, W. (1997). The yeast splice site revisited: new exon consensus from genomic analysis. Cell 91, 739–740.