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 59 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 ªparts listº 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 LuÈ 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- ªRNA worldº (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 ªDExD/H boxº 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 29 hydroxyl of a conserved, proofreading steps of splicing. In addition, we will de- intronic adenosine attacks the phosphate at the 59 splice scribe a spliceosomal analog of EF-2, a ribosomal GTPase site, producing a free 59 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 39 hydroxyl of the 59 exon attacks the phosphate at proteins, ªthingsº that promote RNA rearrangements by the 39 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 LuÈ 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 re- veals 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 transesterifi- cation reactants. The intron is shown as a line with S. cerevisiae highly coordinated and carefully regulated fashion. The consensus sequences. 59 splice site is initially recognized through a base-pair- ing 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.