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222 Structure and assembly of the spliceosomal small nuclear ribonucleoprotein particles Christian Kambach*, Stefan Walke† and Kiyoshi Nagai‡ The spliceosome is a macromolecular assembly that carries out the excision of introns from nuclear pre-mRNAs. It consists of four large RNA–protein complexes, called the U1, U2, U4/U6 and U5 small nuclear ribonucleoproteins (snRNPs), and many protein factors. Crystal structures of seven protein components and fragments of the U1 and U2 small nuclear RNAs have been determined in the form of RNA–protein and protein–protein complexes. Together with electron microscopy studies of the snRNPs, these structures have begun to provide important insights into the architecture of the snRNPs and the mechanisms of RNA–protein and protein–protein recognition. Addresses Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 2QH, UK *e-mail: [email protected] † e-mail: [email protected] ‡ e-mail: [email protected] Correspondence: Kiyoshi Nagai Current Opinion in Structural Biology 1999, 9:222–230 http://biomednet.com/elecref/0959440X00900222 © Elsevier Science Ltd ISSN 0959-440X Abbreviations EF elongation factor LRR leucine-rich repeat 2,2,7-trimethylguanosine m3G N7-monomethylguanosine m7G rmsd root mean square deviation RNP ribonucleoprotein RRM RNA recognition motif snRNA small nuclear RNA snRNP small nuclear RNP particle Introduction Most eukaryotic genes contain noncoding intervening sequences (introns) that have to be removed from the primary mRNA transcript prior to translation into protein. In the nucleus, introns are excised by two successive transesterification reactions within a macromolecular assembly called the spliceosome [1–4]. In the first step, the 5′ splice site is attacked by the 2′ hydroxyl group of a conserved adenosine at a position known as the branch point within the intron, such that the 5′ exon is cleaved off and the 5′ end of the intron is ligated to the 2′ hydroxyl group of the branch point adenosine, resulting in a circular lariat intron intermediate. In the second step, the 3′ hydroxyl group of the 5′ exon attacks the phosphodiester bond at the 3′ intron–exon junction, resulting in the ligation of the two exons and liberation of the intron [1–4]. The major components of the spliceosome are four RNA–protein complexes, the U1, U2, U4/U6 and U5 snRNPs (small nuclear ribonucleoprotein particles). The snRNPs are named after their RNA components. For example, the U1 snRNP contains U1 small nuclear RNA (snRNA). The U4 and U6 snRNAs are found extensively base paired in a single particle (U4/U6 snRNP). These snRNPs assemble onto the pre-mRNA through an ordered pathway [1,2]. In contrast to group II self-splicing introns, which are excised by an analogous two-step trans-esterification reaction through the folding of the well-conserved intron sequences [5], nuclear pre-mRNA introns contain only short conserved sequences at the 5′ and 3′ splice sites and at the branch point (followed by the polypyrimidine tract in metazoan introns) and thus require trans-acting factors in order to splice [1–4]. The U1 and U2 snRNPs bind to the 5′ splice site and the branch point of the pre-mRNA, respectively, and a pre-assembled U4/U6•U5 tri-snRNP then joins the complex. Genetic and biochemical experiments have revealed an intricate network of interactions between pre-mRNA and snRNAs, and between the snRNAs, that undergoes an extensive rearrangement during the course of the splicing reaction. In the spliceosome, the extensive base pairing between the U4 and U6 snRNAs is unwound and the U6 snRNA subsequently base pairs with both U2 snRNA and the 5′ splice site [1–4,6]. A highly conserved loop in the U5 snRNA interacts with the exon sequences at the 5′ and 3′ splice sites and these interactions are important for the second trans-esterification step [7–9]. Thus, nuclear pre-mRNA splicing is a highly dynamic process and protein components play important regulatory roles in the assembly of the snRNPs and the rearrangement of the network of RNA–RNA interactions [2,10,11]. Spliceosomes sediment at 50–60S, corresponding to an approximate molecular weight of 4.8 MDa [12]. This indicates a complexity that is comparable to the ribosome and, to date, some 80–100 protein factors have been shown to be involved in metazoan splicing [10,11,13–15]. Spliceosomal proteins can be divided into those that are tightly associated with snRNPs and the non-snRNP splicing factors (for reviews, see [2,10,11,16–18]. Based on functionality and sequence similarities to known proteins, many spliceosomal proteins have been classified as being ATPases, helicases, protein kinases, GTPases or peptidyl-prolyl cis/trans isomerases and are often related to members of their respective class with known structures [2,4,11]. This suggests that these proteins may be involved in the regulation of the spliceosomal assembly. In fact, the splicing reaction is inhibited by the addition of phosphatase inhibitors or nonhydrolysable ATP analogues [19,20]. Proteins containing helicase motifs are likely to be involved in the rearrangement of the RNA–RNA interaction network [2,4,10,11]. GTPases and peptidyl-prolyl cis/trans isomerases may take part in the Structure and assembly of the spliceosomal small nuclear ribonucleoprotein particles Kambach, Walke and Nagai 223 Figure 1 Structure and assembly of the Sm proteins. (a) Crystal structure of the D3 protein (front view), with the hydrogen-bond network involving Tyr62 and the highly conserved residues Glu36, Asn40, Arg64 and Gly65 shown. The structure is shown in a ribbon representation, with helix A in red, β strands 1, 2 and 3 (blue) are made from residues within the Sm1 motif and β strands 4 and 5 (yellow) are made from residues within the Sm2 motif. (b) The D3 protein (side view), showing the heavily bent strands β2, β3 and β4. Color scheme as in (a). (c) Crystal structure of the D3 (gold) and B (blue) protein dimer. (d) A ribbon model of the Sm protein assembly in the core snRNP domain. (e) A surface representation of the Sm protein assembly, with the electrostatic surface potential shown (blue, positive; red, negative). Reproduced with permission from [32••]. conformational changes that occur within the spliceosome [10]. Protein sequence motifs found in the spliceosomal proteins include the ribonucleoprotein (RNP) motif or RNA recognition motif (RRM), the Sm motif, the GTPase motif, zinc fingers, leucine-rich repeats (LLRs), K homology (KH) domains, doublestranded RNA binding domains (dsRBDs), the DEAD (DEAH) box, the RGG box and the WD repeat. For a comprehensive listing of the motifs found in spliceosomal proteins, see Burge et al. [2]. The discovery of these motifs provoked interesting speculation concerning the origin and evolution of the splicing machinery. detail, both genetically and biochemically [1–4,7–11,16,17]; however, the gap between our current understanding at the biochemical level and our knowledge of the underlying structural requirements at a molecular level has yet to be closed. The recent crystal structure determination of the catalytic core of a group I self-splicing intron illustrates the power of structural analysis in understanding catalytic mechanisms [21,22••]. Of the many components of the nuclear pre-mRNA splicing machinery, the crystal structures of seven snRNP-associated proteins have been determined, three of those as complexes with their snRNA targets. These structures have given considerable insight into the molecular mechanisms of RNA–protein and protein–protein recognition between the spliceosomal components. This review will concentrate on illustrating how this knowledge will help us to understand snRNP assembly and architecture. The multiple RNA–RNA, RNA–protein and protein–protein interactions that are essential for the fidelity and efficiency of the splicing reaction have been studied in great 224 Macromolecular assemblages The core small nuclear ribonucleoprotein domain U snRNPs contain two classes of proteins: those specific to a given snRNP and those that are common to the U1, U2, U4 and U5 snRNPs [2,10]. The latter are called core or Sm proteins and they assemble on the snRNAs into a globular structure called the core snRNP domain. The Sm-proteinbinding site (the Sm site) is a short, conserved uridine-rich sequence present in the U1, U2, U4 and U5 snRNAs. Eight generic Sm proteins have been identified in snRNPs purified from a HeLa cell nuclear extract [17]. They are named, in order of decreasing size, B′/B, D3, D2, D1, E, F and G. The B and B′ proteins arise from a single gene by alternative splicing and differ only in 11 residues at their C termini [23,24]. These Sm proteins contain a conserved sequence motif in two segments, Sm1 and Sm2, which are connected by a linker of variable length [25–27]. The Sm motif is related to no known protein sequence motif and, hence, these proteins form a distinct protein family. Core domain assembly is marked by several distinct intermediates. In the absence of snRNA, the Sm proteins exist as three subcomplexes, D1D2, D3B (or D3B′) and EFG. The EFG subcomplex binds, together with the D1D2 subcomplex, to the U snRNA to form the stable subcore domain, which is then joined by the D3B (or D3B′) heterodimer to complete core domain assembly [28]. Neither the individual Sm proteins nor individual Sm subcomplexes bind to snRNA. Core domain formation is an essential step in U snRNP biogenesis and occurs in the cytoplasm after the nuclear export of newly transcribed U snRNAs containing the N7-monomethylguanosine (m7G) cap [29]. Core assembly triggers hypermethylation of the m7G cap to a 2,2,7-trimethylguanosine (m3G) cap structure. The core domain and the m3G cap act as a bipartite nuclear import signal and the pre-snRNP matures in the nucleus by association with specific proteins. The nuclear import of the U4 and U5 snRNPs depends less on the presence of the m3G cap than the U1 and U2 snRNPs [30,31••]. Recently, the crystal structures of two Sm protein subcomplexes, D1D2 and D3B, have been solved [32••]. The four Sm proteins show a common fold containing a short, N-terminal α helix followed by a five-stranded, antiparallel β sheet (Figure 1a, b). Strands 1–3 of the β sheet are made from residues within the Sm1 motif, the linker of variable length between the two motifs forms a connecting loop and Sm2 motif residues constitute β strands 4 and 5. Strands 2, 3, and 4 are heavily bent. Strand 5 loops back over the bent strands to pair with strand 1. The main interaction interface in both complexes comprises β strand 4 of one partner (D2 or B) pairing with β strand 5 of the other (D1 or D3, respectively), thereby continuing the β sheet throughout the complex (Figure 1c). The D1D2 and D3B subcomplexes reveal a high degree of structural similarity at the level of both the individual protein fold and the dimer architecture: a superposition of the individual Cα backbones atoms within the Sm1 and Sm2 motifs of the two dimers as rigid bodies yields an rmsd of 0.9 Å. The D1D2 and D3B dimer structures show that each Sm protein can have two neighbours: one pairing with its β4 strand and the other pairing with its β5 strand. A model of a higher order structure could be built by adding a monomer one by one using the same subunit interactions. This leads to the conclusion that seven core proteins could form a complete ring [32••]. Figure 2 (a) (b) U1 70K protein m3G cap SF3b protein complex U1A protein U1 snRNA Core domain U1 snRNP m3G cap (c) (d) SF3a protein m3G cap complex U2B′′–U2A′ protein complex Core domain U2 snRNA U2 snRNP U6 snRNA 3′ end Base paired U4/U6 snRNAs Loop 1 Core domain m G cap U4/U6 snRNP 20S U5 snRNP U5 snRNA 3 U4/U6.U5 snRNP Current Opinion in Structural Biology Electron micrographs of negatively stained splicesomal snRNPs with their interpretations. (a) U1snRNP, (b) U2 snRNP, (c) U4/U6 snRNP and (d) U4/U6•U5 tri-snRNP. The electron micrographs were kindly provided by B Kastner. Adpated with permission from [38] Structure and assembly of the spliceosomal small nuclear ribonucleoprotein particles Kambach, Walke and Nagai Co-immunoprecipitation and yeast two-hybrid systems were used to investigate pairwise interactions of the Sm proteins [33•,34•]. Kambach et al. [32••] have been able to arrange all seven Sm proteins within a seven-membered ring (Figure 1d) in a manner that is consistent with all the known pairwise interactions [33•,34•]. The heptameric ring is the only core domain model that is consistent with all the structural, biochemical and genetic data currently available. The inner surface lining the ring bears a high concentration of positive charges (Figure 1e). The central hole (20 Å diameter) is large enough to accommodate the single-stranded RNA of the Sm site. It is therefore probable that the snRNA binds to or is threaded through this hole. The AUU sequence of the Sm site [35] has been shown to cross-link with the G Sm protein by UV light. Core protein binding studies of U1 and U5 snRNAs and their variants showed that, not only the Sm-site sequence, but also the distance between the Sm site and both the flanking stems and the internal loop present in the flanking stem of U5 snRNA can affect the binding of the core protein [36]. This suggests that the flanking stems also interact extensively with the core proteins. Further characterisation of the RNA-binding interface of the core proteins has to await more detailed cross-linking studies and the crystal structure of the assembled complex. The U1, U2, U4/U6 and U5 snRNPs each show a globular domain in negatively stained electron micrographs [37], similar to the appearance of an in vitro reconstituted core domain (Figure 2). This domain remains intact even when the specific proteins are depleted from the snRNPs [37,38]. The overall dimensions of the core domains (about 80 Å in diameter) are in good agreement with the seven subunit model of the proposed core domain [32••]. In addition to the canonical Sm proteins, closely related Smlike proteins have been found in Saccharomyces cerevisiae and man. Two of these proteins (Uss1p and SmX3) have been shown to be associated with U6 snRNA in yeast [25,27]. It is now believed that the U6 snRNP contains a full set of Smlike proteins that form a core-domain-like assembly in the U6 snRNP, both in yeast and in man (J Beggs, B Séraphin, T Achsel, R Lührmann, personal communication). Proteins bearing strong similarity to the Sm proteins have also been found in the archeon Archaeoglobus fulgidus [39]. This shows that ancestral Sm proteins appear early in evolution. Proteins of the Sm family are more widespread than originally thought and may have diverse functions. U1 small nuclear ribonucleoprotein particle Figure 3a shows a schematic representation of the human U1 snRNP [1,3]. The U1 snRNA contains 163 nucleotides with a m3G cap and forms four stem–loops. The ACUUACCU sequence present at the 5´ end is complementary to the conserved sequence (AGGURAGU) at the 5′ splice site of pre-mRNA and plays an important role in binding the U1 snRNP to the 5′ splice site. The Sm site (AUUUGUGG) present in the single-stranded region is the binding site for the core Sm proteins. In addition, human 225 U1 snRNP contains three specific proteins, named U1 70K, U1A and U1C. U1 70K and U1A bind to stem–loops I and II within the U1 snRNA, respectively (Figure 3a). The U1A protein contains two RNP domains (or RRMs) that are linked by a protease-sensitive peptide. The N-terminal RNP domain of U1A is necessary and sufficient for binding to U1 snRNA [40,41]. The crystal structure of the N-terminal fragment of the U1A protein in complex with its U1 snRNA hairpin II binding site provides important insights into RNA recognition by RNP domains [42] (Figure 4a). The RNP domain contains a four-stranded antiparallel β sheet flanked on one side by two α helices [43]. The 10 nucleotide loop of the U1 snRNA hairpin II (stem–loop II) binds on the surface of the β sheet as an open structure. The first seven nucleotides of stem–loop II, AUUGCAC, and the loop-closing C⋅G base pair form an intricate hydrogen-bond network with the sidechains and the mainchain of the U1A protein. These nucleotides show stacking interactions with either an adjacent RNA base or a protein sidechain, or both, that stabilise the hydrogen-bond network with the protein by restricting the orientation of the RNA bases [42]. The U1 70K protein contains a single copy of the RNP motif (or RRM) around residues 100–180, followed by a highly charged C-terminal tail with alternating Arg-(Glu/Asp) and Arg-Ser repeats [44]. A fragment of the U1 70K protein containing the RNP motif is alone capable of binding stem–loop I of U1 snRNA. The N-terminal fragment containing residues 1–97 (with no known sequence motifs) can, however, be incorporated into the core U1 snRNP consisting of the U1 snRNA and the core Sm proteins [45]. The N-terminal fragment of the U1 70K protein preceding the RNP motif is unable to bind U1 snRNA on its own, but it interacts with the core domain through protein–protein interactions. The U1 70K protein can be chemically cross-linked to the B and D2 proteins [45]. The U1C protein binds to the U1 snRNP only in the presence of both the core domain and the U1 70K protein, and does not bind the U1 snRNA on its own. This indicates that the U1C protein probably binds both to the U1 70K protein and to the Sm proteins. The latter contact is corroborated by the observation of a cross-link between U1C and the Sm B protein [46]. U1 snRNPs depleted of the U1C protein fail to bind to pre-mRNA. The U1C protein was proposed to form a noncanonical Cys2-His2-type zinc finger domain near its N terminus. This segment is sufficient to restore the binding of the U1 snRNP to the 5′ splice site [47]. The U1C protein apparently alters the conformation of the 5′ end of U1 snRNA so that it can pair with the 5′ splice site. Electron micrographs of the U1 snRNP show two protuberances emerging from the central globular core snRNP domain [48] (Figure 2a). Antibodies specific to the U1A and U1 70K proteins were used to identify each of the proteins in a single protuberance. The protuberance corresponding to the U1 70K protein was found to be closer to the 226 Macromolecular assemblages Figure 3 U1A protein (a) G U U A C A C C U A 60- U U C G G A U1 70K G protein C G G 50- G A 40 A AG G G U GG U U U U C CC C I A A C C A A G A GG C U AGU U G 20 G 30 A C G G U1C protein 10- U C C A U U C m32, 2, 7 G p p p A U A II C U C C G G A U Core RNP G domain U -80 G C U G 150 A C IV U C C U G C G C C C G U -90 G C 100 G U U CAA CG A U U U C C C C U A III A U G C U A A A G GG U G U C G C A 110 C G C 160 U 120 C 140 G 130 G G C C A U A A U U U G U G G U A G U G U G OH Sm site 5′ss (b) III UU C G G C A U G C I G C AA A UU UA G U U U U U A II 120 A U C G a U 60 U G C U6-I C G-20 G C G C G C A U U A 140 G U AG 50 C G C 10-C A U6-II A U U A U A U2B′′ protein bp G C G A U 2, 2, 7 90 100 110G C 160 C GpppAU CGCUU A AAGUGUAGUAUC U GUUCUU AC UC m3 A AAUAUAUUAAA GGAUUUUUG GAGCAG C U A U UG G 150 30 40 C A U U A GCAUCG CCUGG Sm site C 70 C G IV A C G CGUGGC GGACCU G U A 80 A Core RNP C C AU C G C 180AA COH domain U C 170 A C U IIb SF3b SF3a Schematic representation of the U1 and U2 snRNPs. (a) The interaction between the U1 snRNA and protein components within the U1 snRNP. The Sm proteins (B/B′ D3, D2, D1, E, F and G) assemble around the Sm site, forming the core RNP domain. The U1 70K and U1A proteins bind to stem–loops I and II, respectively. The U1C protein does not bind to U1 snRNA on its own and requires the presence of the U1 70K protein. The nucleotides near the 5′ end of U1 snRNA (5′ss) are known to pair with the conserved sequence at the 5′ splice site of the premRNA. For the crystal structure of U1A bound to hairpin II, see Figure 4a [42]. A model of the core RNP domain is shown in Figure 1e [32••]. (b) The domain structure of the U2 snRNP inferred from biochemical data and electron microscopy. The Sm proteins assemble around the Sm site to form the core RNP domain, as in U1 snRNP (a). The U2B′′–U2A′(LRR) protein complex binds stem–loop IV of U2 snRNA. The crystal structure of this complex is shown in Figure 4b [49•]. The SF3a complex joins the large domain consisting of the core RNP domain and the U2B′′–U2A′(LRR) complex. The SF3b complex is thought form a large domain at the 5′ end of the U2 snRNA. The U6-I and U6-II sequences highlighted are known to pair with U6 snRNA within the spliceosome, after the pairing of U4 and U6 snRNAs is unwound. The branch point (bp) sequence highlighted pairs with the conserved sequence at the branch point within the intron. Adapted with permission from [18]. U2A′ protein Current Opinion in Structural Biology antibody bound to the m3G cap of U1 RNA than that corresponding to the U1A protein [48]. The sizes of the protuberances are consistent with those predicted from the molecular weights of the U1A and U1 70K proteins. U2 small nuclear ribonucleoprotein particle Figure 3b shows a schematic representation of the human U2 snRNP [1,3,17]. The U2 snRNA, containing 187 nucleotides, forms four stem–loop structures. The Sm site and the regions that base pair with the branch point and the U6 snRNA are highlighted. The 12S U2 snRNP purified under high salt conditions contains two U2-specific proteins, the U2B′′ and U2A′ proteins, in addition to the core Sm proteins, whereas the 17S U2 snRNP purified under low salt conditions contains nine additional proteins, including the heteromeric splicing factors SF3a and SF3b [2,10,11]. The role of the U2A′ and U2B′′ proteins in splicing has long remained elusive, but recent evidence from S. cerevisiae indicates that these two proteins are required for the integration of the U2 snRNP into the pre-spliceosome [49••]. The 5′ half of the U2 snRNA is extensively modified (2′-O-methyl groups and pseudouridines). The requirements of these modifications for both the conversion of the 12S U2 snRNP into the (spliceosome assembly competent) 17S U2 snRNP particle and the subsequent splicing activities were addressed by Yu et al. [50••]. The authors found a correlation between the extent of nucleotide modification and U2 snRNP function in splicing and concluded that these modifications within the 27 nucleotides at the 5′ end of the U2 snRNA are essential for the 12S→17S U2 RNP conversion. The crystal structure of the U2B′′–U2A′ protein complex bound to a fragment of U2 snRNA has been determined at 2.4 Å resolution [51••] (Figure 4b). The U2B′′ protein, containing two RNP domains, is closely related to the Structure and assembly of the spliceosomal small nuclear ribonucleoprotein particles Kambach, Walke and Nagai 227 Figure 4 Crystal structure of the U1A protein and the U2B′′–U2A′(LRR) protein complex bound to their cognate RNA hairpins. (a) A surface representation of a complex between the U1A protein (white) and U1 snRNA stem–loop II [42]. Amino acid residues specific to the U1A protein are highlighted in red. (b) A surface representation of the ternary complex between the U2B′′–U2A′(LRR) proteins and a fragment of U2 snRNA stem–loop IV. The U2B′′ protein is in white, with amino acid residues specific to U2B′′ highlighted in yellow. The U2A′(LRR) protein is shown in blue. Reproduced with permission from [51••]. (a) (b) U1A U2B" A11 C12 A11 G12 C10 C10 U2A’ U13 U13 C14 G9 C15 G9 U8 U7 U8 C16 C15 A14 U7 A6 C5•G16 base pair U1A protein [41]. The U1A protein binds to stem–loop II of U1 snRNA on its own, whereas the U2B′′ protein binds to stem–loop IV of U2 snRNA only in complex with the U2A′ protein [41], which is a member of the LRR protein family [52]. A complex between a fragment of the U2B′′ protein containing the N-terminal 96 residues and the U2A′(LRR) protein shows high affinity and specificity for stem–loop IV of U2 snRNA [41]. This U2B′′ fragment differs from the U1A protein in only 25 positions. Stem–loop II of U1 snRNA contains a 10 nucleotide loop, with the AUUGCACUCC sequence closed by a C⋅G base pair, whereas stem–loop IV of U2 snRNA contains an 11 nucleotide loop, with the AUUGCAGUACC sequence closed by a U⋅U base pair [41,51••]. The LRRs in the U2A′(LRR) protein form a solenoid that is similar to, but more irregular than, those found in the porcine ribonuclease inhibitor [52]. Helix A of the U2B′′ protein fits into the concave surface of the parallel β sheet of the LRRs, and the N-terminal and C-terminal arms flanking the LRRs embrace the N-terminal RNP domain of the U2B′′ protein. Scherly et al. [53] showed that two amino acid replacements, Asp24→Glu and Lys28→Arg, in the U1A protein allow it to form a stable complex with the U2A′(LRR) protein. The guanidinium group of Arg28 from the U2B′′ protein protrudes to form hydrogen bonds with Thr69 and Glu92 of the U2A′(LRR) protein. In the crystal structure of the U1A protein in complex with snRNA stem–loop II, the first seven loop nucleotides, AUUGCAC, and the loop-closing C⋅G base pair are recognised by the U1A protein, but the last three loop nucleotide bases are not [42]. In the U2B′′–U2A′(LRR) complex with a fragment of U2 snRNA stem–loop IV, all the loop nucleotides are involved in extensive interactions with the U2B′′ protein. The double-stranded stem of U2 snRNA hairpin IV interacts with the U2A′(LRR) protein and, hence, extends in a different direction from the RNP domain than that of U1 snRNA hairpin II [51••]. The differences between the two RNP proteins (U2B′′ and U1A) and the presence of the U2A′(LRR) protein allow the two A6 U1 RNA U5•U17 base pair cognate complexes to form a distinct network of interactions, even for the first six loop nucleotides that are conserved between the two RNA hairpins. These interactions cannot be formed by the noncognate complexes, resulting in a highly specific complex. Electron micrographs of negatively stained 12S U2 snRNPs show a small domain attached to the core domain that could be identified as being the U2B′′–U2A′ protein complex bound to U2 snRNA hairpin IV [38,54]. In contrast, the larger 17S U2 snRNP particle contains nine additional U2-specific proteins, including the SF3a and SF3b complexes, and, thus, shows a second large domain consisting of SF3b connected to the core domain, with a single-stranded region of U2 snRNA appearing like a filament (Figure 2b) [54]. SF3a associates with the core proteins and the U2B′′–U2A′ protein complex. U4/U6, U5 and tri-(U4/U6•U5) small nuclear ribonucleoproteins The U4/U6 snRNP isolated from a HeLa cell nuclear extract contains U4 and U6 snRNAs, which are extensively base paired, and the core Sm proteins bound to the Sm site of the U4 snRNA. Electron micrographs of the U4/U6 snRNPs show a globular core domain and the Y-shaped filamentous structure of the base paired U4 and U6 snRNAs protruding from the core domain [55] (Figure 2c). Human U5 snRNA contains a long stem–loop structure, with two internal loops followed by a single-stranded region and a short stem–loop structure. The core Sm proteins bind to the Sm site within the single-stranded region. The highly conserved loop I of U5 snRNA plays an important role in aligning the 5′ and 3′ exons for ligation during the second trans-esterification reaction [7,8]. The human 20S U5 snRNP is far more complex than the U1 and U2 snRNPs (Figure 2d). It contains nine U5-specific proteins (220, 200, 116, 110, 102, 100, 52, 40 and 15 kDa), in addition to the core Sm proteins (Figure 2d). The 200 kDa protein contains two 228 Macromolecular assemblages RNA helicase motifs (DEIH and DDAH), whereas the 100 kDa protein contains a single RNA helicase motif (DEAD) [2,4,10]. These proteins have been implicated in the rearrangement of RNA–RNA interactions during the splicing reaction. The 116 kDa protein is required for the second step of splicing and bears high homology to the GTPbinding ribosomal translocase elongation factor (EF)-2 [10]. The 116 kDa protein can be cross-linked to a stable hairpin inserted between the branch point and 3′ splice site and it has been proposed that the 116 kDa protein may be involved in 3′ splice site selection by a scanning mechanism [56••]. Homologues of the 220 kDa (Prp8p), 200 kDa (Snu246p) and 116 kDa (Snu114p) proteins have been identified in yeast. The yeast Prp8 protein and its human counterpart (the 220 kDa protein) can be cross-linked to nucleotides around the 5′ splice site, the branch point and 3′ splice site and are thought to collaborate with the conserved loop I of U5 snRNA in aligning the 5′ exon and the 3′ splice site during the second trans-esterification reaction [57–59]. Dix et al. [60••] cross-linked proteins to U5 snRNA either uniformly or site-specifically labelled with 4-thiouridine, within the reconstituted U5 snRNP and located the RNA contact sites for five yeast proteins. Prp8p is in contact with a wide region of U5 snRNA, including the stem of conserved loop I, whereas Snu114p was cross-linked to a 4-thiouridine that was introduced to the 5′ strand of internal loop I. The U4/U6 and U5 snRNPs associate, together with more than half a dozen tri-snRNP-specific proteins, to form a trisnRNP complex (Figure 2d) [2,4,10]. This complex joins the U1 and U2 snRNPs assembled on the pre-mRNA. Conclusions As the discovery of introns was made only 20 years ago, our understanding of the molecular mechanism of nuclear premRNA splicing has advanced remarkably within the relatively short history of its research. The methods developed to study the ribosome, such as RNA footprinting, cross-linking and immunoelectron microscopy, were applied to and have facilitated the investigation of the splicing machinery. The number of protein components involved in pre-mRNA splicing is far greater than in the ribosome and the splicing process is highly dynamic, involving the assembly, rearrangement and disassembly of many components. Thus, the splicing machinery is less accessible to crystallographic analysis. Neubauer et al. [61••] purified spliceosomes that were fully assembled around a biotinylated, 32P-labelled pre-mRNA substrate and rapidly characterised their protein components using mass spectrometry and an EST (expressed sequence tag) database search after they had been isolated from two-dimensional electrophoresis gels. This new method, together with the complete yeast genome sequence now available, will greatly facilitate the further isolation and characterisation of the components involved in splicing. Fromont-Racine et al. [62•] characterised a network of protein–protein interactions within yeast spliceosomal snRNPs using exhaustive two-hybrid screening. This will complement the biochemical investigation of protein–protein interactions within the splicing machinery. A detailed structural knowledge of the components of the splicing machinery, as well as their interactions, are essential to understanding the architecture of the snRNPs and their assembly. Crystallisation and X-ray analyses of either the snRNPs or their large domains may be possible. The recent progress in ribosome crystallography, providing the first electron density map of a 50S ribosomal subunit at a resolution higher than 10 Å, is extremely encouraging ([63••]; see the review by Agrawal and Frank, this issue, pp 215–221). The vast amount of biochemical data and the 15 high-resolution structures of ribosomal proteins [64] will greatly facilitate the initial interpretation of the electron density map. Crystallographic data at high resolution will eventually provide details of its architecture at or near atomic resolution. The interactions between the ribosome and many essential factors, such as tRNA, mRNA, EF-Tu and EF-G, are being studied in low-resolution maps obtained using (cryo) electron microscopy ([64]; see the review by Agrawal and Frank, this issue, pp 215–221). This hybrid approach has proved to be extremely powerful, creating working models of the ribosome at increasingly higher resolution. Understanding the splicing process requires the structural knowledge of many intermediate states. Such intermediate states may be isolated using mutant substrate pre-mRNAs or mutant protein factors that allow the accumulation of intermediate species. Biochemical heterogeneity or the inherent flexibility of such complexes might preclude single-particle cryoelectron microscopy at high resolution, but even low-resolution structures, together with the crystal structures of their constituents, will provide valuable information. Acknowledgements The authors thank Andy Newman, Chris Oubridge, Richard Bayliss and Phil Evans for critical reading of the manuscript, Berthold Kastner for allowing us to include his electron micrographs and present and past members of the group for their contributions to the project. The work was supported by the Medical Research Council and Human Frontier Science Program. CK was supported by NATO and EU fellowships, and SW by Boehringer Ingelheim and EU Marie Curie studentships. References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest •• of outstanding interest 1. 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