Structure and assembly of the spliceosomal small nuclear ribonucleoprotein particles

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Structure and assembly of the spliceosomal nuclear ribonucleoprotein particles

small

Christian Kambach*, Stefan Walket and Kiyoshi Nagait 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 Ul , U2, U4/U6 and U5 small nuclear ribonucleoproteins (snRNPs), and many protein factors. Crystal structures of seven protein components and fragments of the Ul 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 Cambridge, CB2 2QH, UK *e-mail: ckl @mrc-lmb.cam.ac.uk te-mail: [email protected] Ie-mail: [email protected] Correspondence: Kiyoshi Nagai

Current Opinion

in Structural

Biology

of Molecular

Biology,

Hills Road,

1999,9:222-230

http:Ilbiomednet.comlelecref~O95944OXOO900222 0 Elsevier

Science

Ltd ISSN

0959-440X

Abbreviations EF elongation factor LRR leucine-rich repeat 2,2,7-trimethylguanosine m3G NT-monomethylguanosine m7G rmsd root mean square deviation RNP ribonucleoprotein RRM RNA recognition motif snRNA small nuclear RNA small nuclear RNP particle snRNP

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 [l--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 [l--4]. The major components of the spliceosome are four RNA-protein complexes, the Ill, UZ, LJ4/176 and U5 snRNPs (small nuclear ribonucleoprotein particles). The

snRNPs are named after their RNA components. For example, the Ul snRNP contains Ul small nuclear RNA (snRNA). The U4 and L6 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 [l--4]. The Ul and U2 snRNPs bind to the 5’ splice site and the branch point of the pre-mRNA, respectively, and a pre-assembled U4/U6*US 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 [l-4,6]. A highly conserved loop in the US snRNA interacts with the exon sequences at the 5’ and 3’ splice sites and these interactions are important for the second trans-esterification step [7-91. 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 SO-6OS, 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 [10,11,13-151. Spliceosomal proteins can be divided into those that are tightly associated with snRNPs and the non-snRNP splicing factors (for reviews, see [2,10,11,16-181. 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 ris/traw isomerases may take part in the

Structure

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1

Structure and assembly of the Sm proteins. (a) Crystal structure of the Ds 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, p strands 1, 2 and 3 (blue) are made from residues within the Sml motif and p strands 4 and 5 (yellow) are made from residues within the Sm2

motif. (b) The D, protein (side view), showing the heavily bent strands p2, p3 and p4. Color scheme as in (a). (d Crystal structure of the Ds (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 [lo]. 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. [Z]. The discovery of these motifs provoked interesting speculation concerning the origin and evolution of the splicing machinery.

detail, both genetically and biochemically [14,7-l 1,16,17]; 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

The core small domain

assemblages

nuclear

ribonucleoprotein

U snRNPs contain two classes of proteins: those specific to a given snRNP and those that are common to the LJl, U2. ZJ4 and US snRNPs [&lo]. 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 LIl. U2, 114 and LJ.5 snRNAs. Eight generic Sm proteins have been identified in snRKPs purified from a HeLa cell nuclear extract 1171. They are named, in order of decreasing size, B’/B, D,, D,, D,, 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, Sml and Sm2, which are connected by a linker of variable length [25-271. The Sm motif is related to no known protein sequence motif and, hence, these proteins form a distinct protein family.

to a 2,2.7-trimethylguanosine (mjG) cap structure. The core domain and the miG 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 L4 and US snRNPs depends less on the presence of the m,G cap than the Ul and 112 snRNPs [X),31”]. Recently, the crystal structures of two Sm protein subcomplexes. D,D, and D,B, have been solved [32”]. ‘I’he four Sm proteins show a common fold containing a short, N-terminal a helix followed by a five-stranded, antiparallel p sheet (Figure la, 1,). Strands l-3 of the p sheet are made from residues within the Sml motif, the linker of variable length between the two motifs forms a connecting loop and Sm2 motif residues constitute p 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 inter&e in both complexes comprises fi strand 4 of one partner (D2 or B) pairing with p strand 5 of the other

Core domain assembly is marked by several distinct intermediates. In the absence of snRNA, the Sm proteins exist as three subcomplexes, DID,, D3B (or D,B’) and EFG. The EFG subcomplex binds, together with the D,D, subcomplex, to the lJ snRNA to form the stable subcore domain, which is then joined by the D,B (or D,B’) 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 IT snRNAs containing the NT-monomethylguanosine (m7G) cap [29]. Core assembly triggers hypermethylation of the m7G cap Figure

(D,

or &,

respectively),

thereby

sheet throughout the complex (Figure D,B subcomplexes reveal a high degree ilarity at the level of both the individual the dimer architecture: a superposition Ca backbones atoms within the Sml the two dimers as rigid bodies yields an D,D2 and D,B dimer structures show

continuing

lc). The

the

DID,

of structural

p

and sim-

protein fold and of the individual and SmZ motifs of rmsd of 0.9 ‘4. The that each Sm protein can have two neighhours: one pairing with its 04 strand and the other pairing with its pS 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 co the conclusion that seven core proteins could form a complete ring [.32”].

2

r

(a)

W

Cd)

U6 snRNA

3’end 20s

U5 snRNP

U5 snRNA I Ul snRNP

U4/U6

U2 snRNP

snRNP

U4/U6-U6

snRNP

current opinion I” structural sloiogy j Electron micrographs of negatively stained splicesomal snRNPs (d) U4/U6*U5 tri-snRNP. The electron micrographs were kindly

with their interpretations. provided by B Kastner.

(a) UlsnRNP, (b) U2 snRNP, (c) U4/U6 Adpated with permission from [36]

snRNP

and

Structure

and

assembly

of the spliceosomal

small

Co-immunoprecipitation and yeast two-hybrid systems were used to investigate pairwise interactions of the Sm proteins [33’,34’]. Kambach ef al. [32”] have been able to arrange all seven Sm proteins within a seven-membered ring (Figure Id) 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 te). The central hole (20 w diameter) is large enough to accommodate the single-stranded RNA of rhe 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 LJl and I!5 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 IT5 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 LJl, UZ, 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 A 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 Sucdvzromyes cereuisiue and man. Two of these proteins (Lisslp and SmX3) have been shown to be associated with 116 snRANA in yeast [2.5,27]. It is now believed that the 1;6 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 SCraphin, T Achsel, R Liihrmann, personal communication). Proteins bearing strong similarity to the Sm proteins have also been found in the archeon Archaeoghbus fkgia’us [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.

Ul small

nuclear

ribonucleoprotein

particle

Figure 3a shows a schematic representation of the human Ul snRNP [1,3]. The CT1 snRNA contains 163 nucleotides with a m,G cap and forms four stem-loops. The ACIJLJACCIJ sequence present at the S’end is complementary to the conserved sequence (AGGIJRAGU) at the 5’ splice site of pre-mRNA and plays an important role in binding the Ul snRNP to the 5’ splice site. The Sm site (ALIUUGUGG) present in the single-stranded region is the binding site for the core Sm proteins. In addition, human

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LJl snRNP contains three specific proteins, named 1Jl 7OK, IJIA and UlC. Ul 70K and UlA bind to stem-loops I and II within the IJl snRN.4, respectively (Figure 3a). ‘I-he IilA protein contains two RNP domains (or RRhls) that are linked by a protease-sensitive peptide. The N-terminal RNP domain of IJlA is necessary and sufficient for binding to LJl snRNA [40,41]. The crystal structure of the N-terminal fragment of the IJlA protein in complex with its Lrl snRNA hairpin II binding site provides important insights into RNA recognition by RI%P domains [42] (Figure 4a). The RNP domain contains a four-stranded antiparallel p sheet flanked on one side by two a helices [43]. The 10 nucleotide loop of the LJl snRNA hairpin II (stem-loop II) binds on the surface of the fl sheet as an open structure. The first seven nucleotides of stem-loop II, ALilJGCAC, and the loop-closing C.G base pair form an intricate hydrogen-bond network with the sidechains and the mainchain of the IJlA 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 LJl 70K protein contains a single copy of the RNP motif (or RRM) around residues 100-180, followed by a charged C-terminal tail with alternating highly Arg-(Glu/Asp) and Arg-Ser repeats [44]. A fragment of the 111 70K protein containing the RNP motif is alone capable of binding stem-loop I of Ul snRNA. The N-terminal fragment containing residues l-97 (with no known sequence motifs) can, however, be incorporated into the core Ul snRNP consisting of the IT1 snRNA and the core Sm proteins [45]. The N-terminal fragment of the I!1 70K protein preceding the RNP motif is unable to bind Vl snRNA on its own, but it interacts with the core domain through protein-protein interactions. The LJl 70K protein can be chemically cross-linked to the B and 11, proteins [4.5]. The UlC protein binds to the lJ1 snRNP only in the presence of both the core domain and the LJl 70K protein, and does not bind the Ii1 snRNA on its own. This indicates that the I:lC protein probably binds both to the LJl 70K protein and to the Sm proteins. The latter contact is corroborated by the observation of a cross-link between LJlC and the Sm B protein [461. IT1 snRNPs depleted of the UlC protein fail to bind to pre-mRNA. The LJlC protein was proposed to form a noncanonical Cys2-His,-type zinc finger domain near its N terminus. This segment is sufficient to restore the binding of the I!1 snRNP to the 5’ splice site [47]. The IJlC protein apparently alters the conformation of the 5 end of LJl snRNA so that it can pair with the 5’ splice site. Electron micrographs of the I!1 snRNP show two protuberances emerging from the central globular core snRhP domain [48] (Figure Za). Antibodies specific to the LJlA and CJl 70K proteins were used to identify each of the proteins in a single protuberance. The protuberance corresponding to the Ll 70K protein was found to be closer to the

226

Figure

Macromolecular

assemblages

3 Schematic representation of the Ul and U2 snRNPs. (a) The interaction between the Ul snRNA and protein components within the Ul snRNP. The Sm proteins (B/B’ D3, D2, Dl, E, F and G) assemble around the Sm site, forming the core RNP domain. The Ul 70K and Ui A proteins bind to stem-loops I and II, respectively. The Ul C protein does not bind to Ul snRNA on its own and requires the presence of the Ul 70K protein. The nucleotides near the 5’ end of Ul snRNA (5’s~) are known to pair with the conserved sequence at the 5’ splice site of the premRNA. For the crystal structure of U 1 A bound to hairpin II, see Figure 4a [421. A model of the core RNP domain is shown in Figure 1 e [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 Ul snRNP (a). The UPB”-UPA’(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 UPB”-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 [161.

UlA protein

(a)

Ul 70K protein

Ul C protein

/

,

(W

Current Op~nton m Structural Biology

antibody bound to the m,G cap of Ul ‘RNA than that corresponding to the UlA protein [Ml. The sizes of the protuberances are consistent with those predicted from the molecular weights of the UlA and IT1 7OK proteins.

U2 small

nuclear

ribonucleoprotein

particle

Figure 3b shows a schematic representation of the human 112 snRNP [1,3,17]. The II2 snRNA, containing 1X7 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 172 snRNP purified under high salt conditions contains two ITZ-specific proteins, the LB” and UZA’ proteins, in addition to the core Sm proteins, whereas the 175 112 snRNP purified under low salt conditions contains nine additional proteins. including the heteromeric splicing factors SF3a and SF3b [2,1&l 11. The role of the liZA’ and 11213” proteins in splicing has long remained elusive, but recent evidence from

S. CfreGsae indicates that these two proteins are required for the integration of the UZ snRNP into the pre-spliceosome [49”]. The 5’ half of the U2 snRNA is extensively modified (Z’Omethyl groups and pseudouridines). The requirements of these modifications for both the conversion of the 1% CJ2 snRNP into the (spliceosome assembly competent) 17s U2 snRNP particle and the subsequent splicing activities were addressed by Yu et (I/. [SO”]. The authors found a correlation between the extent of nucleotide modification and LJ2 snRNP function in splicing and concluded that these modifications within the 27 nucleotides at the 5’ end of the 112 snRNA are essential for the 12S+l7S 112 RNP conversion. The crystal structure of the lJ2B”-L2A protein complex bound to a fragment of 1:2 snRNA has been determined at 2.4 A resolution [51”] (Figure 4b). The IJLN” protein, containing two RNP domains, is closely related to the

Structure

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4

Crystal structure of the U 1 A protein and the U2B”-U2A’(LRR) protein complex bound to their cognate RNA hairpins. (a) A surface representation of a complex between the Ul A protein (white) and Ul snRNA stem-loop II [42]. Amino acid residues specific to the Ul A protein are highlighted in red. (b) A surface representation of the ternary complex between the USB”-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 UPA’(LRR) protein is shown in blue. Reproduced with permission from [51**1.

UlA protein [41]. The UlA protein binds to stem-loop II of Ul snRNA on its own, whereas the UZB” protein binds to stem-loop IV of U2 snRNA only in complex with the UZA’ protein [41], which is a member of the LRR protein family [Xl. A complex between a fragment of the CJZB” protein containing the N-terminal 96 residues and the UZA’(LRR) protein shows high affinity and specificity for stem-loop IV of U2 snRNA [41]. This 1JZB” fragment differs from the lJlA protein in only 25 positions. Stem-loop II of Ul snRNA contains a 10 nucleotide loop, with the AIJUGCACUCC 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 UZA’(LRR) protein form a solenoid that is similar to, but more irregular than, those found in the porcine ribonuclease inhibitor [SZ]. Helix A of the IJZB” protein fits into the concave surface of the parallel p sheet of the LRRs, and the N-terminal and C-terminal arms flanking the LRRs embrace the N-terminal RNP domain of the UZB” protein. Scherly et al. [.53] showed that two amino acid replacements, Asp24+Glu and LysZ&-+Arg, in the 1JlA protein allow it to form a stable complex with the UZA’(LRR) protein. The guanidinium group of Arg28 from the UZB” protein protrudes to form hydrogen bonds with Thr69 and Glu92 of the LJZA’(LRR) protein. In the crystal structure of the UlA 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 UlA protein, but the last three loop nucleotide bases are not [42]. In the UZB”-UZA’(LRR) complex with a fragment of U2 snRNA stem-loop IV, all the loop nucleotides are involved in extensive interactions with the UZB” protein. The double-stranded stem of LJ2 snRNA hairpin IV interacts with the UZA’(LRR) protein and, hence, extends in a different direction from the RNP domain than that of IT1 snRNA hairpin II [Sl”]. The differences between the two RNP proteins (UZB” and UlA) and the presence of the UZA’(LRR) protein allow the two

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 1% 1_J2 snRNPs show a small domain attached to the core domain that could be identified as being the IJZB”-LJZA’ protein complex bound to IJZ snRl%A hairpin IV [38,.54]. In contrast, the larger 17s U2 snRNP particle contains nine additional UZ-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 LJ2 snRNA appearing like a filament (Figure 2b) [54]. SF3a associates with the core proteins and the 1JZB”-UZA’ protein complex.

U4N6, nuclear

U5 and tri-(U4/U6*U5) ribonucleoproteins

small

The

U4/116 snRNP isolated from a HeLa cell nuclear contains tJ4 and 116 snRNAs, which are extensively base paired, and the core Sm proteins bound to the Sm site of the tJ4 snRNA. Electron micrographs of the rJ4/U6 snRNPs show a globular core domain and the Y-shaped filamentous structure of the base paired 174 and U6 snRNAs protruding from the core domain [SS] (Figure Zc). extract

Human US 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 [JS 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 US snRNP is far more complex than the Ul and U2 snRNPs (Figure Zd). It contains nine LJS-specific proteins (220, 200, 116, 110, 102, 100,.52,40 and 15 kDa), in addition to the core Sm proteins (Figure Zd). The 200 kDa protein contains two

228

Macromolecular

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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 [lo]. 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 (Snul14p) 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 US snRNA in aligning the 5’ exon and the 3’ splice site during the second tratzx-esterification reaction [S7--591. Dix rt a/. [60”] cross-linked proteins to LJS snRNA either uniformly or site-specifically labelled with 4-thiouridine, within the reconstituted US snRNP and located the RNA contact sites for five yeast proteins. PrpSp is in contact with a wide region of US snRNA, including the stem of conserved loop I, whereas Snul14p was cross-linked to a 4-thiouridine that was introduced to the 5’ strand of internal loop I. The U4/U6 and US 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 Ul and U2 snRNPs assembled on the pre-mRNL4.

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 eta/. [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 eta/. [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 SOS ribosomal subunit at a res0 olution higher than 10 A, is extremely encouraging ([63”]; see the review by Agrdwal 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. [Jnderstanding the splicing process requires the structural knowledge of many intermediate states. Such intermediate states may be isolated using mutant substrate pre-mRNAs or murant 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 I\Tenman. Chris Ouhridge. Richard Bayliss and Phil Evans for critical reading of the manuscript, Berthold Kastncr for allominl: us tn include his electron micrographs and present and past members of the group for their contributions to the project. The work was supported by the Rfrdical Research Council and Human Frontier Science Program. CX was supported by NATO and EC fellowship\. and SW hl: Boehringcr lngelheim and ELi Marie Curie studentships.

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and recommended

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published

within

reading the annual

period

of review,

l of special interest -*of outstanding interest

1.

Moore MJ, Query C, Sharp PA: Splicing messenger RNAs by the spliceosome. by Gesteland RF, Atkins JF. Cold Spring Harbor Laboratory Press; 1993:303-357.

of precursors to In The RNA World. Edited Harbor, NY: Cold Spring

2.

Burge CB, Tuschl TH, Sharp PA: Splicing of precursors to mRNAs by the spliceosome. In The RNA World, edn 2. Edited by Gesteland RF, Cech T, Atkins JF. Cold Spring Harbor, NY: Cold Sprinq Harbor Laboratory Press: 1999:525-560.

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