Available online at www.sciencedirect.com

ScienceDirect

Structural studies of the : zooming into the heart of

the machine

Wojciech P Galej, Thi Hoang Duong Nguyen, Andrew J Newman and

Kiyoshi Nagai

Spliceosomes are large, dynamic ribonucleoprotein complexes canonical subunits: U1, U2, U4, U5 and U6 small nuclear

that catalyse the removal of from messenger RNA ribonucleoprotein particles () and various other

precursors via a two-step splicing reaction. The recent crystal non-snRNP factors [2]. The assembly process begins with

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structure of Prp8 has revealed Reverse Transcriptase-like, the recognition of the 5 splice site (5 SS) and the branch

Linker and Endonuclease-like domains. The branch- point sequence (BP) by U1 and U2 snRNPs, respectively,

point cross-link with the Linker domain of Prp8 in active and further recruitment of the U4/U6.U5 tri-snRNP leads

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and together with suppressors of 5 and 3 splice to the formation of the pre-catalytic complex B. A major

site mutations this unambiguously locates the active site cavity. remodeling event catalyzed by the two DExD/H box

Structural and mechanistic similarities with group II self- Prp28 and Brr2 [3] leads to formation of the

splicing introns have encouraged the notion that the catalytically competent complex B*, in which the first

spliceosome is at heart a ribozyme, and recently the ligands for step of splicing occurs.

two catalytic magnesium ions were identified within U6 snRNA.

They position catalytic divalent metal ions in the same way as The enormous complexity and highly dynamic nature of

Domain V of group II intron RNA, suggesting that the the spliceosome present a formidable challenge for struc-

spliceosome and group II intron use the same catalytic tural and mechanistic studies. Until recently high resol-

mechanisms. ution structural information was mostly restricted to small

proteins or single domains of spliceosomal components

Addresses

[4]. At the same time, electron cryo-microscopy has

MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge

CB2 0QH, United Kingdom provided valuable insights into the global shape of the

splicing machinery, but compositional and conformation-

Corresponding authors: Galej, Wojciech P ([email protected]) ˚

al heterogeneity has limited the resolution to 20–30 A [5].

and Nagai, Kiyoshi ([email protected])

Now substantial progress has been made towards

high-resolution structure determination of large multi-

component spliceosomal assemblies (Table 1). Following

Current Opinion in Structural Biology 2014, 25:57–66

landmark work on the U1 snRNP [6,7] and the U4 snRNP

This review comes from a themed issue on Macromolecular

core domain [8] the structures of the two largest spliceo-

machines

somal proteins, Prp8 [9 ] and Brr2 [10 ,11 ,12 ],

Edited by Karl-Peter Hopfner and Tom Smith

have recently been reported, providing valuable insights

For a complete overview see the Issue and the Editorial into spliceosomal activation and the architecture of the

Available online 28th January 2014 active site.

#

0959-440X 2014 The Authors. Published by Elsevier Ltd.

Open access under CC BY-NC-SA license.

Structure and function of Prp8: pseudo-enzyme http://dx.doi.org/10.1016/j.sbi.2013.12.002

conglomerate

Prp8 is the largest and most highly conserved spliceosomal

protein (280 kDa, 61% identity between yeast and human).

Introduction It interacts extensively with two other integral U5 snRNP

The spliceosome is a large RNA-protein complex, which

components, the EF-2-like GTPase Snu114 and the

catalyzes the excision of non-coding introns and ligation

DExD/H box Brr2 [13,14] and cross-links with

0

of from the precursors of messenger RNAs (pre- all critical sites of chemistry in the pre-mRNA substrate (5 -

mRNAs) via a two-step trans-esterification reaction. Pro- 0

SS, 3 -SS, BP) as well as with U5 and U6 snRNAs [15–18]. A

teomic studies have revealed that the spliceosome is

number of Prp8 alleles have been shown to suppress

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immensely complex and highly dynamic [1]. For each splicing defects caused by mutations in the 5 -SS, 3 -SS

intron, the spliceosome assembles de novo from its five

and BP [19–22]. These data clearly indicate that Prp8 lies at

the heart of the splicing machinery.

Despite intensive study the domain architecture of Prp8

remained elusive until recently. The first structural

insights came from the C-terminal domain, which

www.sciencedirect.com Current Opinion in Structural Biology 2014, 25:57–66

58 Macromolecular machines

Table 1

A list of structures of spliceosomal components reported in the last two years

Structure Organism MW [kDa] Resolution PDB code

U5 snRNP components

1834–2086

Prp8 :Aar2 Yeast 70 1.8 3SBT

402–2125 a

Brr2 Human 197 2.7 4F91

885–2413 a

Prp8 :Aar2 Yeast 218 2.0 4I43

1834–2392

Prp8 :Aar2 Yeast 105 2.1 4ILG

442–2163 2148–2395

Brr2 :Prp8 Yeast 224 3.1 4BGD

404–2125 2067–2335

Brr2 :Prp8 Human 228 3.6 4KIT

1770–1990 a

Prp8 Human 23 1.4 4JK7

U4 snRNP

a

U4 core domain Human 100 3.6 2Y9A

U2 snRNP components

1–389 50–266 87–237

Prp9 :Prp11 :Prp21 Yeast 89 3.1 4DGW

U2/U6 snRNA hybrid Yeast 36 NMR 2LKR

Other factors Cwc2 Yeast 26 2.4 3TP2 Cwc2 Yeast 25 1.9 3U1Ma SF114–132:U2AF65375–475 Human 26 2.3 4FXW SF11–145:U2AF65372–475 Human 29 NMR 2M0Ga U2AF65148–342:oligo(U) Human 24 NMR 2YH1

a

Multiple coordinate files deposited.

revealed similarities to the Jab1/MPN fold, known from Jab1/MPN domains are connected by disordered linkers

some de-ubiquitinating metallo-enzymes [23,24]. How- and represent structurally independent units (in the

ever its putative isopeptidase active site proved to be absence of Aar2). The active site of the Reverse Tran-

impaired. The discovery of an RNaseH-like domain in scriptase-like domain contains only one of three aspar-

2+

Prp8 [25–27] has attracted much attention due to its tates involved in Mg coordination at the canonical

2+

proposed involvement in Mg ion coordination as part polymerase active site, and therefore it is unlikely to

of a composite RNA-protein spliceosomal active site [28]. be involved in metal ion-dependent catalysis

In canonical RNaseH, RNA backbone cleavage proceeds (Figure 1d). The endonuclease domain of Prp8 has pre-

2+

via a two-metal ion mechanism [29] similar to that pos- served all the negatively charged residues that bind Mn

tulated for splicing catalysis [30]. The active site of the ions at the active site of the structurally similar PA

RNaseH-like domain of Prp8 lacks two of the four nega- endonuclease from influenza virus. However in Prp8 they

2+

tively charged residues involved in Mg coordination, do not bind divalent metal ions; instead they interact with

and no magnesium ions were observed in the original a polypeptide loop lying on top of the active site [9 ]

published crystal structures [25–27]. Recently, MacMil- (Figure 1e). This, together with the fact that these

lan and co-workers reported structures of an alternative residues are not essential for yeast viability, makes it

conformation of the RNaseH-like domain of Prp8, in very unlikely that they function in spliceosomal catalysis.

which a distortion of the b-finger unmasked a previously

inaccessible aspartate residue, allowing magnesium ion

binding under crystallization conditions [31]. Mutations The active site of the spliceosome: RNA core in a protein

promoting the second step of splicing stabilise this new shell

0

(‘‘open’’) conformation. Nevertheless, as yet there is no Splicing defects caused by mutations in the 5 -SS, BP and

0

experimental evidence to support a role for this magnes- 3 -SS can be rescued by mutations in Prp8. Over the years

ium ion at the spliceosomal active site [32]. a number of splice site suppressor alleles of Prp8 have

been isolated [19–22]. Most of these suppressor mutations

The structure of a large fragment of Prp8 (residues 885– map to the inner surface of the cavity formed by the RT

2413) in complex with Aar2, a U5 snRNP assembly factor, Thumb, Linker, Endonuclease and RNaseH-like

revealed three novel domains in the middle part of the domains [9 ] (Figure 2). Site-specific RNA-protein

protein (Figure 1). Reverse Transcriptase-like (RT), cross-linking in activated, affinity purified step 2 spliceo-

Linker and type II Restriction Endonuclease-like somes reveals contacts between BP and Prp8, which were

domains interact intimately with each other to form a precisely mapped to a short sequence between residues

compact structural unit, the ‘‘large domain’’ [9 ]. Nota- 1585-1598 (Norman and Newman, unpublished data).

bly, the existence of the Reverse Transcriptase-like This region is disordered in the Prp8-Aar2 structure,

domain in Prp8 was correctly predicted by Dlakic and however its estimated position together with suppressor

Mushegian [33 ]. The large domain, RNaseH-like and mutations unambiguously locate splicing chemistry in the

Current Opinion in Structural Biology 2014, 25:57–66 www.sciencedirect.com

Structural studies of the spliceosome Galej et al. 59

Figure 1

(a) Snu114 interacting region

885 1375 1650 1840 2090 2150 2395 Prp8 NLS RT palm/fingers Th/X Linker Endo RNaseH Jab1/MPN 2413

(b) Reverse Transcriptase-like (c) Reverse Transcriptase-like Jab1/MPN Jab1/MPN (d)

90° Th/X Th/X

Linker RNaseH-like RNaseH-like (e)

Aar2 Aar2

Endonuclease Endonuclease

(d) (e)

D1166 H1658 K1690 Q1737

T1053

D1700 D1735 R1167 E1684 Y1767

T1702 Prp8 Reverse Transcriptase Active Site Prp8 Endonuclease Active Site Current Opinion in Structural Biology

Structure of the yeast Prp8-Aar2 complex [9 ]. (a) Primary domain architecture of yeast Prp8. (b) Three dimensional arrangement of the six domains of

Prp8 and their interactions with Aar2. (c) Orthogonal view of the complex. (d) Impaired active site of the RT domain of Prp8. (e) the active site of Prp8

Endonuclease domain, showing conserved catalytic residues interacting with a polypeptide loop positioned on top of the active site.

cavity formed by the RT Thumb, Linker, Endonuclease magnesium ion binding sites [35,36,37 ]. Using a com-

and RNaseH-like domains [9 ] (Figure 2). bination of NMR and SAXS, Butcher and co-workers

determined the structure of a protein-free yeast U2/U6

The spliceosome active site is organized around the U5 snRNA duplex [38]. The structure revealed a Y-shaped

snRNA loop 1 and the U2/U6 snRNA duplex [34], which three-way junction with the U6 internal stem-loop (U6

are involved in binding and correct positioning of the sites ISL) co-axially stacked with U2/U6 helix Ib. In this

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of chemistry in the pre-mRNA substrate (5 SS, 3 SS and arrangement the two catalytic magnesium binding sites

BP). Furthermore the U6 snRNA harbours the catalytic (U6 U80 and the AGC catalytic triad), are too far apart to

www.sciencedirect.com Current Opinion in Structural Biology 2014, 25:57–66

60 Macromolecular machines

Figure 2

Reverse Transcriptase-like

P986 BP+2 XL

E1450

N1869 K1864 V1870 H1863 5′-SS XL T1861 RT Th/X L1598 M1399

W1575 L1823 T1982 E1960 L1557 Linker F1834 K1563 E1817

RNaseH-like

R1753

Endonuclease Current Opinion in Structural Biology

The active site cavity of the spliceosome [9 ]. Splice site suppressor mutations (red spheres) co-localize mostly to the inner surface of a cavity formed

by RNaseH-like and Large domains of Prp8. Branch-point crosslinks (BP + 2 XL) analysed in stalled and affinity purified, yeast step 2 spliceosomes

map to a short sequence (1585–1598), part of a disordered loop between residues marked with blue spheres (Norman and Newman, unpublished

0

data). A cross-link between 5 -splice site and the RNaseH-like domain in the human system is marked with green spheres [17]. Positions of suppressor

mutations together with site-specific RNA-protein cross-linking sites unambiguously locate the active site cavity of the spliceosome.

form a two-metal active site like that observed in mechan- catalytic core can be fitted neatly into the active site

istically similar group II introns [39], implying that the cavity of the spliceosome [9 ,41]. Recently Fica et al.

structure would be modified in active spliceosomes. [37 ] identified ligands of two catalytic magnesium ions

in the spliceosome for the first and second catalytic steps

Structural and mechanistic similarities between spliceo- by elegant stereospecific phosphorothioate substitutions

somes and group II intron ribozymes strongly suggest and metal rescue experiments. Strikingly, all of the cat-

they may have evolved from a common ancestor [40].The alytic metal ligands in U6 snRNA correspond to those

crystal structure of a self-splicing group IIC intron from observed to position catalytic divalent metal ions in group

Oceanobacillus iheyensis revealed an intricate architectural II intron RNA (Figure 5a; [37 ,42]). This suggests that

RNA scaffold surrounding a tightly packed catalytic core the spliceosome and the group II intron use the same

[39]. Notably, Prp8 and the group II intron scaffold have catalytic mechanisms, providing further support for their

remarkably similar dimensions, so that the group II intron common evolutionary origin.

Current Opinion in Structural Biology 2014, 25:57–66 www.sciencedirect.com

Structural studies of the spliceosome Galej et al. 61

An NTC-related protein, Cwc2 has also been shown to the Prp8 RNaseH-like domain and the authors proposed a

act

contact spliceosomal RNA in B , B* and C complexes novel mechanism whereby Prp8 RNaseH-like domain

[43]. Cwc2 is essential for the formation of catalytically negatively regulates the unwinding activity of Brr2.

active step 1 spliceosomes and it cross-links specifically to

the U6-ISL and the region upstream of the U6 ACA- Recently Hahn et al. [53] showed that the N-terminal

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GAGA box. It was proposed to play a role in remodelling helicase cassette of Brr2 cross-links with the 5 and 3

U6 snRNA into a catalytically active conformation, by splice sites suggesting that it is closely associated with the

bringing distant parts of U6 snRNA into close proximity active site cavity formed by Prp8 prior to the second step

[43]. The structure of Cwc2 revealed a unique combi- of splicing. Suppressors of U4cs-1 and brr2-1 [54] map on

nation of a zinc finger (ZnF) and an RNA Recognition one face of Prp8 [9 ] and this surface might interact with

Motif (RRM) forming a compact toroidal unit [44,45]. Brr2, although this interaction could be transient and

Two subdomains with a potential for nucleic acid binding modulated by other proteins such as Snu114. We pro-

may provide a platform for multipartite RNA recognition posed a plausible model demonstrating how Prp8 and

in the spliceosome. Brr2 might collaborate to shape the active site of the

spliceosome [11 ]. In this model, U6 snRNA unwound

Brr2 structures and implications for spliceosome from the U4 snRNA could be introduced into the active

activation site cavity to base pair with the U2 snRNA.

Brr2, an integral U5 snRNP component, is a large Ski2-like

helicase (250 kDa) responsible for unwinding the U4/U6 U5 snRNP biogenesis: an ordered assembly pathway

snRNA duplex during catalytic activation of the spliceo- The cytoplasmic precursor of yeast U5 snRNP lacks the

some (Figure 3a). It consists of two consecutive helicase mature U5 snRNP component Brr2 but contains Aar2

cassettes, but only the N-terminal cassette is functional instead. Upon nuclear import, Aar2 is replaced by Brr2 in

and essential for cell-viability and U4/U6 unwinding [46]. a phosphorylation-dependent manner [55,56] (Figure 4a).

Recently the crystal structure of a 200 kDa human Brr2 The structure of the Prp8:Aar2 complex revealed a critical

fragment [10 ] revealed two Hel308-like cassettes [47] role for Aar2 in the organization of Prp8 domains [9 ].

forming a compact entity with a large, functionally import- While the main body of Aar2 binds principally to the

ant inter-cassette interface (Figure 3b,c) [10 ]. Previous junction of the Linker and Endonuclease domains, the

yeast two-hybrid and pull-down studies [23,48] suggested very C-terminal tail extends much further and inserts

that the C-terminal cassette might modulate N-terminal between Jab1/MPN and RNaseH-like domains forming a

cassette activity through interactions with Snu114 and the remarkable intermolecular beta-sheet, zipping the two

RNaseH-like and Jab1/MPN domains of Prp8. The EF-2- domains together (Figure 4c). Biochemical experiments

like GTPase Snu114 regulates Brr2 activity in a nucleo- showed that the binding of Aar2 and Brr2 to Prp8 is

tide-dependent manner [49,50] while the Jab1/MPN mutually exclusive [55,56,11 ]. Superposition of the

domain strongly stimulates Brr2 helicase activity in vitro crystal structures of yeast Brr2-Jab1/MPN [11 ] and

[51,52]. Nguyen et al. [11 ] and Mozaffari-Jovin et al. [12 ] Prp8-Aar2 complexes [9 ] (using the Jab1/MPN domain

recently reported the crystal structures of yeast and human of Prp8, present in both structures) provided insights into

Brr2, respectively, in complex with the Prp8 Jab1/MPN the molecular basis of the competition between Aar2 and

domain. Contrary to earlier results [23] the Jab1/MPN Brr2 (Figure 4b). Surprisingly, the Brr2-binding interface

domain interacts exclusively with the N-terminal cassette of the Jab1/MPN domain is still exposed in the Prp8-Aar2

of Brr2 (Figure 3c). Residues in Prp8 whose mutations complex, however the tight domain arrangement,

cause type 13 retinitis pigmentosa are located at or close to enforced by the presence of Aar2, causes a clash between

the interaction surface and they are important for the Brr2 and the RNaseH-like domain of Prp8, disabling Brr2

modulation of yeast Brr2 unwinding in vitro [51,11 ]. binding to the Jab1/MPN domain of Prp8 by a steric

The main difference between the human and yeast com- hindrance mechanism [11 ] (Figure 4d).

plex structures lies in the very C-terminal region of the

Jab1/MPN domain. Residues 2396–2413 (2318–2335 in Evolutionary origin of the spliceosome

human) are disordered in the yeast Jab1/MPN domain, The fact that nuclear pre-mRNA splicing and group II

whereas in the human structure weak, discontinuous intron self-splicing proceed via two successive trans-

density attributed to this region was observed in the esterification reactions with a lariat-intron intermediate

putative RNA-binding tunnel of Brr2. This tail was pro- led to the far-sighted hypothesis that both systems have a

posed to prevent premature U4/U6 unwinding before common evolutionary origin [57,58]. The identification of

activation and to inhibit Brr2 in post-activation steps of structurally and functionally conserved counterparts of

splicing [12 ]. Using various truncated forms of the U4/U6 domains V, VI and the Binding Sites (EBS) of group

snRNA duplex for unwinding assays Mozaffari-Jovin et al. II introns in the RNA core of the spliceosome [59]

0

[52] concluded that Brr2 binds to the 3 end and translo- together with the discovery of trans-splicing suggested

cates along the U4 snRNA. Furthermore unwinding was that the spliceosomal snRNAs might have their origin

inhibited upon addition of micromolar concentrations of in the domains of group II introns having become

www.sciencedirect.com Current Opinion in Structural Biology 2014, 25:57–66

62 Macromolecular machines

Figure 3

(a) Pre-catalytic spliceosomes Activated spliceosomes 5′ U1 snRNA 5′ ACAGAG 3′ ′ 5 ? 3 ′ Snu114:GTP Snu114:GDP exon 5 ′ exon Mg2+ U5 snRNA 2+ U5 snRNA Mg Prp8 U6 snRNA ′ 5′ AGC 3 A 5′ 5′ U2 snRNA A 3′ U2 snRNA 5′ Brr2 Prp28 ′ ′ 3 3 3′ 5′ ′ U6 snRNA 3 exon 3′ exon 12 proteins 35 proteins AGC U4 snRNA ACAGAG 2 snRNAs Prp8 cross-linking sites 5′ 3′

(b) N-terminal cassette C-terminal cassette

1 479 701 901 911 1011 1142 1200 1309 1330 1541 1739 1749 1857 1988 2049 2163 yBrr2 RecA1 RecA2 WH Ratchet HLH FN3 RecA1 RecA2 WH Ratchet HLH FN3

(c) yPrp8Jab yPrp8Jab

N-HLH N-FN3

Linker C-terminus C-WH 90° N-Ratchet

N-WH N-Ratchet C-RecA1 N-RecA2 N-HLH

C-FN3

C-HLH N-WH N-terminus C-RecA2 N-RecA2 N-RecA1 C-Ratchet N-RecA1 Putative RNA binding tunnel N-terminal cassette C-terminal cassette Current Opinion in Structural Biology

Crystal structures of the Brr2 helicase [10 ,11 ,12 ]. (a) RNA interaction network in pre-catalytic and activated spliceosomes. Extensively based-

paired U4/U6 snRNA duplex undergoes remodelling by the action of Brr2 helicase leading to the formation of the catalytically active secondary

structures in the U6 snRNA. Adapted from [2]. (b) Schematic representation of the domain organizations of Brr2. Domains are colour coded: pink, N-

terminal extension; light grey, RecA1; dark grey, RecA2; blue: winged-helix (WH); cyan, ratchet; yellow, helix-loop-helix (HLH) and orange,

fibronectin3-like (FN3). (c) Overall structure of Brr2:Jab1/MPN complex. The structural domains are coloured as in (a) and labelled with N- and C- to

indicate which helicase cassette they belong to.

independent transcription units capable of acting in trans, IV of group II introns encodes a multi-functional protein.

in the course of evolution [60]. However, it remained The thumb/X domain of this Intron Encoded Protein

unclear how ancestral spliceosomes recruited numerous (IEP) facilitates self-splicing activity of group II introns

proteins on the way to their present complexity. Domain whereas the Reverse Transcriptase and Endonuclease

Current Opinion in Structural Biology 2014, 25:57–66 www.sciencedirect.com

Structural studies of the spliceosome Galej et al. 63

Figure 4

(a) Cytoplasm Nucleus

Jab RH Jab RH Snu114 Aar2 Snu114 Aar2 Prp8 Prp8 Aar2 phosphorylation

Jab RH P Snu114 Aar2 Prp8

U5 core domain Jab Brr2 RH Brr2 Snu114 Prp8 P Aar2

(b) (c) Brr2 C-term Prp8 RT Jab1/MPN

Jab1/MPN (c) Aar2 RNaseH

(d)

(d) clash

Aar2 RNaseH

RNaseH Brr2 Prp8 En Brr2 N-term

Current Opinion in Structural Biology

U5 snRNP biogenesis. (a) Schematic representation of the key stages in yeast U5 snRNP biogenesis. The precursor particle, Aar2-U5 snRNP is

transported from the cytoplasm to the nucleus, where after phosphorylation Aar2 is replaced by the helicase Brr2 [55]. (b) Superposition of Jab1/MPN

domain of Prp8-Aar2 and Brr2-Jab1/MPN complexes [9 ,11 ]. (c) Intermolecular beta sheet formed between Aar2 and Jab1/MPN and RNaseH-like

domains of Prp8. (d) A steric clash between Brr2 and the RNaseH-like domain of Prp8 reveals molecular basis of the competitive binding of Brr2 and

Aar2 to Prp8.

www.sciencedirect.com Current Opinion in Structural Biology 2014, 25:57–66

64 Macromolecular machines

Figure 5

(a) A 73 A U A A 370 A C G A G 5′ exon G C G U G U δ- 5’5′ exonexon C A M1 O C G A C C A M1 O δ- U G C U A U R3 C G R3 U A pro-Rp U A G C -O P Splice site U A pro-Rp C G G C -O P Splice site R2 U6-3′ ′ R2 G C δ- U2-5 A U G M2 O G C δ- G C M2 O R1 C G G C U A

388 288 53 R1 3′ AGC-5′ 3′ AGA-5′

Group IIC intron domain V U6 snRNA Internal Stem Loop (ISL)

(Oceanobacillus iheyensis) (Saccharomyces cerevisiae)

(b) Lactococcus lactis LtrA (Intron Encoded Protein)

RT finger/palm thumb/X D En 1 599

Saccharomyces cerevisiae Prp8 (splicing factor)

RT finger/palm thumb/X Linker En 885 1810

Current Opinion in Structural Biology

Similarities between components of group II introns and the spliceosomes. (a) Secondary structures and catalytic metal interactions in the domain V of

group IIC intron [42] and spliceosomal U6 snRNA Internal Stem Loop (ISL); catalytic magnesium ions M1 and M2 are coordinated by RNA nucleotides

0

labeled in red. For the first step reaction, R1 represents 2 hydroxyl of branch point adenosine (or equivalent water molecule in hydrolytic group IIC

0

intron); R2, the intron; R3, the pro-Sp oxygen. For the second step exon ligation reaction, R1 represents 3 oxygen leaving group; R2 the pro-Sp

0

oxygen; R3 the 3 exon. Adapted from [37 ]. (b) Comparison of the domain architecture of Prp8 and Intron Encoded Protein reveals a previously

unknown evolutionary link between the two systems. RT, reverse transcriptase domain; En, endonuclease domain; D, DNA binding domain.

domains are involved in the intron mobility mechanism recombinant spliceosomal subunits (U1 snRNP [6]) as

[59]. Although there is no evidence for such activities in well as large, multi-domain proteins and their complexes

Prp8 from modern spliceosomes the similarities in the (Prp8 [9 ] and Brr2 [10 ,11 ,12 ]). These structures

sequence and domain architecture between IEP and Prp8 have provided invaluable insights into the basic splicing

(Figure 5b) provide a new and compelling evolutionary mechanism and have paved the way for even larger and

link between group II intron self-splicing and nuclear pre- more challenging targets. To fully understand the bio-

mRNA splicing [9 ,33 ]. This suggests that one of the logical implications of these and other emerging struc-

most central proteins in the spliceosome may have been tures it will be necessary to combine data from different

recruited from an ancestral IEP, that has lost its original sources. A hybrid approach combining crystallography

activities and started to function as an assembly platform and electron microscopy may play a very important role

for snRNAs and substrate pre-mRNA in modern spliceo- in the future. The introduction of direct electron detec-

somes. tors and recent developments in image processing have,

in the most favourable cases, allowed 3D reconstructions

˚

Conclusions and future perspectives of asymmetric assemblies at close to 4 A resolution [61]. A

The last few years have seen tremendous progress in combination of crystallography and high-resolution elec-

structural studies of the splicing machinery. As crystal- tron microscopy could allow structural analysis of increas-

lization targets became more complex it has proved ingly complex assemblies, with the ultimate goal of

possible to reconstitute and solve the structure of entirely pseudo-atomic models of the entire spliceosome.

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Structural studies of the spliceosome Galej et al. 65

Acknowledgements 14. Liu S, Rauhut R, Vornlocher H, Lu R: The network of protein–

protein interactions within the human U4/U6.U5 tri-snRNP.

We would like to acknowledge Yasushi Kondo, John Hardin, Christine

RNA 2006, 12:1418-1430.

Norman, Pei-Chun Lin and Israel Sanchez Fernandez for helpful comments

on the manuscript. We would also like to apologize to all our colleagues 15. Turner I, Norman CM, Churcher MJ, Newman AJ: Dissection of

whose work could not be cited here due to space limitations. This work was Prp8 protein defines multiple interactions with crucial RNA

supported by the Medical Research Council. sequences in the catalytic core of the spliceosome. RNA 2006,

12:375-386.

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interactions in the U5 snRNP of Saccharomyces cerevisiae.

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RNA 1998, 4:1239-1250.

have been highlighted as:

17. Reyes JL, Gustafson EH, Luo HR, Reyes JL, Gustafson EH,

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