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Structural Studies of the Spliceosome: Zooming Into the Heart Of Available online at www.sciencedirect.com ScienceDirect Structural studies of the spliceosome: 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 introns from messenger RNA ribonucleoprotein particles (snRNPs) and various other precursors via a two-step splicing reaction. The recent crystal non-snRNP factors [2]. The assembly process begins with 0 0 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 intron 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 0 0 spliceosomes 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- helicases 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 helicase Brr2 [13,14] and cross-links with 0 of exons 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 0 0 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.
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