Crystal structure of Prp8 reveals active site cavity of the spliceosome - PubMed (original) (raw)

. 2013 Jan 31;493(7434):638-43.

doi: 10.1038/nature11843. Epub 2013 Jan 23.

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Crystal structure of Prp8 reveals active site cavity of the spliceosome

Wojciech P Galej et al. Nature. 2013.

Abstract

The active centre of the spliceosome consists of an intricate network formed by U5, U2 and U6 small nuclear RNAs, and a pre-messenger-RNA substrate. Prp8, a component of the U5 small nuclear ribonucleoprotein particle, crosslinks extensively with this RNA catalytic core. Here we present the crystal structure of yeast Prp8 (residues 885-2413) in complex with Aar2, a U5 small nuclear ribonucleoprotein particle assembly factor. The structure reveals tightly associated domains of Prp8 resembling a bacterial group II intron reverse transcriptase and a type II restriction endonuclease. Suppressors of splice-site mutations, and an intron branch-point crosslink, map to a large cavity formed by the reverse transcriptase thumb, and the endonuclease-like and RNaseH-like domains. This cavity is large enough to accommodate the catalytic core of group II intron RNA. The structure provides crucial insights into the architecture of the spliceosome active site, and reinforces the notion that nuclear pre-mRNA splicing and group II intron splicing have a common origin.

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Figures

Figure 1

Figure 1. Structure of the large domain in yeast Prp8 (residues 885-1824)

a, The large domain consists of a group II intron reverse transcriptase-like domain and a type II restriction endonuclease-like domain; b, The palm sub-domain of HCV RNA-dependent RNA polymerase (1NB6). Asp residues (Asp220 in motif A, and Asp318 and Asp319 motif C) coordinate two catalytic Mg2+ ions; c, The corresponding residues in the palm sub-domain of the group II intron reverse transcriptase-like domain of Prp8; d, The catalytic centre of the influenza virus PA endonuclease (2W69). His-41, Glu-80, Asp-108 and Glu119 coordinate two catalytic divalent ions; e, The corresponding residues in the En domain of Prp8.

Figure 2

Figure 2. Overall structure of yeast Prp8885-2413 in complex with Aar2

a, Domain architecture of Prp8885-2413 and Aar2; b, Aar2 organises the arrangement of the RT/En, RNaseH-like and Jab1/MPN domains; c, Orthogonal view of the complex; d, A view (as in c) without Aar2 and the Jab1/MPN domain. The RNaseH-like domain has no direct contact with the RT/En domain.

Figure 3

Figure 3. Overview of the Prp8 active site cavity in an open book view

a, Overview with the suppressors of splice site (5′SS, 3′SS and BP) mutations (red spheres). Green spheres indicate the sequence (1966-SAAMS-1970) corresponding to the cross-linking site of hPrp8 to the 5′ SS (ref 18); b, Stereo view of the RNaseH-like domain surface making up the active site cavity; c, Stereo view of the RT/En domain surface making up the active site cavity. Crosslink of the pre-mRNA branch point (BP+2) nucleotide is located between residues 1585-1598 in sequence (C.M. Norman and A.J.N., unpublished data). This site is found within the disordered loop (blue dotted line) between residues 1575 and 1598 (blue spheres).

Figure 4

Figure 4. Suppressors of U4cs1 and brr2-1 alleles mapped on the Prp8 structure

a, U4cs-1 (blue spheres) and brr2-1 (green spheres) suppressor mutants map on one face of the RT-En domain of Prp8; b, A view rotated by 120° along y axis; c, Both types of suppressor mutants map to the same region of the Prp8 RT domain. Residues that suppress both alleles are marked with star (*).

Figure 5

Figure 5. Comparison between the active site of group II intron and the spliceosome (Prp8)

a, Group II intron from Oceanobacillus iheyensis (PDB: 3IGI). Domain V, red; EBS helix (blue); spliced exons (green); Catalytic Mg2+ ions (yellow sphere); Scaffolding RNA (grey); b, The RT/En domain with the RNaseH-like domain of Prp8 with the active RNA elements of group II intron modeled on its surface for size comparison. At present there are insufficient experimental constraints for the precise position or orientation of the RNA; c, electrostatic potential (±5kTe−1) plotted on the solvent accessible surface of the Prp8 (calculated with APBS).

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