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Crystal structure of Prp8 reveals active site cavity of the spliceosome.

Galej WP, Oubridge C, Newman AJ, Nagai K - Nature (2013)

Bottom Line: The structure reveals tightly associated domains of Prp8 resembling a bacterial group II intron reverse transcriptase and a type II restriction endonuclease.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.

View Article: PubMed Central - PubMed

Affiliation: MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK.

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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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)65.
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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)65.

Mentions: The crystal structure of an Oceanobacillus iheyensis group II intron reveals a tightly packed functional centre consisting of exon-binding loops (EBS), exons and domain V organized by the surrounding RNA scaffold46,47. No structure of a group II intron in complex with IEP has yet been reported but the interaction between the Lactoccocus lactis L1LtrB intron and its IEP has been studied biochemically48-50. The N-terminal region of the RT domain binds intron DIVa with high affinity whereas the Th/X domain makes contact with the catalytic core of the intron including the E1-DI, DII and DVI-E2 regions to promote splicing48,50. The functional RNA core of the spliceosome is postulated to be similar to that of a group II intron. Prp8 and the O. iheyensis group II intron have remarkably similar dimensions and the Prp8 active site cavity is approximately the right size to accommodate the essential RNA domains of group II intron RNA (Fig 5a and 5b). It is tempting to suggest that Prp8 has replaced the RNA scaffold surrounding the group II intron. Notably, the spliceosomal RNA catalytic core crosslinks to the region of Prp8 between RT and En domains (C.M. Norman and A.J.N., unpublished results) and to the RNaseH-like domain18 (Fig 3b and 3c). The surface of this region exhibits extraordinary sequence conservation (Fig S18) and is remarkably electropositive (Fig 5c). This is consistent with its role as the binding site for the RNA catalytic core. Structural analysis of a group II intron at different stages of catalysis has revealed that the intron active site can adopt two alternative conformations46,47. It has been proposed that Prp8 may undergo a transition between two alternative states that facilitate the first and second steps of splicing, respectively35. This transition may be achieved by repositioning of the RNaseH-like domain and the extended polypeptide chains (Fig 3b and 3c), which line the inner surface of the active site cavity.


Crystal structure of Prp8 reveals active site cavity of the spliceosome.

Galej WP, Oubridge C, Newman AJ, Nagai K - Nature (2013)

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)65.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3672837&req=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)65.
Mentions: The crystal structure of an Oceanobacillus iheyensis group II intron reveals a tightly packed functional centre consisting of exon-binding loops (EBS), exons and domain V organized by the surrounding RNA scaffold46,47. No structure of a group II intron in complex with IEP has yet been reported but the interaction between the Lactoccocus lactis L1LtrB intron and its IEP has been studied biochemically48-50. The N-terminal region of the RT domain binds intron DIVa with high affinity whereas the Th/X domain makes contact with the catalytic core of the intron including the E1-DI, DII and DVI-E2 regions to promote splicing48,50. The functional RNA core of the spliceosome is postulated to be similar to that of a group II intron. Prp8 and the O. iheyensis group II intron have remarkably similar dimensions and the Prp8 active site cavity is approximately the right size to accommodate the essential RNA domains of group II intron RNA (Fig 5a and 5b). It is tempting to suggest that Prp8 has replaced the RNA scaffold surrounding the group II intron. Notably, the spliceosomal RNA catalytic core crosslinks to the region of Prp8 between RT and En domains (C.M. Norman and A.J.N., unpublished results) and to the RNaseH-like domain18 (Fig 3b and 3c). The surface of this region exhibits extraordinary sequence conservation (Fig S18) and is remarkably electropositive (Fig 5c). This is consistent with its role as the binding site for the RNA catalytic core. Structural analysis of a group II intron at different stages of catalysis has revealed that the intron active site can adopt two alternative conformations46,47. It has been proposed that Prp8 may undergo a transition between two alternative states that facilitate the first and second steps of splicing, respectively35. This transition may be achieved by repositioning of the RNaseH-like domain and the extended polypeptide chains (Fig 3b and 3c), which line the inner surface of the active site cavity.

Bottom Line: The structure reveals tightly associated domains of Prp8 resembling a bacterial group II intron reverse transcriptase and a type II restriction endonuclease.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.

View Article: PubMed Central - PubMed

Affiliation: MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK.

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.

Show MeSH