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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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Overview of the Prp8 active site cavity in an open book viewa, 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the pre-mRNA branch point (BP+2) nucleotide is located between residues 1585-1598 in sequence (C.M. Norman and A.J.N., unpublished data).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).
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Figure 3: Overview of the Prp8 active site cavity in an open book viewa, 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).

Mentions: The catalytic centre of the spliceosome includes an intricate network of interactions involving U2, U5 and U6 snRNAs and substrate pre-mRNA (Fig S17). Prp8 crosslinks to crucial residues in U6 snRNA and in the invariant exon-binding loop 1 of U5 snRNA as well as to all 3 sites of chemistry in the pre-mRNA (5′-SS, BP and 3′-SS)15-18,33,34. Contacts between yeast Prp8 and catalytic core RNA residues were previously mapped by crosslinking and proteolytic cleavage; almost all of the crosslinks lie within the RT-En domain of Prp820 (Fig S17). We have used substrates with modified 3′-SS and captured spliceosomes via the NTC complex protein Prp19 or the step 2 factor Prp18 in order to focus on crosslinks made just before catalytic step 2 (C.M. Norman and A.J.N, unpublished results). Prp8-BP(+2) crosslinks were mapped to the region between residues 1585 and 1598. This disordered region is located between the blue spheres in Fig 3c. The crosslinking site is located in the mobile loop near the RT Th/X domain and distant from the residues corresponding to the Mg2+-coordinating residues in the RT and En domains.


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

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

Overview of the Prp8 active site cavity in an open book viewa, 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).
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Related In: Results  -  Collection

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Figure 3: Overview of the Prp8 active site cavity in an open book viewa, 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).
Mentions: The catalytic centre of the spliceosome includes an intricate network of interactions involving U2, U5 and U6 snRNAs and substrate pre-mRNA (Fig S17). Prp8 crosslinks to crucial residues in U6 snRNA and in the invariant exon-binding loop 1 of U5 snRNA as well as to all 3 sites of chemistry in the pre-mRNA (5′-SS, BP and 3′-SS)15-18,33,34. Contacts between yeast Prp8 and catalytic core RNA residues were previously mapped by crosslinking and proteolytic cleavage; almost all of the crosslinks lie within the RT-En domain of Prp820 (Fig S17). We have used substrates with modified 3′-SS and captured spliceosomes via the NTC complex protein Prp19 or the step 2 factor Prp18 in order to focus on crosslinks made just before catalytic step 2 (C.M. Norman and A.J.N, unpublished results). Prp8-BP(+2) crosslinks were mapped to the region between residues 1585 and 1598. This disordered region is located between the blue spheres in Fig 3c. The crosslinking site is located in the mobile loop near the RT Th/X domain and distant from the residues corresponding to the Mg2+-coordinating residues in the RT and En domains.

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