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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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Suppressors of U4cs1 and brr2-1 alleles mapped on the Prp8 structurea, 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 (*).
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Figure 4: Suppressors of U4cs1 and brr2-1 alleles mapped on the Prp8 structurea, 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 (*).

Mentions: In the U4-cs1 (cold sensitive) mutant the three nucleotides (AAA) adjacent to helix I of the U4-U6 snRNA duplex are replaced by UUG, extending helix I by three base-pairs and concealing part of the U6 snRNA sequence (ACAGA) which base-pairs with the 5′-SS. At the restrictive temperature (16 °C) the spliceosome stalls before the first trans-esterification because the U4-U6 snRNAs remain base-paired and the ACAGA box fails to base-pair with the 5′-SS. Screens for suppressors of U4-cs1 in Prp8 isolated many single mutations in five regions (Prp8-cat mutants) (Table S6)36,37. Two of the five regions (d and e) are within our crystal structure (Fig 4). Six mutations in region d map within or near the four-stranded β-sheet protruding from the reverse transcriptase fingers domain (Fig 4c), and three map on the loop connecting RTα12 and RTα13. These are the most exposed parts of the fingers and palm domain. In region d and part of region e, three of the mutations are located within or near the exposed β-hairpin of the reverse transcriptase-like domain and two on the exposed loop in the endonuclease domain. Intriguingly all of the suppressor mutations in the RT/En domain map on one face of the RT/En domain. Five mutations of region e map within the RNaseH-like domain (Fig 4a), and four of these are located within the β-finger, which forms a continuous β-sheet with the tail of Aar2 and the β-barrel in the Jab1/MPN domain. It is possible that this β-hairpin is also involved in a protein–protein interaction crucial for positioning the RNaseH-like domain in the U5 snRNP or spliceosome.


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

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

Suppressors of U4cs1 and brr2-1 alleles mapped on the Prp8 structurea, 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 (*).
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Related In: Results  -  Collection

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Figure 4: Suppressors of U4cs1 and brr2-1 alleles mapped on the Prp8 structurea, 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 (*).
Mentions: In the U4-cs1 (cold sensitive) mutant the three nucleotides (AAA) adjacent to helix I of the U4-U6 snRNA duplex are replaced by UUG, extending helix I by three base-pairs and concealing part of the U6 snRNA sequence (ACAGA) which base-pairs with the 5′-SS. At the restrictive temperature (16 °C) the spliceosome stalls before the first trans-esterification because the U4-U6 snRNAs remain base-paired and the ACAGA box fails to base-pair with the 5′-SS. Screens for suppressors of U4-cs1 in Prp8 isolated many single mutations in five regions (Prp8-cat mutants) (Table S6)36,37. Two of the five regions (d and e) are within our crystal structure (Fig 4). Six mutations in region d map within or near the four-stranded β-sheet protruding from the reverse transcriptase fingers domain (Fig 4c), and three map on the loop connecting RTα12 and RTα13. These are the most exposed parts of the fingers and palm domain. In region d and part of region e, three of the mutations are located within or near the exposed β-hairpin of the reverse transcriptase-like domain and two on the exposed loop in the endonuclease domain. Intriguingly all of the suppressor mutations in the RT/En domain map on one face of the RT/En domain. Five mutations of region e map within the RNaseH-like domain (Fig 4a), and four of these are located within the β-finger, which forms a continuous β-sheet with the tail of Aar2 and the β-barrel in the Jab1/MPN domain. It is possible that this β-hairpin is also involved in a protein–protein interaction crucial for positioning the RNaseH-like domain in the U5 snRNP or spliceosome.

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
Related in: MedlinePlus