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Evidence for a group II intron-like catalytic triplex in the spliceosome.

Fica SM, Mefford MA, Piccirilli JA, Staley JP - Nat. Struct. Mol. Biol. (2014)

Bottom Line: Here we show by genetics, cross-linking and biochemistry in yeast that analogous triples form in U6 and promote catalytic-metal binding and both chemical steps of splicing.Because the triples include an element that defines the 5' splice site, they also provide a mechanism for juxtaposing the pre-mRNA substrate with the catalytic metals.Our data indicate that U6 adopts a group II intron-like tertiary conformation to catalyze splicing.

View Article: PubMed Central - PubMed

Affiliation: 1] Graduate Program in Cell and Molecular Biology, University of Chicago, Chicago, Illinois, USA. [2] Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, Illinois, USA. [3].

ABSTRACT
To catalyze pre-mRNA splicing, U6 small nuclear RNA positions two metals that interact directly with the scissile phosphates. U6 metal ligands correspond stereospecifically to metal ligands within the catalytic domain V of a group II self-splicing intron. Domain V ligands are organized by base-triple interactions, which also juxtapose the 3' splice site with the catalytic metals. However, in the spliceosome, the mechanism for organizing catalytic metals and recruiting the substrate has remained unclear. Here we show by genetics, cross-linking and biochemistry in yeast that analogous triples form in U6 and promote catalytic-metal binding and both chemical steps of splicing. Because the triples include an element that defines the 5' splice site, they also provide a mechanism for juxtaposing the pre-mRNA substrate with the catalytic metals. Our data indicate that U6 adopts a group II intron-like tertiary conformation to catalyze splicing.

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Genetic evidence for base-triple interactions between the AGC triad and the ACAGAGA region of the U6 snRNA(a,b) Spot assays showing growth on selective media of equivalent numbers of yeast cells containing combinations of alleles at G52 and G60 (a) or C61 (b). (c,d) Spot assays showing growth on selective media of equivalent numbers of yeast cells containing combinations of alleles at A53 and A59 (c) or C61 (d). Matrices are presented as in Figs. 2a,b. Note that in a yeast also contained a mutation at position 59 in U4 to repair U4/U6 stem I (ref. 19). For additional positional specificity controls see Supplementary Fig. 1. (e,f) Diagrams of observed (group II intron) and predicted (spliceosome) isomorphic base-triple interactions11 involving the first (f) and second (e) residues of the catalytic triad. Diagrams are as in Fig. 2c.
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Figure 3: Genetic evidence for base-triple interactions between the AGC triad and the ACAGAGA region of the U6 snRNA(a,b) Spot assays showing growth on selective media of equivalent numbers of yeast cells containing combinations of alleles at G52 and G60 (a) or C61 (b). (c,d) Spot assays showing growth on selective media of equivalent numbers of yeast cells containing combinations of alleles at A53 and A59 (c) or C61 (d). Matrices are presented as in Figs. 2a,b. Note that in a yeast also contained a mutation at position 59 in U4 to repair U4/U6 stem I (ref. 19). For additional positional specificity controls see Supplementary Fig. 1. (e,f) Diagrams of observed (group II intron) and predicted (spliceosome) isomorphic base-triple interactions11 involving the first (f) and second (e) residues of the catalytic triad. Diagrams are as in Fig. 2c.

Mentions: The other two positions of the AGC triad, U6-G60 and U6-A59, have been predicted to pair with U6-G52 and U6-A53, respectively, of the conserved ACAGAGA sequence. Importantly, these two residues fall between the 5′ splice site binding site of U6 and U2/U6 helix Ia, which is immediately adjacent to the catalytic core, such that triplex formation would promote docking of the 5′ splice site into the catalytic core (Fig. 1a; ref. 11). All three point mutations at the central position of the AGC triad, G60, are lethal and only the conservative substitution G60A can be suppressed by restoring base-pairing in U2/U6 helix Ib (refs. 15,19). Nevertheless, we found that a mutation of the predicted base-triple residue, G52, suppressed G60U, albeit mildly (Fig. 3a). This marks the first observed suppression of G60U (Supplementary Note 3). Remarkably, suppression of G60U did not require restoration of base-pairing in U2/U6 helix Ib (Fig. 3a). As we observed for mutations at U6-C61 (Fig. 2), suppression of G60U was allele- and position-specific (Fig. 3a-b, Supplementary Fig. 1b, Supplementary Note 4), thus providing compelling evidence for an interaction between G52 of the ACAGAGA sequence and G60 of the AGC triad, in the context of a base triple that includes helix Ib base pairing.


Evidence for a group II intron-like catalytic triplex in the spliceosome.

Fica SM, Mefford MA, Piccirilli JA, Staley JP - Nat. Struct. Mol. Biol. (2014)

Genetic evidence for base-triple interactions between the AGC triad and the ACAGAGA region of the U6 snRNA(a,b) Spot assays showing growth on selective media of equivalent numbers of yeast cells containing combinations of alleles at G52 and G60 (a) or C61 (b). (c,d) Spot assays showing growth on selective media of equivalent numbers of yeast cells containing combinations of alleles at A53 and A59 (c) or C61 (d). Matrices are presented as in Figs. 2a,b. Note that in a yeast also contained a mutation at position 59 in U4 to repair U4/U6 stem I (ref. 19). For additional positional specificity controls see Supplementary Fig. 1. (e,f) Diagrams of observed (group II intron) and predicted (spliceosome) isomorphic base-triple interactions11 involving the first (f) and second (e) residues of the catalytic triad. Diagrams are as in Fig. 2c.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4257784&req=5

Figure 3: Genetic evidence for base-triple interactions between the AGC triad and the ACAGAGA region of the U6 snRNA(a,b) Spot assays showing growth on selective media of equivalent numbers of yeast cells containing combinations of alleles at G52 and G60 (a) or C61 (b). (c,d) Spot assays showing growth on selective media of equivalent numbers of yeast cells containing combinations of alleles at A53 and A59 (c) or C61 (d). Matrices are presented as in Figs. 2a,b. Note that in a yeast also contained a mutation at position 59 in U4 to repair U4/U6 stem I (ref. 19). For additional positional specificity controls see Supplementary Fig. 1. (e,f) Diagrams of observed (group II intron) and predicted (spliceosome) isomorphic base-triple interactions11 involving the first (f) and second (e) residues of the catalytic triad. Diagrams are as in Fig. 2c.
Mentions: The other two positions of the AGC triad, U6-G60 and U6-A59, have been predicted to pair with U6-G52 and U6-A53, respectively, of the conserved ACAGAGA sequence. Importantly, these two residues fall between the 5′ splice site binding site of U6 and U2/U6 helix Ia, which is immediately adjacent to the catalytic core, such that triplex formation would promote docking of the 5′ splice site into the catalytic core (Fig. 1a; ref. 11). All three point mutations at the central position of the AGC triad, G60, are lethal and only the conservative substitution G60A can be suppressed by restoring base-pairing in U2/U6 helix Ib (refs. 15,19). Nevertheless, we found that a mutation of the predicted base-triple residue, G52, suppressed G60U, albeit mildly (Fig. 3a). This marks the first observed suppression of G60U (Supplementary Note 3). Remarkably, suppression of G60U did not require restoration of base-pairing in U2/U6 helix Ib (Fig. 3a). As we observed for mutations at U6-C61 (Fig. 2), suppression of G60U was allele- and position-specific (Fig. 3a-b, Supplementary Fig. 1b, Supplementary Note 4), thus providing compelling evidence for an interaction between G52 of the ACAGAGA sequence and G60 of the AGC triad, in the context of a base triple that includes helix Ib base pairing.

Bottom Line: Here we show by genetics, cross-linking and biochemistry in yeast that analogous triples form in U6 and promote catalytic-metal binding and both chemical steps of splicing.Because the triples include an element that defines the 5' splice site, they also provide a mechanism for juxtaposing the pre-mRNA substrate with the catalytic metals.Our data indicate that U6 adopts a group II intron-like tertiary conformation to catalyze splicing.

View Article: PubMed Central - PubMed

Affiliation: 1] Graduate Program in Cell and Molecular Biology, University of Chicago, Chicago, Illinois, USA. [2] Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, Illinois, USA. [3].

ABSTRACT
To catalyze pre-mRNA splicing, U6 small nuclear RNA positions two metals that interact directly with the scissile phosphates. U6 metal ligands correspond stereospecifically to metal ligands within the catalytic domain V of a group II self-splicing intron. Domain V ligands are organized by base-triple interactions, which also juxtapose the 3' splice site with the catalytic metals. However, in the spliceosome, the mechanism for organizing catalytic metals and recruiting the substrate has remained unclear. Here we show by genetics, cross-linking and biochemistry in yeast that analogous triples form in U6 and promote catalytic-metal binding and both chemical steps of splicing. Because the triples include an element that defines the 5' splice site, they also provide a mechanism for juxtaposing the pre-mRNA substrate with the catalytic metals. Our data indicate that U6 adopts a group II intron-like tertiary conformation to catalyze splicing.

Show MeSH
Related in: MedlinePlus