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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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The U6 triplex promotes exon ligation in vitroa, Configuration of the C288*C358-G385 base triple in the group II intron (PDB 4FAQ, ref. 8), equivalent to the spliceosomal U6-A53*U6-A59/U2-U23 base triple; left, side view; right, top view. b, Denaturing PAGE analysis of splicing of ACT1 pre-mRNA in extracts reconstituted with the indicated U6 variants. No dep., no depletion; no rec., no reconstitution. Upper case indicates wild-type allele. c, Quantification of exon ligation for the indicated U6 variants, normalized to wild-type U6; exon ligation was calculated as mRNA/lariat intermediate (ref. 19). Error bars represent s.d. of two independent experiments and two technical replicates for each experiment. The efficiency of branching was within 15% of wild type for all U6 variants (quantification not shown). For full gel see Supplementary Fig. 8i.
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Figure 8: The U6 triplex promotes exon ligation in vitroa, Configuration of the C288*C358-G385 base triple in the group II intron (PDB 4FAQ, ref. 8), equivalent to the spliceosomal U6-A53*U6-A59/U2-U23 base triple; left, side view; right, top view. b, Denaturing PAGE analysis of splicing of ACT1 pre-mRNA in extracts reconstituted with the indicated U6 variants. No dep., no depletion; no rec., no reconstitution. Upper case indicates wild-type allele. c, Quantification of exon ligation for the indicated U6 variants, normalized to wild-type U6; exon ligation was calculated as mRNA/lariat intermediate (ref. 19). Error bars represent s.d. of two independent experiments and two technical replicates for each experiment. The efficiency of branching was within 15% of wild type for all U6 variants (quantification not shown). For full gel see Supplementary Fig. 8i.

Mentions: Because our crosslinking experiments implied that the triplex is present during exon ligation (Fig. 6d), we tested whether exon ligation required the triplex. Of the bases involved in the triplex, only mutations at A59 show a specific defect in exon ligation18. Our in vivo results suggest that A59 forms a base-triple interaction with A53, similar to that observed for the equivalent positions in the group II intron (Fig. 3c, Fig. 8a). Thus, we asked whether the exon ligation defects conferred by U6-A59 mutations could be suppressed by mutations at U6-A53. Strikingly, the A53C mutation strongly improved exon ligation efficiency for both A59U and A59G (Fig. 8b,c). Suppression was allele-specific because A59G was enhanced rather than suppressed by other A53 mutations and A59U was only mildly suppressed by other A53 mutations. Importantly, the observed suppression was also position-specific, as neither A59G nor A59U were substantially suppressed by a mutation at the neighboring G52 (Fig. 8b,c), which nevertheless suppressed the exon ligation defect conferred by a mutation at its base-triple partner G60 (Supplementary Fig. 7a,b). A53C also suppressed the exon ligation defect of compromised pre-mRNA reporters both in vitro and in vivo (Supplementary Fig. 7d,e). Overall the allele- and position-specific suppression of exon ligation defects we observed in vitro explicitly paralleled the suppression we observed in vivo (Fig. 3a,c). As in vivo, we infer that the in vitro suppression reflects the formation of a group II-like base-triple that includes an interaction between A53C and the backbone of A59 (Fig. 8a). Together, these results indicate that the U6 triplex not only forms at the exon ligation stage (Fig. 6d) but also functions at this catalytic stage both in vivo and in vitro.


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)

The U6 triplex promotes exon ligation in vitroa, Configuration of the C288*C358-G385 base triple in the group II intron (PDB 4FAQ, ref. 8), equivalent to the spliceosomal U6-A53*U6-A59/U2-U23 base triple; left, side view; right, top view. b, Denaturing PAGE analysis of splicing of ACT1 pre-mRNA in extracts reconstituted with the indicated U6 variants. No dep., no depletion; no rec., no reconstitution. Upper case indicates wild-type allele. c, Quantification of exon ligation for the indicated U6 variants, normalized to wild-type U6; exon ligation was calculated as mRNA/lariat intermediate (ref. 19). Error bars represent s.d. of two independent experiments and two technical replicates for each experiment. The efficiency of branching was within 15% of wild type for all U6 variants (quantification not shown). For full gel see Supplementary Fig. 8i.
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Figure 8: The U6 triplex promotes exon ligation in vitroa, Configuration of the C288*C358-G385 base triple in the group II intron (PDB 4FAQ, ref. 8), equivalent to the spliceosomal U6-A53*U6-A59/U2-U23 base triple; left, side view; right, top view. b, Denaturing PAGE analysis of splicing of ACT1 pre-mRNA in extracts reconstituted with the indicated U6 variants. No dep., no depletion; no rec., no reconstitution. Upper case indicates wild-type allele. c, Quantification of exon ligation for the indicated U6 variants, normalized to wild-type U6; exon ligation was calculated as mRNA/lariat intermediate (ref. 19). Error bars represent s.d. of two independent experiments and two technical replicates for each experiment. The efficiency of branching was within 15% of wild type for all U6 variants (quantification not shown). For full gel see Supplementary Fig. 8i.
Mentions: Because our crosslinking experiments implied that the triplex is present during exon ligation (Fig. 6d), we tested whether exon ligation required the triplex. Of the bases involved in the triplex, only mutations at A59 show a specific defect in exon ligation18. Our in vivo results suggest that A59 forms a base-triple interaction with A53, similar to that observed for the equivalent positions in the group II intron (Fig. 3c, Fig. 8a). Thus, we asked whether the exon ligation defects conferred by U6-A59 mutations could be suppressed by mutations at U6-A53. Strikingly, the A53C mutation strongly improved exon ligation efficiency for both A59U and A59G (Fig. 8b,c). Suppression was allele-specific because A59G was enhanced rather than suppressed by other A53 mutations and A59U was only mildly suppressed by other A53 mutations. Importantly, the observed suppression was also position-specific, as neither A59G nor A59U were substantially suppressed by a mutation at the neighboring G52 (Fig. 8b,c), which nevertheless suppressed the exon ligation defect conferred by a mutation at its base-triple partner G60 (Supplementary Fig. 7a,b). A53C also suppressed the exon ligation defect of compromised pre-mRNA reporters both in vitro and in vivo (Supplementary Fig. 7d,e). Overall the allele- and position-specific suppression of exon ligation defects we observed in vitro explicitly paralleled the suppression we observed in vivo (Fig. 3a,c). As in vivo, we infer that the in vitro suppression reflects the formation of a group II-like base-triple that includes an interaction between A53C and the backbone of A59 (Fig. 8a). Together, these results indicate that the U6 triplex not only forms at the exon ligation stage (Fig. 6d) but also functions at this catalytic stage both in vivo and in vitro.

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