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Crystal structure of a eukaryotic group II intron lariat.

Robart AR, Chan RT, Peters JK, Rajashankar KR, Toor N - Nature (2014)

Bottom Line: On the basis of structural and biochemical data, we propose that π-π' is a dynamic interaction that mediates the transition between the two steps of splicing, with η-η' serving an ancillary role.The structure also reveals a four-magnesium-ion cluster involved in both catalysis and positioning of the 5' end.Given the evolutionary relationship between group II and nuclear introns, it is likely that this active site configuration exists in the spliceosome as well.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093, USA.

ABSTRACT
The formation of branched lariat RNA is an evolutionarily conserved feature of splicing reactions for both group II and spliceosomal introns. The lariat is important for the fidelity of 5' splice-site selection and consists of a 2'-5' phosphodiester bond between a bulged adenosine and the 5' end of the intron. To gain insight into this ubiquitous intramolecular linkage, we determined the crystal structure of a eukaryotic group IIB intron in the lariat form at 3.7 Å. This revealed that two tandem tetraloop-receptor interactions, η-η' and π-π', place domain VI in the core to position the lariat bond in the post-catalytic state. On the basis of structural and biochemical data, we propose that π-π' is a dynamic interaction that mediates the transition between the two steps of splicing, with η-η' serving an ancillary role. The structure also reveals a four-magnesium-ion cluster involved in both catalysis and positioning of the 5' end. Given the evolutionary relationship between group II and nuclear introns, it is likely that this active site configuration exists in the spliceosome as well.

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The position of DVI within the intron structure. a, DII forms an inverted Y-shaped structure that engages in four distinct tetraloop-receptor interactions with the IC stem (orange), DIII (yellow), and DVI (purple). DVI interacts with DII via two tandem tetraloop-receptor interactions, η-η’ and π-π’. b, In vitro self-splicing assays of the P.li.LSUI2 intron. The wild-type (WT) intron efficiently catalyzes both steps of splicing and forms intron lariat and ligated exons. Mutagenesis of either η-η’ (Δη’)20 or π-π’ (Δπ) inhibits the second step, resulting in the accumulation of lariat-3′ exon and 5′ exon. A combination of both mutations (Δη’Δπ) nearly blocks the second step of splicing with predominantly lariat-3′ exon present. c, Fo–Fc density for the 2′-5′ lariat phosphodiester bond contoured at 3σ. The nucleobase of A615 is disordered and not visualized. This map was calculated using a model deleted for A615, G1, and U2 (shown in stick format) to avoid model bias. The 5′ (G1) and 3′ (A620) ends form a non-canonical base pair.
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Figure 3: The position of DVI within the intron structure. a, DII forms an inverted Y-shaped structure that engages in four distinct tetraloop-receptor interactions with the IC stem (orange), DIII (yellow), and DVI (purple). DVI interacts with DII via two tandem tetraloop-receptor interactions, η-η’ and π-π’. b, In vitro self-splicing assays of the P.li.LSUI2 intron. The wild-type (WT) intron efficiently catalyzes both steps of splicing and forms intron lariat and ligated exons. Mutagenesis of either η-η’ (Δη’)20 or π-π’ (Δπ) inhibits the second step, resulting in the accumulation of lariat-3′ exon and 5′ exon. A combination of both mutations (Δη’Δπ) nearly blocks the second step of splicing with predominantly lariat-3′ exon present. c, Fo–Fc density for the 2′-5′ lariat phosphodiester bond contoured at 3σ. The nucleobase of A615 is disordered and not visualized. This map was calculated using a model deleted for A615, G1, and U2 (shown in stick format) to avoid model bias. The 5′ (G1) and 3′ (A620) ends form a non-canonical base pair.

Mentions: In the O. iheyensis structure, DII was drastically truncated to a small stem loop structure, and in many previous biochemical studies of the aI5γ intron, DII was similarly shortened to study the first step of splicing. We can now visualize the intact DII substructure and find that it serves as a central hub for four different tetraloop receptor interactions (Fig. 3a). DII makes contacts with domains I, III, and VI to organize a large portion of the intron structure.


Crystal structure of a eukaryotic group II intron lariat.

Robart AR, Chan RT, Peters JK, Rajashankar KR, Toor N - Nature (2014)

The position of DVI within the intron structure. a, DII forms an inverted Y-shaped structure that engages in four distinct tetraloop-receptor interactions with the IC stem (orange), DIII (yellow), and DVI (purple). DVI interacts with DII via two tandem tetraloop-receptor interactions, η-η’ and π-π’. b, In vitro self-splicing assays of the P.li.LSUI2 intron. The wild-type (WT) intron efficiently catalyzes both steps of splicing and forms intron lariat and ligated exons. Mutagenesis of either η-η’ (Δη’)20 or π-π’ (Δπ) inhibits the second step, resulting in the accumulation of lariat-3′ exon and 5′ exon. A combination of both mutations (Δη’Δπ) nearly blocks the second step of splicing with predominantly lariat-3′ exon present. c, Fo–Fc density for the 2′-5′ lariat phosphodiester bond contoured at 3σ. The nucleobase of A615 is disordered and not visualized. This map was calculated using a model deleted for A615, G1, and U2 (shown in stick format) to avoid model bias. The 5′ (G1) and 3′ (A620) ends form a non-canonical base pair.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4197185&req=5

Figure 3: The position of DVI within the intron structure. a, DII forms an inverted Y-shaped structure that engages in four distinct tetraloop-receptor interactions with the IC stem (orange), DIII (yellow), and DVI (purple). DVI interacts with DII via two tandem tetraloop-receptor interactions, η-η’ and π-π’. b, In vitro self-splicing assays of the P.li.LSUI2 intron. The wild-type (WT) intron efficiently catalyzes both steps of splicing and forms intron lariat and ligated exons. Mutagenesis of either η-η’ (Δη’)20 or π-π’ (Δπ) inhibits the second step, resulting in the accumulation of lariat-3′ exon and 5′ exon. A combination of both mutations (Δη’Δπ) nearly blocks the second step of splicing with predominantly lariat-3′ exon present. c, Fo–Fc density for the 2′-5′ lariat phosphodiester bond contoured at 3σ. The nucleobase of A615 is disordered and not visualized. This map was calculated using a model deleted for A615, G1, and U2 (shown in stick format) to avoid model bias. The 5′ (G1) and 3′ (A620) ends form a non-canonical base pair.
Mentions: In the O. iheyensis structure, DII was drastically truncated to a small stem loop structure, and in many previous biochemical studies of the aI5γ intron, DII was similarly shortened to study the first step of splicing. We can now visualize the intact DII substructure and find that it serves as a central hub for four different tetraloop receptor interactions (Fig. 3a). DII makes contacts with domains I, III, and VI to organize a large portion of the intron structure.

Bottom Line: On the basis of structural and biochemical data, we propose that π-π' is a dynamic interaction that mediates the transition between the two steps of splicing, with η-η' serving an ancillary role.The structure also reveals a four-magnesium-ion cluster involved in both catalysis and positioning of the 5' end.Given the evolutionary relationship between group II and nuclear introns, it is likely that this active site configuration exists in the spliceosome as well.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093, USA.

ABSTRACT
The formation of branched lariat RNA is an evolutionarily conserved feature of splicing reactions for both group II and spliceosomal introns. The lariat is important for the fidelity of 5' splice-site selection and consists of a 2'-5' phosphodiester bond between a bulged adenosine and the 5' end of the intron. To gain insight into this ubiquitous intramolecular linkage, we determined the crystal structure of a eukaryotic group IIB intron in the lariat form at 3.7 Å. This revealed that two tandem tetraloop-receptor interactions, η-η' and π-π', place domain VI in the core to position the lariat bond in the post-catalytic state. On the basis of structural and biochemical data, we propose that π-π' is a dynamic interaction that mediates the transition between the two steps of splicing, with η-η' serving an ancillary role. The structure also reveals a four-magnesium-ion cluster involved in both catalysis and positioning of the 5' end. Given the evolutionary relationship between group II and nuclear introns, it is likely that this active site configuration exists in the spliceosome as well.

Show MeSH