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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We thank S. Banerjee and the staff of the NE-CAT beamlines at the Advanced Photon Source (APS) of Argonne National Laboratory. We thank P. Ghosh, S. Joseph, G. Ghosh, R. Doolittle, Y. Tor, D. Donoghue and T. Wiryaman for comments on the manuscript. We thank R. Das and F.-C. Chou for assistance with phenix.erasser for structure refinement and G. Bricogne for advice on Buster refinement. We also thank N. T. Schirle for preliminary biochemical characterization of the P.li.LSUI2 intron. R.T.C. was supported by the Cell, Molecular, and Genetics Training Program funded by NIH predoctoral training grant 5T32GM007240. J.K.P. was supported by the UCSD Molecular Biophysics Training Program funded by NIH predoctoral training grant 5T32GM008326. NE-CAT is supported by NIH grant 8P41GM103403-10 and APS is supported by the US DOE under contract number DE-AC02-06CH11357. This work was supported by a Hellman Foundation Fellowship and NIH grant 5R01GM102216 awarded to N.T.
The authors declare no competing financial interests.
Extended data figures and tables
Tertiary interactions are indicated with Greek letters and domains are labelled with Roman numerals. Colouring of the individual domains is consistent with the overall view of the tertiary structure shown in Fig. 1.
Extended Data Figure 2 The Yb-MAD experimental, density-modified map of the portion of DV containing the catalytic triad contoured at 1.8σ.
The EBS1–IBS1 and EBS2–IBS2 interactions position the 5′ exon. They do not form a continuous binding interface with the presence of a highly distorted backbone at the junction between these two motifs. As a result, the helical axes of the EBS1–IBS1 and EBS2–IBS2 pairings are positioned ∼90° relative to each other. The EBS3–IBS3 interaction places the 3′ exon in the active site.
Individual domains and subdomains are depicted in different colours. Domain names are labelled with Roman numerals. a and b show different rotations of the intron structure.
Extended Data Figure 5 Companion to Fig. 2 showing the location of the individual tertiary interactions relative to the overall structure.
a, κ–κ′. b, ε–ε′. c, ρ–ρ′.
Extended Data Figure 6 Splicing assays for the DVI mutants showing the proportion of branched product.
Blue and orange bars correspond to lariat 3′ exon and lariat, respectively. The Δη′ and Δπ mutants accumulate large amounts of lariat 3′ exon, thus indicating a second-step splicing defect. The Δη′Δπ double mutant is almost completely blocked before the second step. The A620C mutant shows 2.4-fold greater accumulation of lariat 3′ exon compared to the wild-type intron, indicating that the interaction between G1 and A620 is important for the second step. In the yeast aI5γ intron, G1 instead interacts with the penultimate residue44, indicating a certain degree of flexibility for this pairing. There is evidence for a similar interaction between the termini of nuclear introns45 involving nearby (but not exactly equivalent) residues, which also has a significant effect upon the second step of splicing. Therefore, the 5′ and 3′ ends of nuclear introns may have a similar arrangement within the spliceosome.
a, b, Depiction of the RNA ligands surrounding metals M3 and M4, respectively. Yb3+ anomalous map contoured at 9σ. c, Yb3+ anomalous map for wild type contoured at 9σ. d, Compared with the wild-type intron, the Yb3+ anomalous map for the G79A mutant (contoured at 4σ) is lacking the peaks corresponding to M3 and M4, even at a lower contour level. e, Tl+ was used as a probe for monovalent ions in the RNA structure46. The Tl+ anomalous map (purple mesh contoured at 5.5σ) revealed a strong peak located 3.8 Å from M1 that coordinates to the nucleobase of J2/3 residue G421 and the backbone of DV nucleotide G550. This sodium ion Na1 (purple sphere) is significantly closer to M1 than the equivalent K+ ion found in O. iheyensis25. Otherwise, this monovalent ion binding site is relatively conserved between these two introns.
The η–η′ interaction persists throughout the splicing reaction and is visible in the pre-catalytic state. The weaker density for the central region of DVI suggests a partially disordered, dynamic region with possible helical remodelling in the conserved internal loop during splicing. The general pattern of side-by-side packing of domains II and VI persists between the two steps. Catalytic triad mutation consisted of an AGC→GAU substitution.
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Robart, A., Chan, R., Peters, J. et al. Crystal structure of a eukaryotic group II intron lariat. Nature 514, 193–197 (2014). https://doi.org/10.1038/nature13790
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