Now on display: a gallery of group II intron structures at different stages of catalysis
© Marcia et al.; licensee BioMed Central Ltd. 2013
Received: 31 January 2013
Accepted: 8 April 2013
Published: 1 May 2013
Group II introns are mobile genetic elements that self-splice and retrotranspose into DNA and RNA. They are considered evolutionary ancestors of the spliceosome, the ribonucleoprotein complex essential for pre-mRNA processing in higher eukaryotes. Over a 20-year period, group II introns have been characterized first genetically, then biochemically, and finally by means of X-ray crystallography. To date, 17 crystal structures of a group II intron are available, representing five different stages of the splicing cycle. This review provides a framework for classifying and understanding these new structures in the context of the splicing cycle. Structural and functional implications for the spliceosome are also discussed.
KeywordsRetrotransposition Spliceosome X-ray structures RNA catalysis Metal ions
Group II introns are mobile ribozymes capable of self-splicing and retrotransposition . As retrotransposable elements, group II introns have invaded the genomes of most life forms and enhanced genomic diversity in all domains of life. In this way, they have played a crucial role in the evolution of modern organisms [2, 3]. At the present time, they remain important in archaea, bacteria, and unicellular and multicellular eukaryotes because they ensure the correct expression of certain housekeeping genes and because they hinder the distribution of other harmful mobile genetic elements [4, 5]. Of particular interest to the field of RNA processing, group II introns are considered evolutionary ancestors of the spliceosome, which is the ribonucleoprotein complex essential for pre-mRNA processing in higher eukaryotes, including humans [6–8]. Finally, group II introns are potentially useful medical tools, because they can be artificially reprogrammed to insert into desired DNA or RNA sites [9–11]. Consequently, they are macromolecules of great microbiological, biotechnological and pharmacological interest.
Available 3-D X-ray structures of the group II intron
5′-exon hydrolysis (pre)
No (active site mutant)
5′-exon hydrolysis (pre)
No (nonfunctional divalent ion)
5′-exon hydrolysis (post)
Partial (nonfunctional monovalent ion)
Partial (nonfunctional monovalent ion)
No (nonfunctional divalent ion)
No (nonfunctional monovalent ion)
Partial (active site mutant)
No (nonfunctional divalent ion)
No (nonfunctional divalent ion)
The purpose of this review is to provide a framework for classifying these new structures and understanding them in the context of the splicing cycle. After providing a brief summary of all available 3-D structures of the group II introns, the catalytic cycle will be outlined in a step-by-step fashion. Each catalytic event will be presented in a way that highlights structural details while describing the experimental strategy used to capture each state crystallographically. Finally, the implications of all group II intron structures for interpreting spliceosomal function will also be discussed.
Overview of available group II intron structures
Five different constructs have been used for crystallizing the group II introns to date. They all correspond to the Oceanobacillus iheyensis group II intron. Its wild type sequence was initially modified by adding a GAAA tetraloop to the terminus of the D2 stem, by inserting an RNA hairpin in place of D4, by truncating the D6 stem to about half its length, and by supplying native exons at the 5′- and 3′-ends . These modifications resulted in the construct named here OiD1-6. From OiD1-6, two other constructs were derived, specifically by mutating the catalytic residue G359 to adenosine (construct OiD1-6-G359A, ), or by removing D6 and the flanking exons (construct OiD1-5, ). Finally, from OiD1-5 the construct Oi5eD1-5 was obtained by adding the short 5′-exon sequence UUAU at the 5′-end, and the construct OiD1-5-C377G was obtained by a point mutation at the catalytic position 377 .
Two structures describe conformational rearrangements that occur between the first and second splicing steps. These are 4FAR (2.86 Å resolution) and 4FAU (2.87 Å resolution) .
The postcatalytic state of the intron is represented by structure 3IGI (3.13 Å resolution) .
Seven structures reflect the ligand-free, linear form of the intron. These mimic the state of the ribozyme that is released after exon ligation, and were obtained using construct OiD1-5 crystallized in the presence of different metal ions: K+/Mg2+ (4E8M, 3.50 Å resolution), Rb+/Mg2+ (4E8P, 3.28 Å resolution), Tl+/Mg2+ (4E8Q, 2.84 Å resolution), Cs+/Mg2+ (4E8R, 3.36 Å resolution), NH4 +/Mg2+ (4E8N, 2.96 Å resolution), Na+/Mg2+ (4FAX, 3.10 Å resolution), and K+/Ba2+ (4E8V, 3.99 Å resolution) . A ligand-free form was also obtained for the functionally impaired C377G mutant (4FB0, 3.22 Å resolution). Most of the ligand-free structures represent active (K+/Mg2+, Rb+/Mg2+, Tl+/Mg2+, NH4 +/Mg2+) or partially active (Cs+/Mg2+) states that mimic the retrotransposable form of the intron before it binds target substrates .
Four structures correspond to the retrotransposable form of the intron after target substrate binding. These structures were obtained by crystallizing the spliced (OiD1-6) or ligand-free (OiD1-5) intron with oligonucleotides that mimic ligated exons. They are 3EOG (3.39 Å resolution) , 4E8K (3.03 Å resolution) , 4E8T (3.34 Å resolution) , and 4FAW (2.70 Å resolution), respectively .
The precatalytic state
Upon transcription, the O.i. group II intron folds spontaneously into a stable tertiary structure, forming a ribozyme that is highly reactive in the presence of Mg2+. Therefore, to trap the intron in its precatalytic state crystallographically (Figure 1A1), it was necessary to deactivate the intron and prevent hydrolysis at the 5′-splice site. Two different inactivation methods have been used, namely site-directed mutagenesis  and metal-ion replacement .
The first approach (structure 4DS6) involves mutation of an invariant residue (G359) belonging to the catalytic triad motif in D5 [32–36]. Since G359 is part of a helix, in which it forms a G•U wobble pair with the partner strand, adenosine was chosen to substitute guanosine and form an A-U pair. Considering that the atoms shaping the intron active site are primarily backbone oxygen atoms, the G359A mutation was expected to cause only minimal modification of the RNA structure . Indeed, in comparison to the wild-type intron, the structural perturbation in the mutant is very limited (overall RMSD = 1.2 Å). As expected, the mutation permits visualization of the 5′-splice junction. Constrained by the tight base pairing of the 5′-exon to EBS1, the junction adopts a sharp kink and forms an unusually small angle of approximately 50° between the two phosphate groups that flank the scissile phosphate . Surprisingly, however, the perturbation of the active site induced by the G359A mutation was sufficient to prevent binding of catalytic metals, which explains why activity is abolished almost completely . The cause of this loss of metal ion binding was explained by later studies, which elucidated the network of interactions that anchor metals in the core .
Taken together, the structures of the precatalytic state establish how the intron mediates two essential attributes of splicing, namely efficiency and fidelity, using the EBSs and a four-metal heteronuclear center.
By contrast, splicing fidelity is linked to the appropriate pairing of EBS-intron binding site (IBS) elements. Structure 4DS6 shows that the formation of the EBS1-IBS1 interaction is sufficient to place the 5′-splice junction correctly in the active site even if other elements, including the metal cluster, are not well positioned. Also the intron structures solved using OiD1-5 in a ligand-free state (vide infra) provide an illustrative example of how splicing fidelity is achieved. Specifically, OiD1-5 possesses a short poly-G sequence (GGG) at its 5′-end, and this fails to interact with the EBS1 site. This sequence is artificially inserted immediately downstream of the T7 promoter in order to enhance the yield of in vitro transcription by T7 RNA polymerase [46–48]. Because the GGG sequence is different from that of the native 5′-exon (UUAU) and therefore does not possess any complementarity to EBS1 (AUAA, Figure 2), the 5′-splice junction in those structures is flexible and completely excluded from the active site, even when the catalytic metal center is intact . Thus, EBS1 is highly specific in choosing its partner nucleotides at the 5′-splice site, as supported also by biochemical evidence .
Putative position of the branching nucleotide
A conformational transition to the second splicing step
After the first splicing step, the intron active site becomes rearranged before performing the second transesterification reaction. Specifically, D5 is known to become rearranged, thanks to the flexibility of its two-nucleotide bulge motif [12, 56], while D6 toggles between an active state coordinated to the κ-coordination loop or the D1C helix, and a silent state forming the η-η' interaction with D2 [21, 28, 57]. However, biochemical experiments such as crosslinking studies  and all available crystal structures suggest that a group II intron possesses only one catalytic site for both the first and second splicing steps [12, 24, 58].
Given these observations, one might assume that the reactants for the second splicing step, which remains crystallographically uncharacterized, are already aligned correctly for catalysis in the precatalytic state. However, this is not the case, as long-range interactions involving the second splicing step reactants have been shown to form only between the first and the second splicing steps, or to affect selectively the second and not the first splicing step (that is, the γ-γ' interaction, the interaction between the first and the penultimate intron nucleotides, the IBS3-EBS3 interaction and the η-η' interaction [59–61]). Furthermore, in the structures, the nucleophile of the first splicing step is located near the EBS3 site, in the identical position that must be occupied by the 3′-splice junction during the second splicing step . Therefore, there is also a structural incompatibility that prohibits accommodation of all reactants in the same active site at once. Consequently, a rearrangement of the active site between the splicing steps is likely to happen.
In light of recent structures, more detailed hypotheses about such a rearrangement can be proposed. The structures suggest two types of conformational rearrangements, one involving a movement of the hydrolyzed scissile phosphate (Figure 1A1), the other a movement of the J2/3 junction and the two-nucleotide bulge (Figure 1B). The first conformational rearrangement, which directly follows 5′-exon cleavage, was visualized by crystallizing Oi5eD1-5 in the presence of the physiological, catalytically functional ions Mg2+ and K+ (structure 4FAR, reference  and Figures 1 and S1 therein). Upon hydrolysis, which occurs during the crystallization process, the 5′-exon maintains coordination to M1 through its 3′-OH group and is not displaced significantly from its binding site, as expected since the 5′-exon is the nucleophile of the second splicing step. Instead, hydrolysis induces relaxation of the kinked RNA backbone at the 5′-splice junction and the hydrolyzed scissile phosphate is released from the active site. Specifically, the free phosphate is displaced by about 4 Å, where it interacts directly with the K2 site, which evidently plays a direct role in organizing, and potentially liberating, splicing products. The second conformational rearrangement was visualized in a structure of Oi5eD1-5 solved in the presence of Li+/Mg2+ (4FAU) . In this structure, the 5′-exon has undergone hydrolysis and one observes an equilibrium between two conformations in the active site: the catalytic triple helix conformation and an inactive toggled conformation. The conformational change involves two residues in the J2/3 junction (G288-C289) and one residue in the two-nucleotide bulge (C377, D5), all known to be dynamic elements of group II introns [12, 58]. In the inactive toggled conformation, which is visualized most clearly when the intron is crystallized in a buffer of Na+/Mg2+ (structure 4FAX, see reference  and Figure 4 therein), G288 rotates by about 90° around an axis connecting its C5′ and C3′ backbone atoms, while the cytosine moiety of C377 rotates by about 70° around the glycosidic bond. Both residues in the inactive toggled conformation are stabilized by a new network of interactions. Among these, two involve the 2′-OH groups of both residues, which do not form any interactions in the triple helix conformation typical of the precatalytic state. These interactions are particularly interesting because the two hydroxyl groups had previously been demonstrated to be important in catalysis using biochemical methods, but their role was unclear until now [32, 34]. In addition to disrupting the triple helix, the conformational rearrangement also moves the RNA ligands that are essential for anchoring the M1-M2-K1-K2 metal center. This causes the interactions between catalytic ions and the 5′-splice junction to be broken and facilitates the release of the latter.
In summary, therefore, a concerted conformational rearrangement appears likely to promote the transition to the second splicing step. Considering the central role of the residues involved in the rearrangement, we cannot exclude the notion that the inactive toggled intron conformation might also occur at other points of the splicing cycle, and we would like to suggest two scenarios to support this hypothesis. First, the inactive toggled conformation may represent an intermediate conformation that occurs while the intron folds into its active, precatalytic state. This hypothesis is supported by the fact that a mutant designed to stabilize the inactive toggled conformation (C377G) shows a tenfold reduction in the rate of the first splicing step in addition to its pronounced defect in the second splicing step (see reference  and Figure S5 therein). Second, the opening of the triple helix and the consequent disruption of the active site metal cluster may be important for successfully terminating the splicing cycle, when the ligated exons must be released from the active site to form a free intron. The inactive toggled conformation would prevent ligated exons from being rehydrolyzed through SER, which is a prevalent in vitro side-reaction that represents a major problem for productive splicing in vivo.
Second splicing step
The second splicing step remains an important area for future structural studies, as it has not been fully elucidated by existing structures. Two sets of structures would be required to describe its mechanism at a molecular level, namely the structure of the state preceding cleavage of the 3′-splice junction and the structure of the postcatalytic state. While the latter can be represented by structure 3IGI (Figure 1C), which corresponds to the postcatalytic linear intron harboring products of the splicing reaction in its active site [26, 27]; the former structure is not yet available and can only be deduced from modeling exercises (Figure 1C).
Postcatalytic ligand-free state
Upon completion of the splicing reaction, the ligated exons are released from the active site and the free intron is liberated in a linear or lariat form. While the structure of the lariat form is not yet available, many structures have been obtained for the linear form (4E8M, 4E8P, 4E8R, 4E8Q, 4E8N, 4E8V, 4FAX and 4FB0; see Figure 1D) .
To obtain the structures of the excised intron in a ligand-free state (that is, without any bound exons or ligated exons), it was necessary to prevent co-crystallization of exon-like fragments deriving from the splicing reaction and from self-degradation of the intron . To this end, we utilized construct OiD1-5, which folds spontaneously during transcription in vitro, and adopts a homogeneous, active conformation after purification, yielding a free, multiple-turnover ribozyme that is a good mimic for the postcatalytic state of the intron . The ligand-free intron structures are nearly identical to the available ligand-bound ones, which is a fairly typical case for protein enzymes and ribozymes that catalyze two-metal ion phosphodiester cleavage reactions . All residues are visible in the electron density and only the EBS1 site is slightly disordered, as expected given the absence of base-pairing with a corresponding IBS1 sequence. Despite their overall similarity to the ligand-bound states of the intron, the ligand-free intron structures show remarkable features, particularly in terms of the catalytic metal ions.
First, the ligand-free structures show that, even in the absence of K+, monovalent ions like Tl+, Rb+, Cs+, Na+ and NH4+, and divalent ions like Ba2+ can support the correct folding of the intron scaffold. Therefore, these structures unambiguously reveal the identity of numerous important metal binding sites. These observations demonstrate a remarkable adaptability of group II introns, and potentially other large RNA molecules, to different metal ions. This is important given that metal ions are very useful tools for studying large RNAs, not only crystallographically [30, 64], but also spectroscopically [65, 66] and biochemically .
Second, the ligand-free structures show that the catalytic metal center M1-M2-K1-K2 is correctly bound within the active site when the intron is crystallized in the presence of physiological ions (Mg2+/K+), or any other ions that support chemical catalysis. This observation is surprising considering that the metals – in particular M1 and M2 – are less tightly coordinated and more exposed to the solvent in the absence of the exons. Indeed, in the ligand-free structures M1-M2 are bridged by a water molecule that occupies the position of the scissile phosphate oxygen . This water molecule is therefore likely to represent an important element in the ligand-free active site, because it completes the KMgO cluster. The integrity of the active site in the ligand-free intron supports the observation that this ribozyme is a highly efficient retrotransposable element.
SER and retrotransposition
The structure of the empty, ligand-free intron sets the stage for understanding the mechanism of its retrotransposition into genomic DNA or into RNA (Figure 1E) . The first retrotransposition step (which is a reverse-splicing reaction) is thought to be approximated in vitro by the spliced-exon reopening reaction, where ligated exons are bound and then attacked by the free intron, because the chemistry of the two reactions is known to be identical [13–15]. Both the precatalytic and the postcatalytic states of the SER reaction have now been characterized crystallographically using RNA substrates (structures 3EOG, 4E8K, 4E8T and 4FAW [30, 31]).
The precatalytic state of SER was first visualized in 2008, when a self-spliced intron was co-crystallized with an oligonucleotide mimicking ligated exons (structure 3EOG) . In another approach to visualizing the precatalytic state of SER, construct OiD1-5 was co-crystallized in the presence of Ca2+ with an oligonucleotide that corresponds to the sequence of native ligated exons (structures 4E8K and 4E8T) . These latter structures revealed the presence of an intact active site, whose geometry is highly reminiscent of that of the precatalytic state preceding 5′-exon hydrolysis. The scissile phosphate of the substrate is located between the M1 and M2 sites, presenting the pro-S oxygen atom at approximately 2 Å from each of the two metals. The stereochemistry of the scissile phosphate in the structure is thus in perfect agreement with previous biochemical experiments that had predicted a preference for the pro-S configuration on the basis of phosphorothioate substitutions . Moreover, the 5′-exon portion of the oligonucleotide tightly binds to the EBS1 site, while the 3′-exon nucleotide shows a well-defined Watson–Crick base-pairing only for the uridine at the scissile position (IBS3) with the corresponding EBS3 adenosine. M1 coordinates to the leaving group (the 3′-OH of the nucleotide in 5′ to the scissile phosphate), while M2 coordinates to the scissile phosphate oxygen, in agreement with the two-metal ion mechanistic hypothesis . By contrast, the structure of the post-hydrolytic state of SER was obtained using the OiD1-5 construct, bound to the same oligonucleotide used for solving 4E8K and 4E8T, but co-crystallized in the presence of physiological ions Mg2+ and K+ (structure 4FAW) . This structure currently represents the structure of the intron at the highest resolution ever achieved (2.7 Å) and so far the highest resolution structure of a noncoding RNA longer than 200 nucleotides, except for the ribosomal subunits. In this structure, the 5′-exon portion of the oligonucleotide is visible in the electron density, as it forms base pairs with the EBS1 binding site in the same position as in the pre-hydrolytic state. By contrast, the 3′-end has been released and, as happens for ligand-free structures, the KMgO cluster is completed by a water molecule bound between M1 and M2.
The structures of the IBS-EBS interactions and of the metal center of the SER reaction are particularly significant, because they help in understanding the mechanism of the second splicing step, as discussed above. Furthermore, a solvent molecule coordinated by C358 in the catalytic triad and by M2 can also be identified in the precatalytic state (structures 4E8K and 4E8T) at about 3.2 Å from the scissile phosphate, in a direct line with the scissile P-O bond . This positioning, which is identical to that of the nucleophile for the first splicing step, suggests that this solvent molecule likely represents the reaction nucleophile of the SER reaction. Therefore, it represents the most likely location occupied by the nucleophile of the first reverse-splicing step, namely the 3′-OH group of the last intron nucleotide. These observations further corroborate the hypothesis of a single major active site for group II introns  and shed light on the molecular mechanism of the retrotransposition event. Certainly, to obtain a more complete visualization of the reverse-splicing reaction, it will be necessary to crystallize the intron in complex with DNA substrates.
Implications for the spliceosome
Besides revealing the molecular mechanism of different stages of the intron splicing cycle, the structures described so far also provide new evidence to support the idea that group II introns may be functionally and structurally related to the spliceosome [6–8]. Therefore, we will briefly discuss how the intron structures contribute to a deeper understanding of spliceosomal architecture and function.
Group II introns and the spliceosome have many strong analogies. Sequence conservation analyses revealed precise correspondence of active site motifs in the two systems . Specifically, the catalytic triad is well conserved within intron D5 and in the spliceosomal snRNA subunit U6 , the J2/3 junction (intron D2-3) corresponds to residues in the conserved spliceosomal ACAGAGA box (U6) , and the two-nucleotide bulge motif (intron D5) is likely to correspond to bulged residues either in the internal stem loop of U6 (U80, [71, 73]) or in U2-U6 helix I (A25, [30, 74]). Mutations at any of these conserved positions have similar effects in the two systems [14, 58, 75, 76]. Besides sequence similarities, the two macromolecules also share similar preferences for the stereochemical configuration of the scissile phosphate in the two splicing steps [15, 63, 77]. Moreover, the metal ion requirements are strikingly similar in both the intron and the spliceosome. Not only are both machineries selectively dependent on magnesium as a divalent ion [4, 78], but they are also both tightly controlled by monovalent ions, that is, potassium [50, 79]. Finally, both macromolecules are known to pause in transiently inactive states to regulate the transitions between the different splicing steps [30, 80].
In the light of these analogies, it seems plausible to believe that the mechanistic details learned from the new intron structures may be pertinent for spliceosomal splicing. In particular, the structural arrangement of the active site motifs and the reactants, the identity and the coordination of the metal ions in the catalytic heteronuclear center, and possibly the dynamics of the conformational toggling observed for the group II intron may have similar correspondence also in the spliceosome. Two specific hypotheses have been proposed, each agreeing with different sets of experimental data and differing in the choice of the toggling residues and in how the spliceosomal elements are positioned in the active site . Other scenarios are also possible and further studies on the spliceosome are required to obtain a more detailed representation of its active site.
Certainly, at present, it is very hard to picture with atomic precision a similarity between the approximately 150-kDa monomeric group II intron ribozyme and the approximately 12-MDa, heteromultimeric spliceosomal ribonucleoprotein. Recently, though, a significant milestone in this direction has been achieved with the determination of the crystal structure of Prp8, a spliceosomal component that interacts directly with all active site elements . Importantly, the Prp8 structure suggests that none of the protein motifs possess catalytic activity, thus reinforcing the current belief that spliceosomal chemistry is performed by the RNA subunits . Even more interestingly, the structure reveals that Prp8 folds around an overall positively charged cavity whose dimensions exactly correspond to the conserved RNA components within the group II intron active site . Evolution seems to have substituted the group II intron scaffold, which is provided by the noncatalytic intron domains (predominantly D1), with the protein scaffold of Prp8, presumably to achieve a finer regulation of splicing fidelity and a more elaborate coordination of the interaction network with other spliceosomal components and regulatory factors. Within this shell, catalytic elements similar to those of a group II intron (for example, U6) are still believed to reside in the core of the spliceosome, suggesting that an RNA element similar to the group II intron D5 is conserved from bacteria to humans.
Overall, the combination of all new structures of group II introns and spliceosomal components reinforces the hypothesis that the two systems may share a common catalytic core and a common mechanism for arranging their reactants and controlling the transitions between the chemical splicing steps.
The large collection of available group II intron structures has recently brought our understanding of splicing mechanism to a new level.
Future work is now likely to focus on the characterization of D6, and the structure of conformational states that participate in branching. Hopefully, these types of structures will reveal the position of the branching nucleotide involved in the mechanism of the first splicing step and will pave the way for visualizing the structures of the branched intron/3′-exon intermediate and of the ligand-free lariat intron. Furthermore, structures containing D6 will reveal the conformation of the 3′-splice junction in the precatalytic state, and in the state that immediately precedes the second splicing step.
Eventually, all these structural snapshots will enable the creation of a movie that depicts each stage of the splicing cycle at high resolution. These pieces of structural information will be valuable not only for understanding the reaction mechanism of group II introns, but for understanding pre-mRNA splicing in general, as group II introns share many structural and mechanistic features with their spliceosomal cousins.
MM and SS are currently postdoctoral associates at Yale University. AMP is the William Edward Gilbert Professor of Molecular, Cellular and Developmental Biology and Professor of Chemistry at Yale, and a Howard Hughes Medical Institute Investigator.
Exon binding site
Intron binding site
Protein Data Bank
We acknowledge the beamline scientists at 24-ID-C and E, NE-CAT, APS, Argonne (IL), USA, and Dr. Maximilian Bailor, Dr. Isabel Chillón Gázquez, Dr. Olga Fedorova, Nathan Pirakitikulr, and all members of the Pyle lab for constructive discussion and critical reading of the manuscript. This project was supported by the National Institute of Health (RO1GM50313).
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