Modular engineering of a Group I intron ribozyme

All Group I intron ribozymes contain a conserved core region consisting of two helical domains, P4–P6 and P3–P7. Recent studies have demonstrated that the elements required for catalysis are concentrated in the P3–P7 domain. We carried out in vitro selection experiments by using three newly constructed libraries on a variant of the T4 td Group I ribozyme containing only a P3–P7 domain in its core. Selected variants with new peripheral elements at L7.1, L8 or L9 after nine cycles efficiently catalyzed the reversal reaction of the first step of self-splicing. The variants from this selection contained a short sequence complementary to the substrate RNA without exception. The most active variant, which was 3-fold more active than the parental wild-type ribozyme, was developed from the second selection by employing a clone from the first selection. The results show that the P3–P7 domain can stand as an independent catalytic module to which a variety of new domains for enhancing the activity of the ribozyme can be added.     

A peripheral element assembles the compact core structure essential for group I intron self-splicing

The presence of non-conserved peripheral elements in all naturally occurring group I introns underline their importance in ensuring the natural intron function. Recently, we reported that some peripheral elements are conserved in group I introns of IE subgroup. Using self-splicing activity as a readout, our initial screening revealed that one such conserved peripheral elements, P2.1, is mainly required to fold the catalytically active structure of the Candida ribozyme, an IE intron. Unexpectedly, the essential function of P2.1 resides in a sequence-conserved short stem of P2.1 but not in a long-range interaction associated with the loop of P2.1 that stabilizes the ribozyme structure. The P2.1 stem is indispensable in folding the compact ribozyme core, most probably by forming a triple helical interaction with two core helices, P3 and P6. Surprisingly, although the ribozyme lacking the P2.1 stem renders a loosely folded core and the loss of self-splicing activity requires two consecutive transesterifications, the mutant ribozyme efficiently catalyzes the first transesterification reaction. These results suggest that the intron self-splicing demands much more ordered structure than does one independent transesterification, highlighting that the universally present peripheral elements achieve their functional importance by enabling the highly ordered structure through diverse tertiary interactions.     

Fast formation of the P3-P7 pseudoknot: a strategy for efficient folding of the catalytically active ribozyme

Formation of the P3-P7 pseudoknot structure, the core of group I ribozymes, requires long-range base pairing. Study of the Tetrahymena ribozyme appreciates the hierarchical folding of the large, multidomain RNA, in which the P3-P7 core folds significantly slower than do the other domains. Here we explored the formation of the P3-P7 pseudoknot of the Candida ribozyme that has been reported to concertedly fold to the catalytically active structure with a rate constant of 2 min(-1). We demonstrate that pseudoknot formation occurs during the rapid ribozyme compaction, coincident with formation of many tertiary interactions of the ribozyme. A low physiological concentration of magnesium (1.5 mM) is sufficient to fully support the pseudoknot formation. The presence of nonnative intermediates containing an unfolded P3-P7 region is evident. However, catalysis-based analysis shows these nonnative intermediates are stable and fail to convert to the catalytically active structure, suggesting that rapid pseudoknot formation is essential for folding of the active ribozyme. Interestingly, RNAstructure predicts no stable Alt P3 structure for the Candida ribozyme, but two stable Alt P3s for the Tetrahymena ribozyme, explaining the dramatic difference in folding of the P3-P7 core of these two ribozymes. We propose that rapid formation of the P3-P7 pseudoknot represents a folding strategy ensuring efficient production of the catalytically active structure of group I ribozymes, which sheds new light on the mechanism of effective ribozyme folding in vivo.     

Structural Rearrangements Linked to Global Folding Pathways of the Azoarcus Group I Ribozyme

Stable RNAs must fold into specific three-dimensional structures to be biologically active, yet many RNAs form metastable structures that compete with the native state. Our previous time-resolved footprinting experiments showed that Azoarcus group I ribozyme forms its tertiary structure rapidly (τ < 30 ms) without becoming significantly trapped in kinetic intermediates. Here, we use stopped-flow fluorescence spectroscopy to probe the global folding kinetics of a ribozyme containing 2-aminopurine in the loop of P9. The modified ribozyme was catalytically active and exhibited two equilibrium folding transitions centered at 0.3 and 1.6 mM Mg²⁺, consistent with previous results. Stopped-flow fluorescence revealed four kinetic folding transitions with observed rate constants of 100, 34, 1, and 0.1 s^− 1 at 37 °C. From comparison with time-resolved Fe(II)-ethylenediaminetetraacetic acid footprinting of the modified ribozyme under the same conditions, these folding transitions were assigned to formation of the I_C intermediate, tertiary folding and docking of the nicked P9 tetraloop, reorganization of the P3 pseudoknot, and refolding of nonnative conformers, respectively. The footprinting results show that 50-60% of the modified ribozyme folds in less than 30 ms, while the rest of the RNA population undergoes slow structural rearrangements that control the global folding rate. The results show how small perturbations to the structure of the RNA, such as a nick in P9, populate kinetic folding intermediates that are not observed in the natural ribozyme.     

Preparation of Group I Introns for Biochemical Studies and Crystallization Assays by Native Affinity Purification

The study of functional RNAs of various sizes and structures requires efficient methods for their synthesis and purification. Here, 23 group I intron variants ranging in length from 246 to 341 nucleotides—some containing exons—were subjected to a native purification technique previously applied only to shorter RNAs (<160 nucleotides). For the RNAs containing both exons, we adjusted the original purification protocol to allow for purification of radiolabeled molecules. The resulting RNAs were used in folding assays on native gel electrophoresis and in self-splicing assays. The intron-only RNAs were subjected to the regular native purification scheme, assayed for folding and employed in crystallization screens. All RNAs that contained a 3′ overhang of one nucleotide were efficiently cleaved off from the support and were at least 90% pure after the non-denaturing purification. A representative subset of these RNAs was shown to be folded and self-splicing after purification. Additionally, crystals were grown for a 286 nucleotide long variant of the Clostridium botulinum intron. These results demonstrate the suitability of the native affinity purification method for the preparation of group I introns. We hope these findings will stimulate a broader application of this strategy to the preparation of other large RNA molecules.     

Specific contacts between protein S4 and ribosomal RNA are required at multiple stages of ribosome assembly

Assembly of bacterial 30S ribosomal subunits requires structural rearrangements to both its 16S rRNA and ribosomal protein components. Ribosomal protein S4 nucleates 30S assembly and associates rapidly with the 5′ domain of the 16S rRNA. In vitro, transformation of initial S4–rRNA complexes to long-lived, mature complexes involves refolding of 16S helix 18, which forms part of the decoding center. Here we use targeted mutagenesis of Geobacillus stearothermophilus S4 to show that remodeling of S4–rRNA complexes is perturbed by ram alleles associated with reduced translational accuracy. Gel mobility shift assays, SHAPE chemical probing, and in vivo complementation show that the S4 N-terminal extension is required for RNA binding and viability. Alanine substitutions in Y47 and L51 that interact with 16S helix 18 decrease S4 affinity and destabilize the helix 18 pseudoknot. These changes to the protein–RNA interface correlate with no growth (L51A) or cold-sensitive growth, 30S assembly defects, and accumulation of 17S pre-rRNA (Y47A). A third mutation, R200A, over-stabilizes the helix 18 pseudoknot yet results in temperature-sensitive growth, indicating that complex stability is finely tuned by natural selection. Our results show that early S4–RNA interactions guide rRNA folding and impact late steps of 30S assembly.     

Protein Roles in Group I Intron RNA Folding: The Tyrosyl-tRNA Synthetase CYT-18 Stabilizes the Native State Relative to a Long-Lived Misfolded Structure without Compromising Folding Kinetics

The Neurospora crassa CYT-18 protein is a mitochondrial tyrosyl-tRNA synthetase that also promotes self-splicing of group I intron RNAs by stabilizing the functional structure in the conserved core. CYT-18 binds the core along the same surface as a common peripheral element, P5abc, suggesting that CYT-18 can replace P5abc functionally. In addition to stabilizing structure generally, P5abc stabilizes the native conformation of the Tetrahymena group I intron relative to a globally similar misfolded conformation that has only local differences within the core and is populated significantly at equilibrium by a ribozyme variant lacking P5abc (E^ΔP5abc). Here, we show that CYT-18 specifically promotes formation of the native group I intron core from this misfolded conformation. Catalytic activity assays demonstrate that CYT-18 shifts the equilibrium of E^ΔP5abc toward the native state by at least 35-fold, and binding assays suggest an even larger effect. Thus, similar to P5abc, CYT-18 preferentially recognizes the native core, despite the global similarity of the misfolded core and despite forming crudely similar complexes, as revealed by dimethyl sulfate footprinting. Interestingly, the effects of CYT-18 and P5abc on folding kinetics differ. Whereas P5abc inhibits refolding of the misfolded conformation by forming peripheral contacts that must break during refolding, CYT-18 does not display analogous inhibition, most likely because it relies to a greater extent on direct interactions with the core. Although CYT-18 does not encounter this RNA in vivo, our results suggest that it stabilizes its cognate group I introns relative to analogous misfolded intermediates. By specifically recognizing native structural features, CYT-18 may also interact with earlier folding intermediates to avoid RNA misfolding or to trap native contacts as they form. More generally, our results highlight the ability of a protein cofactor to stabilize a functional RNA structure specifically without incurring associated costs in RNA folding kinetics.     

Mutagenesis of a light-regulated psbA intron reveals the importance of efficient splicing for photosynthetic growth

The chloroplast-encoded psbA gene encodes the D1 polypeptide of the photosystem II reaction center, which is synthesized at high rates in the light. In Chlamydomonas reinhardtii, the psbA gene contains four self-splicing group I introns whose rates of splicing in vivo are increased at least 6–10-fold by light. However, because psbA is an abundant mRNA, and some chloroplast mRNAs appear to be in great excess of what is needed to sustain translation rates, the developmental significance of light-promoted splicing has not been clear. To address this and other questions, potentially destabilizing substitutions were made in several predicted helices of the fourth psbA intron, Cr.psbA4, and their effects on in vitro and in vivo splicing assessed. Two-nucleotide substitutions in P4 and P7 were necessary to substantially reduce splicing of this intron in vivo, although most mutations reduced self-splicing in vitro. The P7-4,5 mutant, whose splicing was completely blocked, showed no photoautotrophic growth and synthesis of a truncated D1 (exons 1–4) polypeptide from the unspliced mRNA. Most informative was the P4′-3,4 mutant, which exhibited a 45% reduction in spliced psbA mRNA, a 28% reduction in synthesis of full-length D1, and an 18% reduction in photoautotrophic growth. These results indicate that psbA mRNA is not in great excess, and that highly efficient splicing of psbA introns, which is afforded by light conditions, is necessary for optimal photosynthetic growth.     

A conformational switch in the DiGIR1 ribozyme involved in release and folding of the downstream I-DirI mRNA

DiGIR1 is a group I-like cleavage ribozyme found as a structural domain within a nuclear twin-ribozyme group I intron. DiGIR1 catalyzes cleavage by branching at an Internal Processing Site (IPS) leading to formation of a lariat cap at the 5′-end of the 3′-cleavage product. The 3′-cleavage product is subsequently processed into an mRNA encoding a homing endonuclease. By analysis of combinations of 5′- and 3′-deletions, we identify a hairpin in the 5′-UTR of the mRNA (HEG P1) that is formed by conformational switching following cleavage. The formation of HEG P1 inhibits the reversal of the branching reaction, thus giving it directionality. Furthermore, the release of the mRNA is a consequence of branching rather than hydrolytic cleavage. A model is put forward that explains the release of the I-DirI mRNA with a lariat cap and a structured 5′-UTR as a direct consequence of the DiGIR1 branching reaction. The role of HEG P1 in GIR1 branching is reminiscent of that of hairpin P-1 in splicing of the Tetrahymena rRNA group I intron and illustrates a general principle in RNA-directed RNA processing.     

Folding of the group I intron ribozyme from the 26S rRNA gene of Candida albicans

Preincubation of the group I intron Ca.LSU from Candida albicans at 37°C in the absence of divalent cations results in partial folding of this intron. This is indicated by increased resistance to T1 ribonuclease cleavage of many G residues in most local helices, including P4-P6, as well as the non-local helix P7, where the G binding site is located. These changes correlate with increased gel mobility and activation of catalysis by precursor RNA containing this intron after preincubation. The presence of divalent cations or spermidine during preincubation results in formation of the predicted helices, as indicated by protection of additional G residues. However, addition of these cations during preincubation of the precursor RNA alters its gel mobility and eliminates the preincubation activation of precursor RNA seen in the absence of cations. These results suggest that, in the presence of divalent cations or spermidine, Ca.LSU folds into a more ordered, stable but misfolded conformation that is less able to convert into the catalytically active form than the ribozyme preincubated without cations. These results indicate that, like the group I intron of Tetrahymena, multiple folding pathways exist for Ca.LSU. However, it appears that the role cations play in the multiple folding pathways leading to the catalytically active form may differ between folding of these two group I introns.     

Role of a conserved J8/7 X P4 base-triple in the Tetrahymena ribozyme

The Tetrahymena group I intron ribozyme folds into a complex three dimensional structure for performing the self-splicing reaction. Catalysis depends on its core structure comprising two helical domains, P4-P6 and P3-P7. The two domains are joined by three sets of conserved base-triple(s) and other tertiary interactions. We found that the disruption of J8/7 X P4, one such conserved base-triple, causes the catalytic ability to deteriorate without altering the folding rate. This suggests that the base-triple stabilizes the active structure of the ribozyme but plays no significant role in RNA folding. By combining the present and previous results, it can be concluded that three sets of conserved base-triples play distinct roles in the Tetrahymena ribozyme.     

Dual roles for the Mss116 cofactor during splicing of the ai5γ group II intron

The autocatalytic group II intron ai5γ from Saccharomyces cerevisiae self-splices under high-salt conditions in vitro, but requires the assistance of the DEAD-box protein Mss116 in vivo and under near-physiological conditions in vitro. Here, we show that Mss116 influences the folding mechanism in several ways. By comparing intron precursor RNAs with long (∼300 nt) and short (∼20 nt) exons, we observe that long exon sequences are a major obstacle for self-splicing in vitro. Kinetic analysis indicates that Mss116 not only mitigates the inhibitory effects of long exons, but also assists folding of the intron core. Moreover, a mutation in conserved Motif III that impairs unwinding activity (SAT → AAA) only affects the construct with long exons, suggesting helicase unwinding during exon unfolding, but not in intron folding. Strong parallels between Mss116 and the related protein Cyt-19 from Neurospora crassa suggest that these proteins form a subclass of DEAD-box proteins that possess a versatile repertoire of diverse activities for resolving the folding problems of large RNAs.     

Minihelix-loop RNAs: minimal structures for aminoacylation catalysts

We report here an in vitro selected ribozyme, KL17, which is active in charging amino acids on its own 5′-OH group. The ribozyme consists of two catalytic domains, one of which (consisting of P5/P6/L6) recognizes amino acid substrates based on the steric environment of the side chain, whereas the other recognizes an aminoacylated oligonucleotide. The secondary structure of this ambidextrous ribozyme arranges into a pseudoknot, where L6 docks onto the 3′-terminal single-stranded region. The formation of this pseudoknot structure brings the P6 region, in which the essential catalytic core is most likely embedded, into the proximity of the 5′-OH group. Our studies show that the P6–L6 domain can be separated from the main body of KL17 and the derived P6–L6 minihelix-loop RNA can act as a trans-aminoacylation catalyst. In this report, we also compare this ribozyme with an analogous aminoacylation system previously characterized in our laboratory and illuminate the similarities and differences between these catalytic systems.     

Toward predicting self-splicing and protein-facilitated splicing of group I introns

In the current era of massive discoveries of noncoding RNAs within genomes, being able to infer a function from a nucleotide sequence is of paramount interest. Although studies of individual group I introns have identified self-splicing and nonself-splicing examples, there is no overall understanding of the prevalence of self-splicing or the factors that determine it among the >2300 group I introns sequenced to date. Here, the self-splicing activities of 12 group I introns from various organisms were assayed under six reaction conditions that had been shown previously to promote RNA catalysis for different RNAs. Besides revealing that assessing self-splicing under only one condition can be misleading, this survey emphasizes that in vitro self-splicing efficiency is correlated with the GC content of the intron (>35% GC was generally conductive to self-splicing), and with the ability of the introns to form particular tertiary interactions. Addition of the Neurospora crassa CYT-18 protein activated splicing of two nonself-splicing introns, but inhibited the second step of self-splicing for two others. Together, correlations between sequence, predicted structure and splicing begin to establish rules that should facilitate our ability to predict the self-splicing activity of any group I intron from its sequence.     

A remarkably stable kissing-loop interaction defines substrate recognition by the Neurospora Varkud Satellite ribozyme

Kissing loops are tertiary structure elements that often play key roles in functional RNAs. In the Neurospora VS ribozyme, a kissing-loop interaction between the stem–loop I (SLI) substrate and stem–loop V (SLV) of the catalytic domain is known to play an important role in substrate recognition. In addition, this I/V kissing-loop interaction is associated with a helix shift in SLI that activates the substrate for catalysis. To better understand the role of this kissing-loop interaction in substrate recognition and activation by the VS ribozyme, we performed a thermodynamic characterization by isothermal titration calorimetry using isolated SLI and SLV stem–loops. We demonstrate that preshifted SLI variants have higher affinity for SLV than shiftable SLI variants, with an energetic cost of 1.8–3 kcal/mol for the helix shift in SLI. The affinity of the preshifted SLI for SLV is remarkably high, the interaction being more stable by 7–8 kcal/mol than predicted for a comparable duplex containing three Watson–Crick base pairs. The structural basis of this remarkable stability is discussed in light of previous NMR studies. Comparative thermodynamic studies reveal that kissing-loop complexes containing 6–7 Watson–Crick base pairs are as stable as predicted from comparable RNA duplexes; however, those with 2–3 Watson–Crick base pairs are more stable than predicted. Interestingly, the stability of SLI/ribozyme complexes is similar to that of SLI/SLV complexes. Thus, the I/V kissing loop interaction represents the predominant energetic contribution to substrate recognition by the trans-cleaving VS ribozyme.     

A conserved 3′ extension in unusual group II introns is important for efficient second-step splicing

The B.c.I4 group II intron from Bacillus cereus ATCC 10987 harbors an unusual 3′ extension. Here, we report the discovery of four additional group II introns with a similar 3′ extension in Bacillus thuringiensis kurstaki 4D1 that splice at analogous positions 53/56 nt downstream of domain VI in vivo. Phylogenetic analyses revealed that the introns are only 47–61% identical to each other. Strikingly, they do not form a single evolutionary lineage even though they belong to the same Bacterial B class. The extension of these introns is predicted to form a conserved two-stem–loop structure. Mutational analysis in vitro showed that the smaller stem S1 is not critical for self-splicing, whereas the larger stem S2 is important for efficient exon ligation and lariat release in presence of the extension. This study clearly demonstrates that previously reported B.c.I4 is not a single example of a specialized intron, but forms a new functional class with an unusual mode that ensures proper positioning of the 3′ splice site.     

Novel RNA structural features of an alternatively splicing group II intron from Clostridium tetani

Group II introns are ribozymes in bacterial and organellar genomes that function as self-splicing introns and as retroelements. Previously, we reported that the group II intron C.te.I1 of Clostridium tetani alternatively splices in vivo to produce five distinct coding mRNAs. Accurate fusion of upstream and downstream reading frames requires a shifted 5′ splice site located 8 nt upstream of the usual 5′ GUGYG motif. This site is specified by the ribozyme through an altered intron/exon-binding site 1 (IBS1–EBS1) pairing. Here we use mutagenesis and self-splicing assays to investigate in more detail the significance of the structural features of the C.te.I1 ribozyme. The shifted 5′ splice site is shown to be affected by structures in addition to IBS1–EBS1, and unlike other group II introns, C.te.I1 appears to require a spacer between IBS1 and the GUGYG motif. In addition, the mechanism of 3′ exon recognition is modified from the ancestral IIB mechanism to a IIA-like mechanism that appears to be longer than the typical single base-pair interaction and may extend up to 4 bp. The novel ribozyme properties that have evolved for C.te.I1 illustrate the plasticity of group II introns in adapting new structural and catalytic properties that can be utilized to affect gene expression.