Pre-splicing conformation and stepwise circularization of a group I intron in Azoarcus pre-tRNA

Domain: biology
Published: 2026-03-20
Editorial outcome: minor_revision

Abstract
Group I introns are catalytic RNAs capable of self-splicing, yet structural insights into full-length precursor RNAs and post-splicing circularization have been limited. Here, we present cryo-EM structures of the Azoarcus pre-tRNAIle system across key catalytic stages, including the full-length precursor in Pre-1S conformations, the linear intron, and the circular introns, at 2.8-3.3 Å resolution. These structures reveal a preformed P1 helix, register shifts during splicing, and a conformational flip of G37 that stabilizes the circularization site. Biochemical analyses confirm a two-step circularization mechanism, generating a secondary circular product via an alternative ligation site. Together, our results provide the atomic-level view of a group I intron system through splicing and circularization. This work uncovers structural principles governing RNA conformational dynamics, catalysis, and circular RNA formation, with broad implications for ribozyme engineering.

Introduction
Group I introns are a class of catalytic RNAs capable of self-splicing through two consecutive transesterification reactions, excising themselves from precursor RNAs and ligating the flanking exons1,2. This process is facilitated by a conserved catalytic core and complex tertiary interactions, often modulated by divalent metal ions and exogenous guanosine3–5. Owing to their robust catalytic capacity and ancient evolutionary origin, group I introns have served as valuable models for understanding RNA structure, catalysis, and the evolution of ribozymes6–12.

While extensive structural studies, particularly on the Tetrahymena thermophila and Azoarcus group I introns, have provided snapshots of ribozymes in splicing-relevant conformations, most available data are limited to constructs with truncated exons or inactive precursors13–18. As a result, the structural basis underlying pre-tRNA folding and the post-splicing fate of the intron, particularly circularization, remains poorly defined. Circular forms of Group I introns have been observed in various systems, where the terminal guanosine initiates an internal nucleophilic attack to produce 5′-truncated circular RNAs19–22. These circular RNAs are thought to regulate RNA turnover, avoid harmful reverse splicing, or serve as templates for homing endonucleases23,24.

The Group I intron from Azoarcus sp. BH72 pre-tRNAIle is a model system with robust self-splicing and circularization abilities25–27. It s compact 205-nt intron resides in the anticodon loop region of the tRNA, and lacks several peripheral domains found in Tetrahymena introns8. Previous crystal structures have captured a catalytically primed state of the Azoarcus ribozyme with short exons, yet failed to represent the full precursor architecture or subsequent conformational transitions following exon ligation15–17.

Here, we employ cryo-EM to resolve high-resolution structures of three key intermediates in the Azoarcus group I intron splicing pathway: the full-length precursor at the Pre-1S state, the linear intron, and the circular introns. Together with biochemical validation, these structures reveal a two-step circularization mechanism in which the P1ext helix undergoes a defined 2-nucleotide register shift after exon ligation, repositioning the catalytic core for intron circularization. This work provides the unprecedented near atomic-resolution view of a complete Azoarcus group I intron splicing and circularization trajectory, offering new mechanistic insights into RNA conformational plasticity, catalysis, and post-splicing processing.

Results
Cryo-EM revealed the Full-Length Azoarcus Pre-tRNAIle at Pre-1S states
Previous crystallographic studies of the Azoarcus group I intron have provided important insights into ribozyme catalysis, especially at stages mimicking the second step of self-splicing16,17. However, these structures typically relied on constructs containing truncated exons and lacked visualization of the full-length pre-tRNA substrate, particularly its structural organization before the first transesterification event. Moreover, the post-splicing fate of the intron, namely its circularization, remained structurally uncharacterized. To bridge this knowledge gap, we investigated the full-length Azoarcus Pre-tRNAIle (284 nt), which contains a catalytic group I intron (205 nt) located in the anticodon loop region of the tRNA, aiming to capture both the pre-splicing and post-splicing states (Fig. 1a-b). Using in vitro splicing assays at physiological temperature (37°C)26,28, we confirmed that this RNA undergoes efficient self-splicing, generating three major products over time: mature tRNA, linear intron (L intron), and circular intron (C intron), with a corresponding reduction in precursor RNA abundance (Fig. 1c). These results verified the system’s robust ability to perform both exon ligation and intron circularization, highlighting its suitability for structural investigation.

We initially conducted single-particle cryo-EM analysis of full-length pre-tRNA following incubation at 37°C for 30 minutes to capture structural intermediates during self-splicing. However, likely due to the conformational heterogeneity of the sample, the resulting maps lacked sufficient resolution for reliable model building. To overcome this limitation, we prepared three distinct RNA samples representing key stages in the splicing pathway: (1) the pre-splicing state using full-length pre-tRNA without added GTP (Fig. S1a); ( 2) the linear intron after splicing; and (3) the circular intron after post-splicing circularization. Cryo-EM analysis of the pre-tRNA sample yielded two distinct conformational states at 3.11 Å and 3.06 Å resolutions, respectively, representing early pre-catalytic intermediates (Pre-1S) (Fig. 1d, Fig. S1b-g, Supplementary Table 1, and Supplementary Movie 1). In both structures, the 5′ exon has already aligned with the internal guide sequence (IGS) to form the canonical P1 helix, while the 3′ exon remains unpaired, positioning the molecule for the first transesterification reaction. Notably, even though no exogenous G was added during sample preparation, weak density corresponding to guanosine was observed in the G-binding pocket, possibly originating from RNA degradation (Fig. 1d-e, Fig. S2a). Comparative analysis of the two Pre-1S states revealed notable conformational flexibility in the tRNA anticodon domain, characterized by an approximately 45° rotational difference between Pre-1S_a and Pre-1S_b (Fig. S2b-d, S3a). This motion likely reflects intrinsic flexibility at the intron–tRNA junction, which contains a single-stranded region (Fig. 1d). These two states likely represent distinct local minima on the RNA’s energy landscape, reflecting dynamic sampling of catalytically permissive conformations prior to exon ligation.

Besides the tRNA exons, both the Pre-1S structures consist of the canonical RNA elements of group I intron (P2, P3, P4, P5, P6, P7, P8, P9, and P9.0) and adopts the canonical architecture seen in previously reported Azoarcus ribozyme structures16,29(Fig. S3b-c). This conservation underscores the structural stability of the catalytic domain even in the context of full-length tRNA exons and emphasizes the conservation of the group I intron fold. Together, our structures provide a high-resolution view of a full-length pre-tRNA substrate poised near the onset of catalysis, offering insight into early structural events associated with tRNA maturation and intron excision.

2-nt sliding of P1 helix between Linear Intron and Pre-tRNA
To investigate the structural transitions underpinning post-splicing and circularization of the Azoarcus group I intron, we determined high-resolution cryo-EM structures of the excised linear intron (L intron; Post-2S) and the subsequent circular introns (C and C′ introns; Post-C) at 3.01 Å resolution for L, 3.27 Å and 2.86 Å resolutions for C and C′ introns (Figs. 2-5, Figs. S4-6, Supplementary Table 1, and Supplementary Movie 1). These reconstructions allowed us to examine in detail how the intron architecture evolves following exon ligation and prepares for circularization.

Structural comparison between the Pre-1S and L intron states revealed that while the global scaffold of the ribozyme, including peripheral domains and the G-binding pocket, remained largely conserved, the P1/P1ext region underwent a notable conformational shift (Fig. 2b-e, Fig. S3d-e). In the Pre-1S state, the P1/P1ext helix is formed via base pairing between the 5′ exon and the internal guide sequence (IGS), aligning the 5′ splice site for nucleophilic attack. Following exon ligation and tRNA release, the P1ext helix is reformed within the intron alone, through intramolecular pairing between the free 5′ end and IGS. Importantly, the newly formed P1ext helix in the linear intron undergoes a 2-nt register shift relative to its position in the Pre-1S state, bringing the circularization reaction nucleophile, the 3′-OH of G205 (ωG, the 3′ terminal G) into planar alignment with the phosphodiester bond between U2 and U3, thus positioning the molecule in a circularization-ready conformation (Fig. 2f).

This finding highlights a key mechanism by which the ribozyme repurposes the same structural framework to carry out two distinct catalytic reactions: exon ligation and subsequent intron circularization. The observed register shift in the P1ext helix underscores the dynamic adaptability of group I introns, allowing them to reconfigure their base-pairing interactions to execute multiple reaction steps with high precision. In addition, we noticed that the density near the G-1 position in the L intron closely resembles the “pseudo-triple-helix” configuration recently described in another group I intron30. This region shows a continuous strand folding back onto the P1ext helix through non-canonical hydrogen bonds and stacking interactions, forming a pseudo-triple-helix–like tertiary contact (Fig. S4g). This interaction likely helps stabilize P1ext during circularization.

G37 reorientation anchors the circularization site and enhances efficiency
High-resolution cryo-EM structures of the L intron and the C intron revealed striking conservation across most of the ribozyme scaffold, particularly within the P1ext helix, which remains compact and nearly identical between the two states (Fig. 3a-b, Fig. S3f, S4, S5, S7, and Supplementary Movie 1). Despite this structural similarity, a single pronounced conformational change was observed during circularization: nucleotide G37 undergoes a distinct reorientation, flipping inward from its original outward-facing configuration while forming a Hoogsteen pair with G11 (Fig. 3c).

To assess the functional significance of G37, we performed site-directed mutagenesis to substitute this residue with A, C, or U, followed by in vitro circularization assays. Among the variants, the G37C mutant displayed a markedly increased yield of circular intron relative to the wild type (Fig. 3d), suggesting enhanced stabilization of the circularization site. This is likely due to the increased thermodynamic stability of the C37-G11 base pair compared to the original G-G pairing. In contrast, the G37A and G37U substitutions failed to promote circularization, indicating that the base-pairing potential and positioning of G37 are critical for productive catalysis. Together, these results identify G37 as a key conformational determinant in the circularization pathway of group I introns. Its dynamic reorientation not only marks a structural transition point but also fine-tunes the local architecture to optimize catalytic efficiency. This underscores a broader principle wherein single-nucleotide conformational switches can exert outsized influence on RNA processing outcomes by stabilizing transient but functionally important RNA configurations.

Identification and Validation of a Secondary Circularization Product
During prolonged incubation of Azoarcus pre-tRNA at 37°C, we observed the emergence of a lower-migrating RNA band beneath the canonical C intron, most clearly visible after 180 minutes (Fig. 1c). This unexpected product, designated as the C′ intron, suggested the possibility of an additional circular RNA species arising through a secondary processing event. The appearance of this smaller circular RNA recalls similar two-step circularization mechanisms reported in Tetrahymena group I introns31–34, prompting us to explore whether the Azoarcus system follows a comparable pathway. To investigate the origin of C′ intron, we conducted in vitro circularization assays using linear intron RNA. Gel electrophoresis revealed the presence of three distinct products: the C intron, the L-3 intron, and the C′ intron. This observation suggests that the C intron may undergo ring opening to form the L-3 linear intron33,34, which subsequently undergoes a second circularization event to generate the C′ intron, thereby supporting a model of sequential processing (Fig. 4a).

To validate the circular nature of both C and C′ introns, we treated the products with RNase R, a 3′-5′ exonuclease that selectively degrades linear RNAs while sparing circular forms. As expected, RNase R treatment led to complete degradation of the linear intron, while the C and C′ introns remained intact (Fig. 4b), confirming their circular topology35. We then employed reverse transcription followed by high-throughput sequencing to precisely map the ligation junctions of both products. Sequencing results revealed that the C intron is circularized at the L-3 position, resulting in the loss of the 5′-terminal GAU trinucleotide (Fig. 4c), whereas the C′ intron is circularized at the L-5 position, involving the removal of a GAUUU pentanucleotide (Fig. 4d). To test whether C′ intron formation occurs via a ring-opened intermediate, we purified C intron and incubated it under standard circularization conditions. Time-course experiments showed efficient conversion of C to C′ (Fig. 4e), confirming that C′ formation proceeds through secondary circularization following C intron opening33,34. Supporting this, 5′-FAM labeling detected only a 4-nt GGAU fragment corresponding to C intron formation, with no 6-nt GGAUUU fragment indicative of a direct conversion from L intron to C′ intron (Fig. 4f). Furthermore, inclusion of a recombinant RNase inhibitor to suppress potential enzymatic cleavage did not prevent the formation of the C′ intron (Fig. S8a). To further assess whether this conversion reflects a water driven, non-enzymatic process, we compared reactions performed under the original condition (pH 7.5) with those carried out under acidic conditions (pH 6.0). Under the neutral condition, hydrolysis of the C intron produced the linear L 3 intron and a clear C′ intron band was detected. In contrast, under acidic conditions, no detectable C′ intron was observed (Fig. S8b). Together, these observations support the interpretation that C′ intron formation is consistent with a water driven, non-enzymatic reaction that is intrinsic to the catalytic RNA.

To gain structural insight into the second circularization product, we determined the cryo-EM structure of the C′ intron at 2.86 Å resolution (Fig. 5a). The overall fold of the ribozyme remains largely conserved relative to the canonical C intron, however, pronounced structural remodeling occurs in the P1ext region (Fig. S3g). In contrast to the compact and fully base-paired P1ext observed in the C intron, the C′ intron displays extensive local unwinding, where nearly all base pairs in this region are disrupted except for the G11-G37 interaction, leaving the segment predominantly single-stranded (Fig. 5b-c and Fig. S7c). This partial melting appears to expand the solvent-exposed cavity adjacent to the catalytic center and may increase local flexibility near the ωG. The observed relaxation of P1ext is consistent with the biochemical evidence for a second, water-driven transesterification step, suggesting that such unwinding could facilitate repositioning of the phosphodiester bond between ωG and C5 for the subsequent ligation event. Together, these results reveal that the Azoarcus group I intron undergoes a two-step circularization process, highlighting a sequential and dynamic pathway reminiscent of Tetrahymena introns33,34.

Coordination of Catalytic Metal Ions Across Splicing and Circularization States
Metal ions play essential roles in group I intron catalysis, where five specific ions (M -M ) have been proposed to form the essential catalytic center. Through cryo-EM analysis of five functional states, including Pre-1S_a, Pre-1S_b, the L intron, the C intron, and the C′ intron, we identified and characterized three metal ion sites, designated M ,M and M (Fig. S9). The M ion displays state-dependent coordination geometry. In Pre-1S conformations, M associates with non-bridging phosphate oxygens of C87, G169, and A171 within the intron core. In the same states, M is positioned near the catalytic center and coordinates the nonbridging phosphate oxygens of G127 and A171, together with the 2′ and 3′ hydroxyl groups of ExoG. This coordination geometry suggests that M may contribute to stabilizing the leaving group and may act in concert with M to support a catalytically competent configuration. During the first circularization step, the key nucleotide U3 at the circularization site undergoes a conformational rearrangement that places its phosphate group within the coordination sphere of both M and M . This rearrangement likely anchors the circularization site within the catalytic core. Concurrently, M coordinates with the 2′ and 3′ hydroxyl groups of ωG, which may facilitate proper positioning of ωG during catalysis. The M ion lies peripherally to the active site and maintains stable coordination with the non-bridging phosphate oxygens of U172 and A173 in all observed states. This conserved geometry likely stabilizes the tertiary architecture of the P9.0 domain and contributes to maintaining the integrity of the ribozyme’s scaffold.

Beyond these established metal sites, we identified an additional ion in the catalytic core occupying the M position previously reported in Tetrahymena ribozymes14. This M coordinates the non-bridging phosphate oxygen of A86 and the 3′-OH of G127. The conservation of this position among group I introns suggests a possible catalytic or structural role, although its precise function remains to be determined. Together, these findings reveal that while the metallo-active core maintains a conserved overall architecture, the coordination geometries of individual ions adjust dynamically in response to transitions between splicing and circularization. This structural plasticity provides a mechanistic basis for how group I introns accommodate distinct chemical reactions while preserving catalytic precision.

Proposed Two-Step Circularization Mechanism
Integrating our structural and biochemical findings, along with previously established models such as the Tetrahymena IVS RNA circularization pathway31–34, we propose a two-step circularization mechanism for the Azoarcus pre-tRNA intron that operates through a “conformational rearrangement-sequential cleavage” framework (Fig. 5d). Following canonical self-splicing, the excised linear intron adopts a catalytically competent conformation through structural reorganization of the P1ext helix. This rearrangement aligns the phosphodiester bond between U2 and U3 within the ribozyme active site. The 3′-hydroxyl group of the conserved catalytic residue ωG then initiates a site-specific intramolecular transesterification reaction at U2, resulting in cleavage at the L-3 site and release of a 3-nt 5′ fragment (GAU). This first ligation event yields the primary circular species, termed the C intron. Subsequently, under near-neutral pH, the C intron undergoes spontaneous hydrolysis at the ligated junction, generating an L-3 linear intermediate33,34. This event promotes register shift of the P1ext helix, which in turn exposes a second phosphodiester bond between U4 and C5. A second transesterification reaction is then catalyzed by ωG, leading to cleavage at the L-5 site and release of a UU dinucleotide, ultimately forming the second circular product, C′ intron.

While this sequential pathway is supported by biochemical data, we cannot exclude the possibility of an alternative route. Specifically, a subset of linear introns may adopt a flexible P1ext conformation de novo, enabling ωG to bypass the L-3 reaction and directly cleave at the U4-C5 site32. This single-step mechanism would yield the C′ intron via release of a 5-nt fragment (GAUUU), reflecting a shortcut within the broader circularization pathway (Fig. 5d). However, as described above, our 5′-FAM labeling experiments detected no 6-nt GGAUUU product under standard in vitro conditions, confirming that direct conversion from the L intron to the C′ intron does not occur in our reconstituted system. While we cannot exclude the possibility that cellular factors or specific physiological conditions might enable this alternative route in vivo, our results indicate that the C intron-mediated sequential pathway represents the predominant mechanism of C′ intron formation under the tested conditions. Together, these results reveal a mechanistically versatile circularization program within group I introns, governed by dynamic P1ext remodeling and catalyzed through the strategic positioning of reactive residues and substrates. This work not only expands our understanding of intron self-processing but also provides a conceptual framework for engineering programmable RNA circularization systems.

Methods
RNA sample preparation
Full-length Azoarcus pre-tRNAIle and L intron were individually cloned into the pUC19 vector. A T7 promoter sequence (5′-TAATACGACTCACTATA-3′) was appended to the 5′ end to enable in vitro transcription by T7 RNA polymerase. DNA templates were amplified via PCR using PrimeSTAR Max DNA Polymerase (TaKaRa), followed by ethanol precipitation and resuspension in DEPC-treated water. Purified DNA was stored at -20°C. In vitro transcription was performed in reactions containing 40 mM Tris-HCl (pH 7.9), 5 mM MgCl₂, 2 mM spermidine, 0.01% Triton X-100, 5 mM DTT, 2 mM of each NTP, 0.2 U/μL recombinant RNase inhibitor (TaKaRa), 50 ng/μL linearized DNA template, and lab-purified T7 RNA polymerase. Reactions were incubated at 37°C for 4 hours. RNA transcripts were purified by denaturing urea–polyacrylamide gel electrophoresis (urea-PAGE). For pre-tRNAIle and L intron, transcripts were resolved on 6% urea-PAGE gels (acrylamide:bisacrylamide = 19:1) at 300 V for 4 hours. For C intron and C′ intron generated from L intron, transcripts were resolved on 15% urea-PAGE at 300 V for 72 hours. Gels were briefly visualized under UV light, and target bands were excised. RNA was eluted from gel slices in 300 mM sodium acetate (pH 5.2) containing 1 mM EDTA, rotating at 4°C for 12 hours. Eluates were concentrated using 30 kDa centrifugal filters (Millipore), mixed with an equal volume of isopropanol, and flash-frozen at -80°C for 10 minutes. Precipitated RNA was washed twice with 75% ethanol, air-dried, dissolved in DEPC-treated water, and stored at -80°C. Sequences of RNA constructs and related DNA primers are provided in Supplementary Data 1.

To generate the G37 point mutation, site-directed mutagenesis was performed using the ClonExpress® II One Step Cloning Kit (Vazyme), following the manufacturer’s protocol. Mutation-specific primers were designed accordingly, and PCR was carried out with PrimeSTAR Max DNA Polymerase (TaKaRa). The amplified product was treated with DpnI (New England Biolabs) to digest the methylated parental plasmid. The resulting product was ligated using the supplied recombination mix and transformed into competent E. coli cells. Mutant RNAs were transcribed and purified following the identical in vitro transcription and PAGE purification protocols established for the wild-type construct.

Cryo-EM sample preparation
Cryo-electron microscopy (cryo-EM) samples were prepared using an in vitro refolding strategy. RNA samples were first denatured by heating at 90°C for 3 min, then immediately diluted in refolding buffer to final concentrations of 40 mM Tris-HCl (pH 7.5) and 15 mM MgCl₂. The mixture was incubated at 37°C for 30 min to refold. For grid preparation, 4 μL of the RNA solution (20μM) was applied to glow-discharged 200-mesh R2/1 Quantifoil® copper grids. Grids were blotted once (blot force 0, blot time 4 s) after a 5-second wait time and rapidly cryocooled in rapid plunging into liquid ethane using a Vitrobot Mark IV (Thermo Fisher Scientific).

Cryo-EM data collection and processing
For pre-tRNAIle,L intron and C intron, the grids were imaged on a Titan Krios G3i cryo-EM microscope (Thermo Fisher Scientific) at a magnification of 105,000x, corresponding to a calibrated pixel size of 0.82 Å. Micrographs were recorded by EPU software (Thermo Fisher Scientific) with a K3 camera. Each image consisted of 30 frames with an exposure time of 2.5 s and a total dose of 57.5-64 electrons per Å2. A total of 2,911 movie stacks for pre-tRNA, 2,888 movie stacks for the L intron, and 3,602 movie stacks for the C intron were collected, with a defocus range of -0.8 to -2.7 μm. For C′ intron, the grids were imaged on a Titan Krios G4 cryo-EM microscope (Thermo Fisher Scientific) at a magnification of 130,000x, corresponding to a calibrated pixel size of 0.96 Å. Micrographs were recorded by EPU software (Thermo Fisher Scientific) with a Falcon4i camera. Each image consisted of 30 frames with an exposure time of 5.88 s and a total dose of 50 electrons per Å2. A total of 8,147 movie stacks for C′ intron were collected, with a defocus range of -0.8 to -1.5 μm. The details of data collection are summarized in Supplementary Table 1.

Data processing was performed using the software package cryoSPARC45. Micrographs were motion corrected with patch motion correction. The CTF parameters of micrographs were estimated using patch CTF estimation. All particles were autopicked using the template picker in cryoSPARC. After 2D classification, 100,0 00 random particles in good 2D classes were used for ab initio map generation followed by 3D heterogeneous refinement. The particles of the good 3D classes were then refined into higher resolution by non-uniform refinement. Resolution for the final map was estimated with the 0.143 criterion of the Fourier shell correlation curve. The resolution map was calculated in cryoSPARC using the “Local Resolution Estimation” option. The processing details are shown in Fig S1, 4, 5, 6 and listed in Supplementary Table 1.

Model building
The initial structural model of the full-length Azoarcus pre-tRNA was generated using AlphaFold346 and rigidly docked into the cryo-EM density of the pre-1S state. To achieve optimal fit, multiple iterations of molecular dynamics flexible fitting (MDFF)47 were performed until convergence. The resulting models were further corrected and refined using Coot48 and phenix.real_space_refine49. For the linear and circularized intron structures, the intron coordinates from the pre-1S model served as templates, which were similarly fitted into their respective cryo-EM maps, manually adjusted by Coot, and subsequently refined. The quality of the final atomic models was assessed using MolProbity50, with statistics provided in Supplementary Table 1. Structural figures were prepared using Chimera51 or ChimeraX52, and secondary structure schematics were generated with RiboDraw, followed by manual adjustments (https://github.com/ribokit/RiboDraw).

RNase R digestion assay
RNase R digestion was used to distinguish circular RNAs from linear species. Reactions (10 μL) were assembled on ice and contained 500 ng RNA, 1× RNase R reaction buffer, and 2 U RNase R (Beyotime). RNase R is a magnesium-dependent 3′→5′ exoribonuclease that selectively degrades linear RNAs while sparing circular forms. Samples were incubated at 37°C for 15 minutes, and the reaction was terminated by heating at 70°C for 10 minutes. An equal volume of 2× RNA loading buffer (95% formamide, 5 mM EDTA, 0.025% SDS, 0.02% xylene cyanol, 0.02% bromophenol blue) was added, followed by denaturation at 95°C for 5 minutes. Digestion products were resolved on an 8% denaturing urea–polyacrylamide gel (acrylamide:bis-acrylamide = 19:1) at 200 V for 3 hours. Gels were stained with 0.5× SuperRed nucleic acid stain (Biosharp) for 1 minute and visualized under 254-nm UV illumination using a gel documentation system (Peiqing Biotech).

Self-splicing Assays
Self-splicing reactions were performed through in vitro refolding followed by incubation at 37°C. For each reaction, 15 pmol pre-tRNAIle RNA were first denatured by heating at 90°C for 3 min. The samples were then immediately diluted in a refolding buffer to final concentrations of 40 mM Tris-HCl (pH 7.5), 15 mM MgCl₂, and 25 μM GTP. Separate reaction mixtures were then incubated at 37°C for 0, 5, 10, 30, 60, 120, and 180 min, respectively. At each designated time point, the corresponding reaction was quenched by adding an equal volume of 2× RNA loading buffer (95% formamide, 5 mM EDTA, 0.025% SDS, 0.02% xylene cyanol, and 0.02% bromophenol blue), followed by denaturation at 95°C for 5 min. Reaction products were resolved by 8% denaturing urea–polyacrylamide gel electrophoresis (acrylamide:bis-acrylamide = 19:1) under constant voltage (300 V) for 8 hours. Gels were stained for 1 min with 0.5× SuperRed nucleic acid stain (Biosharp) and visualized under 254-nm UV transillumination using a gel documentation system (Peiqing Biotech).

Circularization Assays
Two-step and second-step circularization reactions were performed through in vitro refolding followed by incubation at 37°C. For each reaction, 15 pmol of the appropriate RNA substrate (L intron for two-step circularization; C intron for second-step circularization) were first denatured by heating at 90°C for 3 min. The samples were then immediately diluted in a refolding buffer to final concentrations of 40 mM Tris-HCl (pH 7.5) and 15 mM MgCl₂. Separate reaction mixtures for two-step circularization were incubated at 37°C for 0, 10, 20, 30, 60, and 120 min, while those for second-step circularization were incubated for 0, 2, 4, and 6 h. At each designated time point, the corresponding reaction was quenched by adding an equal volume of 2× RNA loading buffer (95% formamide, 5 mM EDTA, 0.025% SDS, 0.02% xylene cyanol, and 0.02% bromophenol blue), followed by denaturation at 95°C for 5 min. Reaction products were resolved by 8% denaturing urea–polyacrylamide gel electrophoresis (acrylamide:bis-acrylamide = 19:1) under constant voltage (200 V) for 40 min. Gels were stained for 1 min with 0.5× SuperRed nucleic acid stain (Biosharp) and visualized under 254-nm UV transillumination using a gel documentation system (Peiqing Biotech).

Circularization of the L Intron with RNase Inhibitor
Circularization reactions of the L intron were performed in the presence or absence of RNase inhibitor. For each reaction, 12 pmol L intron were first denatured by heating at 90°C for 3 min. The samples were then immediately diluted in the refolding buffer to final concentrations of 40 mM Tris-HCl (pH 7.5) and 15 mM MgCl₂. Two parallel sets of reactions were set up: one group supplemented with 10% (v/v) TaKaRa Recombinant RNase Inhibitor (Cat. No. 2313A) (RRI, +), and the other group without RNase inhibitor (-). All reaction mixtures were then incubated at 37°C for 0-60 min. At designated time points (0, 30, and 60 min), equal aliquots were withdrawn and quenched by adding an equal volume of 2× RNA loading buffer (95% formamide, 5 mM EDTA, 0.025% SDS, 0.02% xylene cyanol, and 0.02% bromophenol blue), followed by denaturation at 95°C for 5 min. Reaction products were resolved by 8% denaturing urea–polyacrylamide gel electrophoresis (acrylamide:bis-acrylamide = 19:1) under constant voltage (200 V) for 3h. Gels were stained for 1 min with 0.5× SuperRed nucleic acid stain (Biosharp) and visualized under 254-nm UV transillumination using a gel documentation system (Peiqing Biotech).

Second-step Circularization of the C Intron under Different pH Conditions
Circularization reactions of the C intron were carried out under two different pH conditions (pH 7.5 and pH 6.0) to assess the effect of buffer acidity on the second-step ligation. For each reaction, 8 pmol C intron substrates were first denatured by heating at 90°C for 3 min, followed by immediate dilution into refolding buffers containing 15 mM MgCl₂ and either 40 mM Tris-HCl (pH 7.5) or 40 mM Tris-HCl (pH 6.0). All reaction mixtures were then incubated at 37°C for 0–6 h. At the designated time points (0, 3 h, and 6 h), equal aliquots were removed and quenched by addition of an equal volume of 2× RNA loading buffer (95% formamide, 5 mM EDTA, 0.025% SDS, 0.02% xylene cyanol, and 0.02% bromophenol blue), followed by denaturation at 95°C for 5 min. Reaction products were resolved by 8% denaturing urea–polyacrylamide gel electrophoresis (acrylamide:bis-acrylamide = 19:1) at a constant voltage of 200 V for 3 h. Gels were stained with 0.5× SuperRed nucleic acid stain (Biosharp) for 1 min and imaged under 254-nm UV transillumination using a gel documentation system (Peiqing Biotech).

Circularization Analysis Using Split L Intron Fragments
Circularization reactions were performed using split L intron fragments through in vitro refolding. For each reaction, 4 pmol of Fragment A (1-69 nt, containing two guanosine residues at the 5′ end and FAM-labeled) and 8 pmol of Fragment B (70-205 nt) were co-refolded. As a control, a separate reaction containing only 4 pmol of Fragment A without Fragment B was included. The RNA fragment mixtures were first denatured by heating at 90°C for 3 min. The samples were then immediately diluted in the refolding buffer to final concentrations of 40 mM Tris-HCl (pH 7.5) and 15 mM MgCl₂. The reaction mixtures were incubated at 37°C for 0, 2 and 4 h, while the control reaction (Fragment A only) was incubated for 2 h. All reactions were quenched by adding an equal volume of 2× RNA loading buffer (95% formamide, 5 mM EDTA, 0.025% SDS, 0.02% xylene cyanol, and 0.02% bromophenol blue), followed by denaturation at 95°C for 5 min. Reaction products were resolved using a 10%-20% gradient denaturing urea-polyacrylamide gel (acrylamide:bis-acrylamide = 19:1) under constant voltage (300 V) for 25 min. Following electrophoresis, gels were simultaneously subjected to two detection methods: GelRed staining (Biosharp) was used to detect longer RNA fragments using a gel documentation system (Peiqing Biotech), and FAM imaging was performed to detect short drop-off fragments using a Fast Laser Imager (Typhoon FLA 7000).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Azoarcus pre-tRNA intron is the model system to establish the group I intron self-splicing, circularization and two-metal ion\ncatalysis model in the early days of RNA biochemistry and structural biology. In the manuscript by Hong et al. described a\ntotal of 4 new structures of Azoarcus pre-tRNA intron in the unspliced (pre-1S_a and pre-1S_b), spliced (L) and circularized\n(C) forms, and biochemically characterized a minor yet new form of circularized intron (C’). The structural studies in the\nmanuscript are complemented by biochemical experiments to arrive at the proposed 2-step model for intron circularization\npost splicing.\nWe have two aspects of comments of the manuscript. The first perspective is the limited biochemical characterization\nperformed in this study. As a model system in the field, Azoarcus intron has been the subject for extensive biochemical\ncharacterization (Tanner et al. RNA, 1996). There are some observations regarding circularization efficiency from the\nmanuscript that appear to be inconsistent with literature results that warrants further investigation (see major points below for\ndetails). Moreover, the insights into the newly reported C’ intron is limited by the biochemical data presented in the\nmanuscript, which would benefit from additional experimental validation.\nThe second perspective is regarding structural data processing and modelling. We appreciate the authors for supplementing\nthe electron density maps and models for evaluation. Upon inspection, we have some concerns on the structural modeling,\nespecially when it comes to the cryptic densities present in the pre-1S and L intron electron maps. Additionally, we have\ncomments to improve the data processing and the overall presentation of their findings. The authors should address the\ncomments listed below before publication.\nMajor points:\n1. Changes should be made to Fig. 1 to make it more appropriate to the general readership of Nat Commun.\na. The manuscript would benefit from a cartoon diagramming the splicing reactions referred to in the introduction, perhaps as\npart of Figure 1, instead of in Fig S1\nb. Appreciate the effort to maximize information content in Fig. 1D, however, the dizzying array of arrows and symbols,\nmakes the figure difficult to follow. Please consider simplifying or altering the figure for clarity.\n2. In Fig. 1b, the authors present a self-splicing gel showing the full-time course up to 3 hours using fluorescent RNA\nstaining. The gel looks different from the original results using phosphor-imaging with radio-labelled precursor RNA in\nTanner et al. RNA, 1996 (Fig. 2a), where the C intron is dominant over the L intron after 0.5-1 hour of incubation. The author\nshould explain why there are differences in the circularization efficiency of the same intron. Is it because of different\nconstructs, different reaction conditions, or different detection methods?\n3. The author should test under the literature condition whether the C’ intron band is still present. It is unclear if the C’ intron\nformation is condition-dependent or if the C’ band was missed in the literature due to limited resolution of gel imaging three\ndecades ago.\n4. In Fig. 3d, the gel is not annotated, and the meaning for “ctrl” is unclear - does it mean time 0? Assuming the top band is\nthe C intron and the bottom band is the precursor, the splicing outcome at 40 min does not match the time course provided in\nFig. 1b. Were the reaction conditions different? The authors should show the results for the wt and mutants under the optimal\nself-splicing condition (ideally the literature condition mentioned earlier which strongly favors circularization) and verify the\neffect of the G37C mutant.\n5. For cryoEM data processing, did refinement stop with ‘Non-Uniform Refinement’? Were there additional jobs, such as\nlocal refinement or CTF corrections, that were run? There is often an increase in resolution seen with further processing that\nmight be worthwhile to consider.\n6. The RNA has been incubated at 37C for 30 min to prepare the pre-1S sample. However, with bound guanosine visual\nfrom the map, it is unclear if the intron remains unspliced. It is important to check on denaturing PAGE gel to make sure that\nthe sample giving rise to the pre-1S_a and pre-1S_b structures are indeed reflecting the two unspliced forms instead of one\nbeing unspliced while the other one being spliced.\n7. Related, if the authors can show the two pre_1S structures are truly in unspliced form. Have the two states referred to as\nPre-1S_a and Pre-1S_b been observed in other studies before? Is there a biochemical significance to either state and do\nthe particle populations give any further insights?\n8. In all the structural models, no metal ions were modeled despite sufficient resolution in the core and electron density\nevidence for the presence of metals. The author will also benefit from existing Azoarcus crystal structures in modelling metal\nions. The author should model visible and relevant metal ions, which are key parts of RNA structure and vital to RNA\ncatalysis, and comment on the involvement of metal ions along the Azoarcus intron splicing pathway in the manuscript.\n9. Line 84, ‘Notably, even though no exogenous G was added during sample preparation, clear density corresponding to\nguanosine was observed in the G-binding pocket, possibly originating from RNA degradation’. The density for the exoG is\nrather weak compared to surrounding RNA nucleotides. The author should change the statement of “clear density” and\nacknowledge the weak density. What is the rationale for modeling this density as guanosine since this density is not that\nefficient?\n10. Related, the authors modeled two consecutive G nucleotides (G0 and G-1 in the model) in the L intron model. With an\neven weaker electron density of the second “G” (G-1), the identity of this density is unclear.\na. The additional density folded back onto the P1ext helix is reminiscent of a so-called “pseudo triple helix” recently\nobserved in another group I intron with intact exons (PMID 40339124), suggesting a more general structural phenomenon in\ngroup I intron P1 helices.\n11. Additionally, in the C intron model, the intron has undergone circularization and there is a covalent bond between 3’-OH\nof wG and U3 and should be modeled as such and update the corresponding figure. The numbering of the C intron model is\nincorrect.\n12. Line 130, ‘positions G37 within the active site’ - this nucleotide appears to be far from wG and the site of nucleophilic\nattack for circularization. What is the distance of G37 to the catalytic site?\n13. Line 143, ‘functionally essential RNA configurations’ may be too strong, circularization is still observed in all G37\nmutants as seen by a band present at 40 minutes for each mutant in Fig. 3d, and is essentially the same as wt.\n14. The work on C’ intron appears to be superficial with only limited characterization of its circular nature and sequence.\nWere there any attempts to gain more mechanistic insights into the C’ intron as well? Biochemically, the author should seek\nto start from the purified C intron to identify a condition that gives rise to the C' intron to support their proposed route. If the\nauthors can obtain sufficient C’ intron samples, is it possible to structurally characterize the newly identified C’ intron to\ncomplete the story?\n15. In the discussion section, the authors discussed the relevance of the study to the PIE system (line 228). With the PIE\nsystem focusing on circularization of the “exon” sequence, the author should clarify how intron self-circularization is relevant\nto the PIE application.\nMinor points:\n16. In Fig. 1B, C’ should also be defined in the figure legend in addition to the results section.\n17. In Fig. S1c, which orientation does the first 2D average (top left) correspond to?\n18. On a related note, there is no angular distribution plot as part of the cryoEM workflow. It is unclear if there is any preferred\norientation issue of the samples. The authors should add such information to the workflow figures.\n19. The different coloring of the tRNA in the first two subpanels of Fig. 1C and Fig. S2A is confusing as the left shows it in\ncolor, and the right has it in gray. Please consider another way to depict the structures for clarity.\n20. Fig.3c the numbering of the secondary cartoon diagrams are wrong,\n21. Fig.4f the authors should consider to number the nucleotides as it will be easy for readers to follow.\n22. Have there been previous studies that similarly observed a register shift in group I introns post-splicing?\n23. Lines 94-96: ‘This conservation underscores the structural stability of the catalytic domain even in the context of full-\nlength tRNA exons and emphasizes the modularity of the group I intron fold.’ The phrases ‘conservation of the catalytic\ndomain’ and the ‘modularity of the group I intron fold’ seem to be at odds with each other\n24. Line 115, language: “bringing the circularization reaction nucleophile, 3’-OH of G205 …”\n25. For cryoEM sample preparation, line 279-280, it is unclear what concentration of RNA samples is used to prepare grids.\nAre there differences in concentration for different RNA samples?\n26. For all structural models, it is recommended to model the intron in a single chain while additional exon sequences in\nanother chain (or with zero or negative numbers) to keep the intron-centric nucleotide numbering in the manuscript\nconsistent with the numbering in the structural model.\n27. Combining Tables S1, 2 and 3 into a single table would allow for easier comparison of the datasets and models in this\nmanuscript.\n28. The authors might consider slowing down the animations in the supplementary movie to allow readers to more easily\nfollow the structural findings.", "reviewer_2": "Azoarcus Nat Comm review\nThis manuscript describes an elegant investigation of a model self-splicing ribozyme isolated from Azoarcus pre-tRNA. The\nauthors use cryogenic to solve the structures of the pre-splicing conformation, which includes the intact, but unsliced pre-\ntRNA visible in two stable conformations with respect to the ribozyme. This is the most important finding of the work–the fact\nthat in this system, as opposed to the twice-larger Tetrahymena ribozyme, the exons only base-pair with the internal guide\nsequence, as predicted by phylogeny and biochemical studies, but never before visualized. Thus, this ribozyme is an ideal\ncatalytic RNA to be used for synthetic biology applications. In addition, this ribozyme folds via an apparent two-state\nmechanism, avoiding complicated intermediates and misfolded states (perhaps the authors could point this out in the\ndiscussion at the end of their manuscript where potential synth bio applications are mentioned). So in many ways, this the\nsimplest of the complicated ribozymes and now the authors show us why this is so from a structural biology perspective.\nIn addition, the authors provide two other states of the ribozyme post splicing – a major and a minor circularization product.\nAs expected, but here shown explicitly, the ribozyme does not undergo any major conformational changes, beyond\nrearrangements in the P1 region where changes are needed for splicing and circularization. And a mechanism for the\nselection of the circularization sites is presented, with three mutants supporting the model of a specific interaction.\nOverall, this a well executed and written up study. I only have a few relatively minor\nissues/suggestions:\n1. The title could use a noun after “pre-splicing” such as “Pre-splicing conformation ..”\n2. The gels and kinetics do not really support a sequential mechanism for C and C’ formation. Yes, if a specific state is\nprepared (as in Fig 4b), C’ will be the new product, but that does not prove the sequential circularization. Instead, it looks\nequally likely that C’ is simply a minor product resulting from a statistical distribution of the 5’ terminal sequences in the\nactive site. This ribozyme has no energy driving any of these reactions - they are all basically in equilibrium because they\nare transesterifications, with the exception of the exogenous guanosine driving the first splicing reaction through chemical\npotential. Everything else is reversible. The band intensities suggest (but the authors should do the measurements and\nshow the kinetics and provide the rate constants) that the rate at which C and C’ appear are similar, it’s just that the\namplitudes are vastly different. This issue should be resolved experimentally or through analysis of the existing kinetics.\n3. Along point #2, it would help to resolve the small products - the 5’ cleaved sequences, for example by labeling the\nribozyme at the 5’ end and then running the kinetics to prove the relative kinetics and amplitudes of product formation.", "reviewer_3": "Hong et al., present a comprehensive, high-resolution structural analysis of Azoarcus pre-tRNA-embedded Group I intron\nself-splicing and circularization. By solving a series of pre-catalytic and post-catalytic structures along the sequential\nreaction progression path, the authors uncover, visualize, and characterize several novel steps of pre-tRNA rotation,\nanticodon remodeling, intron P1 helix remodeling, and a secondary intron circularization pathway. The structural work is\nfurther buttressed by biochemical and mutational analyses. Together, this study substantially advances our understanding of\nthe Group I intron splicing mechanisms, with an emphasis on the elusive intron circularization step. The structural work is of\nhigh quality, the illustrations clear, and the text very well and concisely written. The study provides general, novel insights\ninto RNA catalysis, structural dynamics, and will inform engineering efforts towards improved RNA circularization and\nprocessing. Based on these analyses I recommend this work for publication in Nat. Commun. with great enthusiasm. I do\nhave a few minor comments and suggestions below, with the aim of stimulating discussions and further clarifying the very\ncomplex topic for enhanced readability by the broad readership of the journal.\n1. Fig. S1a. Given the complexity of the splicing and circulation reaction, I suggest the authors move this into the main figure.\nIt is quite helpful in orientating readers who are not intimately familiar with this topic.\n2. Fig. S2. and the animation. The authors do a nice job illustrating how the body of pre-tRNA rotates. However, this rotation\nis evidently driven by the structural remodeling of the base anticodon region connected to the intron, which is not locally\nillustrated. I would suggest the authors add a new visual to show how the exon-intron junction changes conformation which\ncauses the pre-tRNA body to rotate. Although generally useful, figures such as Fig. S3 do not clearly show the exact nature\nof the local remodeling.\n3. Fig. 2, 3. The authors show the “extra G”s but don’t seem to mention them in the text or figure legends. Is this a free\ndinucleotide from degraded RNA, or G35-G36 of P2? Is one of the two Gs the “ExoG”? Please clarify.\n4. In the various secondary structure figures, I would suggest the authors add even more numeric labels, particularly near the\nP1 helix and catalytic regions, to ensure readers can follow the chain connectivity and understand the reactions.\n5. Fig. 4f, can solution RNase action be ruled out for the production of C’ intron, as opposed to water-driven hydrolysis? Is\nthere any potential advantage of producing the C’ intron compared to the C intron?", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts.", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts."}, "author_response": "Response: We sincerely appreciate the reviewer’s constructive suggestion. As recommended, we\nhave moved the schematic diagram of the splicing reactions (Previously Fig. S1a) into Fig. 1a to\nmake the figure more accessible to the general readership of Nature Communications.\nResponse: we sincerely appreciate your valuable feedback on Figure 1D. In response, we have\noptimized the figure by simplifying the representation of secondary structures to enhance the\nclarity of the backbone trace. Additionally, we have highlighted the P1 domain and the catalytic\ncore domain to improve visual guidance (Revised Fig. 1e, Fig. S7).\nResponse: We thank the reviewer for pointing out the differences in circularization efficiency\ncompared with Tanner et al., 1996 [1].Our experimental setup differs in both buffer composition\nand reaction temperature (40 mM Tris–HCl pH 7.5 vs 25 mM HEPES pH 7.5, 37 °C vs 32 °C).\nWe believe the higher temperature and Tris buffer accelerate splicing kinetics, resulting in faster\naccumulation of the linear intron and earlier saturation of the circular intron. These factors likely\nexplain the distinct gel patterns observed.\nResponse: We sincerely appreciate the reviewer's insightful observation regarding the differences\nbetween our results and those reported in Tanner et al [1].\nUpon careful re-examination, we confirmed that the C' intron band persists under our experimental\nconditions. Interestingly, we noticed that in Tanner et al.'s Fig 4 (lane 5, 75°C for 5 min), there\nappears to be an unidentified band below the C intron that migrates at a position similar to our\nobserved C' intron (Fig. R1). This suggests that the C' intron may have also been present in their\nsystem, though not previously characterized.\nFig. R1: Splicing of the Azoarcus pre-tRNA at high temperature[1]. The area pointed to by the red\narrow might be the C' intron.\nResponse: We thank the reviewer for their insightful comments. We would like to clarify that “ctrl”\nin Fig. 3d refers to control samples that did not undergo the in vitro circularization reaction. These\ncorrespond to the denatured linear intron RNAs of either the wild type (WT) or the three mutants,\nwhich were directly loaded onto the gel.\nCompared to Fig. 1b is primarily due to the different RNA substrates used. Fig. 1b utilized the full\npre-tRNA substrate, where the P1 helix formed by the exon and the IGS may significantly\naccelerate the refolding of the catalytic core. In contrast, the original Fig. 3d employed a\ntranscribed L intron with a single G at its 5' end, whose refolding kinetics are potentially slower;\nthis could account for its lower circularization efficiency compared to the intron derived from pre-\ntRNA splicing. In accordance with the reviewer's suggestion, we have repeated the experiments\nunder the optimal literature condition that strongly favors circularization, using the L intron with\ntwo Gs at its 5' end. This construct likely promotes in vitro refolding and stabilizes P1ext through\nthe formation of a pseudoknot-like triplex, thereby markedly enhancing circularization. Under\nthese optimized conditions, the G37C mutant consistently demonstrates a clear promotive effect\non circularization, as robustly shown in the revised Fig. 3d.\nResponse: Thank you for your suggestion regarding additional refinement steps in cryo-EM\nprocessing. We fully agree that approaches such as local refinement and CTF corrections often\nimprove map resolution. In fact, we tested several of these commonly used strategies during our\nprocessing pipeline. However, in our case, they did not yield significant improvements and were\ntherefore not emphasized in the workflow. One additional step, “reference-based motion\ncorrection”, applied prior to the final round of Non-Uniform Refinement, did result in an\nimprovement in resolution. We had inadvertently omitted this detail and have now included it in\nthe data processing workflow (revised Fig.S1b-g、Fig.S4-6).\nResponse: Thank you for raising this critical point. We fully appreciate your concern regarding\nwhether the intron remains unspliced in the pre-1S sample, given the presence of bound guanosine\nobserved in the map. To address this directly, we have performed the suggested denaturing PAGE\ngel analysis under conditions identical to those used for cryo-EM sample preparation (the same\nrefolding buffer, without exogenous GTP, incubated at 37°C for 30 min). The results clearly\ndemonstrate that the pre-tRNA substrate remains predominantly unspliced under this specific\ncondition, providing biochemical evidence that the sample is indeed in the unspliced state (Fig R2\n& revised Fig S1a). Furthermore, both of our cryo-EM structures (pre-1S_a and pre-1S_b)\nunequivocally show that the phosphodiester backbone at the 5' splice site remains intact and\nuncleaved. Therefore, based on this supplementary biochemical evidence and direct structural\nobservation, we conclude that both the pre-1S_a and pre-1S_b conformations represent\nthe unspliced pre-RNA state.\nFig. R2: Pre-tRNA splicing and cyclization assay in the presence and absence of GTP under\ndenaturing conditions. Time-course self-splicing reactions of pre-tRNA were performed at 37°C\nin the absence (-) or presence (+) of 25 μM exogenous GTP, as analyzed by denaturing PAGE.\nResponse: We sincerely thank the reviewer for raising these important questions. To the best of\nour knowledge, the coexistence of two distinct conformational states (Pre-1S_a and Pre-1S_b) at\nthe pre-1S stage has not been previously reported. We speculate that this phenomenon may result\nfrom limited long-range interactions between the intron and exons, which confers considerable\nflexibility to the distal exon regions. The two structures we resolved likely reflect such intrinsic\nstructural dynamics. Functionally, the spatial separation between the intron and exons may\nfacilitate their independent folding, thereby minimizing mutual structural interference.\nRegarding the particle distribution, Pre-1S_b does exhibit a larger particle count compared to Pre-\n1S_a (Fig. S2b-d). However, we currently lack robust evidence to conclude that Pre-1S_b\nrepresents a more dominant or functionally superior conformation. Instead, we propose that the\ntwo states may exist in a dynamic equilibrium, collectively constituting the conformational\nlandscape of the pre-1S assembly.\nResponse: We sincerely thank the reviewer for this important suggestion. We have included a\nparagraph to describe the metal ions in our revised manuscript (Page 7).\n“Coordination of Catalytic Metal Ions Across Splicing and Circularization States\nMetal ions play essential roles in group I intron catalysis, where five specific ions (M -M ) have\nA E\nbeen proposed to form the essential catalytic center. Through cryo-EM structures of five functional\nstates (Pre-1S_a, Pre-1S_b, L intron, C intron, and C′ intron), we have identified and characterized\ntwo of these metal ions: M and ME (Fig. S9). The M ion displays state-dependent coordination\nA A\ngeometry. In Pre-1S conformations, M associates with non-bridging phosphate oxygens of C87,\nA\nG169, and A171 within the intron core. However, its distances to both the 3′-OH of u (–1) and the\nscissile phosphate non-bridging oxygen exceed 4.0 Å, suggesting these structures represent pre-\ncatalytic conformations sampling the first splicing step. Notably, during circularization, U3—the\nkey nucleotide at the circularization site—undergoes a conformational rearrangement that\npositions its phosphate group within the coordination sphere of the MA, thereby anchoring the\ncircularization site inside the catalytic core. The M ion lies peripherally to the active site and\nE\nmaintains stable coordination with the non-bridging phosphate oxygens of U172 and A173 in all\nobserved states. This conserved geometry likely stabilizes the tertiary architecture of the P9.0\ndomain and contributes to maintaining the integrity of the ribozyme’s scaffold.\nBeyond these established metal sites, we identified an additional ion in the catalytic core occupying\nthe M position previously reported in Tetrahymena ribozymes (cite paper). This M coordinates\n2 2\nthe non-bridging phosphate oxygen of A86 and the 3′-OH of G127. The conservation of this\nposition among group I introns suggests a possible catalytic or structural role, although its precise\nfunction remains to be determined. Together, these findings reveal that while the metallo-active\ncore maintains a conserved overall architecture, the coordination geometries of individual ions\nadjust dynamically in response to transitions between splicing and circularization. This structural\nplasticity provides a mechanistic basis for how group I introns accommodate distinct chemical\nreactions while preserving catalytic precision.”\nFig. R3: Coordination of catalytic metal ions in the group I intron active site during splicing and\ncircularization. Close-up views show the active site in five key states: Pre-1S_a，Pre-1S_b, L\nintron, C intron, and C′ intron. Cryo-EM densities reveal the coordination states of the catalytic\nmetal ions M , M , and M across these states.\nA E 2\nResponse: We sincerely thank the reviewer for this thoughtful comment regarding the density\nobserved in the G-binding pocket. We have carefully revised the description in the manuscript to\nindicate that the density is weak rather than clear, to more accurately reflect our observation.\nAlthough the density is weak, it aligns precisely with the canonical guanosine-binding site seen in\npreviously characterized Group I intron structures and fits well with the expected geometry of a\nguanosine base. For this reason, we modeled it as a guanosine with partial occupancy, which we\nbelieve provides the most reasonable structural interpretation while acknowledging the limited\ndensity.\nResponse: We thank the reviewer for this insightful comment and for pointing out the recent work\n(PMID: 40339124)[2] describing the pseudo–triple-helix configuration. We carefully re-examined\nthe corresponding region in our L intron map and compared it with the structural features reported\nin that study. Despite the weaker density observed for the second guanosine (G–1), the overall\nspatial arrangement of this region closely resembles the pseudo–triple-helix architecture described\npreviously.\nTo further evaluate this possibility, we performed a detailed interaction analysis on the P1ext helix,\nwhich confirmed that it indeed satisfies the definition of a pseudo triple helix, wherein a continuous\nnucleotide strand engages a double helix via non-canonical pairings (Fig. R4 & revised Fig. S4g).\nWe have accordingly revised the manuscript to explicitly identify this feature as a pseudo–triple\nhelix. We sincerely thank the reviewer for this valuable suggestion, which has helped us refine and\nstrengthen our structural interpretation.\nFig. R4: A pseudo triple helix in the L intron P1ext.The cryo-EM structure shows the P1ext\nadopting a folded-back architecture, forming a stable pseudo triple helix.\nResponse: We sincerely thank the reviewer for this valuable suggestion. We have modeled the\ncovalent linkage between 𝜔G and U3 to reflect intron circularization, and corrected the nucleotide\nnumbering. The updated model has been re-uploaded to the PDB, and the corresponding figure\n(Fig. 3a) has been revised.\nResponse: We thank this reviewer for this pointing out. Our structural analysis shows that in the L\nintron, nucleotide G37 is approximately 20.3 Å away from the phosphate at the circularization site\n(Fig. R5), indicating that it is not directly positioned within the catalytic center. Accordingly, we\nhave revised the text to remove the claim that G37 is “within the active site.”\nFig. R5: G37 is located ~20.3 Å away from the catalytic site.\nResponse: We sincerely thank the reviewer for the insightful suggestion. We have toned it down\nby revising the word “essential” to “important”.\nResponse: We sincerely thank you for your insightful comments regarding the need for deeper\nmechanistic and structural characterization of the C’ intron. Your feedback provided crucial\nguidance for significantly improving our study.\nIn our initial investigations, the yield of highly purified C intron obtained directly from natural\nsources was extremely limited, sufficient only for RNase R digestion and reverse transcription\nsequencing but inadequate for detailed biochemical validation. Therefore, we utilized the\nhydrolytic linearized product of C intron (L-3 intron) to model the formation pathway of C’\nintron[1].\nIn direct response to your comments, we dedicated effort to optimizing our purification strategy\nand have now successfully obtained a preparation of high-purity C intron free from C’ intron\ncontamination. Using this critical material, we performed the key validation experiments you\nsuggested. As shown in Fig. R6 (Revised Fig. 4e), our biochemical data clearly demonstrate that\nC intron first undergoes hydrolytic opening to produce the linear L-3 intron, which subsequently\nre-circularizes to form the C’ intron. This evidence strongly supports our previously proposed two-\nstep circularization model.\nFurthermore, and most significantly, we have now successfully prepared sufficient quantities of\nhighly pure C’ intron. Using single-particle cryo-electron microscopy, we have determined its\nhigh-resolution three-dimensional structure (Fig R7 & revised Fig. 5a). This novel structural\ninformation provides a direct molecular basis for understanding its conformational differences\nfrom the canonical C intron and its unique properties.\nThese crucial supplementary experiments, mechanistic validation, and the new structural findings\nhave all been incorporated into the revised manuscript (Page 5-6). We are grateful again for your\nvaluable suggestions, which have profoundly enhanced the quality and depth of our work.\nFig. R6 Time-course analysis demonstrating the production of C′ intron, concomitant with a\ndecrease in C intron.\nFig. R7 Cryo-EM map and model of the C’ intron at 2.86 Å resolution.\nResponse: We sincerely thank the reviewer for raising this important point. We fully agree that the\nPIE (permuted intron–exon) system is primarily designed to circularize exon sequences, while our\nstudy focuses on the self-circularization of the excised intron. Although the substrates differ, both\nprocesses share the same Group I intron catalytic core and depend on similar structural\nrearrangements, including the alignment of the P1 helix and ωG residue that enable site-specific\nligation. The structural insights from our study, such as the conformational change of G37 and the\nrepositioning of P1/P1ext, illustrate how the catalytic geometry of Group I introns can be tuned to\npromote efficient circularization. These mechanistic principles are directly applicable to the\nengineering of PIE systems to enhance the yield and precision of exon circularization.\nTo address this comment, we have revised the Discussion section (Page 8) to clarify this\nconnection and explain that our findings provide a structural and mechanistic framework that can\nguide further optimization of PIE-based RNA circularization strategies.\nResponse: As suggested, we have now included a clear definition of the C' intron in the legend of\nFig. 1B (Revised Fig. 1c).\nResponse: We sincerely thank the reviewer for pointing this out. To clarify the orientation of\nFigure S1c (Revised Fig. S1c), we have added a schematic directly below (Fig. R8), with an arrow\nindicating the viewing direction—from the bottom, along the path from P8 and P3 toward the\ncatalytic core. The short arm on the left corresponds to the tRNA domain. This rotated view aligns\nwell with the 2D schematic, allowing a clearer correspondence between the spatial structure and\nits simplified representation.\nFig. R8: Structural interpretation of the representative 2D class average. The top-left 2D class\naverage from Fig. S1c is shown alongside its corresponding orientation within the three-\ndimensional cryo-EM density map, illustrating the projection direction from which the 2D image\nwas derived.\nResponse: We thank the reviewer for this comment. As suggested, we have now included the\nangular distribution plot of particle orientations in our cryo-EM workflow figure (Fig. S1f, Fig.\nS4e, Fig. S5e, Fig. S6e) in the Supplementary figures. As shown (Fig. R9), the particles are well-\ndistributed across all orientations, indicating that there is no preferred orientation issue in our\ndataset.\nFig. R9: Particle distribution map, showing a uniform and isotropic pattern without preferred\norientation.\na, Particle distribution map of Pre-1S_a.\nb, Particle distribution map of Pre-1S_b.\nc, Particle distribution map of L intron.\nd, Particle distribution map of C intron.\ne, Particle distribution map of C’ intron.\nResponse: We sincerely appreciate the reviewer's valuable feedback regarding the color usage in\nFigure 1C (Revised Fig. 1d) and Figure S2A (Revised Fig. S2a). We have revised the color scheme\nin these subpanels. The tRNA is now depicted using a consistent and coordinated color scheme\nacross all relevant panels, which significantly enhances the visual consistency and readability of\nthe figures and facilitates structural comparison.\nResponse: We sincerely thank the reviewer for pointing this out. Upon rechecking, we found that\nthe inconsistency was due to an error in the tertiary structure numbering, which did not match the\nsecondary structure. This has now been corrected, and the revised figure has been updated\naccordingly (Fig. 3c).\nResponse: We thank the reviewer for this valuable suggestion. To achieve an optimal balance\nbetween informational clarity and visual aesthetics, we have revised Fig. 4f by specifically\nnumbering the key nucleotides directly involved in the circularization site. We believe this targeted\nlabeling will effectively assist readers in following the critical structural features without\ncompromising the figure's clarity through overcrowding (Revised Fig. 5d).\nResponse: This register shift results from two sequential rearrangements of the IGS region during\nthe splicing process—namely, the formation of P10 and the release of the 5’ exon—between the\npre-1S, pre-2S, and post-2S states. A similar phenomenon has been reported in the Tetrahymena\ngroup I intron. In the study “Cryo-EM reveals dynamics of Tetrahymena group I intron self\nsplicing”[3], the P1/P1ext duplex in the Pre-Tet-S1 (Pre-1S) state (Fig.2d) and the P1’ duplex in\nthe Tet-C (Post-2S) state (Fig.1j) exhibit a 14-nt register shift (Fig. R10).\nFig. R10: Comparison of the secondary structures of the nuclear enzymes Pre-1S and Post-2S P1\nof Tetrahymena[3]\nResponse: We thank the reviewer for this insightful comment. We agree that the original phrasing\ncreated an apparent contradiction. The sentence has been revised to state: \"This conservation\nunderscores the structural stability of the catalytic domain even in the context of full-length tRNA\nexons and emphasizes the conservation of the group I intron fold.\" This change more accurately\nreflects the intended meaning and removes any potential confusion (Page 3).\nResponse: We thank the reviewer for pointing out this imprecise wording. We have revised it as\nthis reviewer suggested.\nResponse: We thank the reviewer for pointing this out. All RNA samples were prepared at a\nconcentration of 20 μM for cryo-EM grid preparation, and this uniform concentration was used\nacross all samples to ensure consistency. We have added this information in the revised manuscript.\nResponse: We thank the reviewer for this valuable recommendation regarding our structural\nmodels. We have followed the suggestion to model the intron and exon sequences separately in all\nour structural models. The specific implementation is as follows:\n1. The intron was modeled as a single continuous chain, which ensures that its nucleotide\nnumbering is entirely consistent with the intron-centric numbering scheme used throughout the\nmanuscript. 2. For the exons, our initial intention was to also model them as a single chain.\nHowever, during modeling, the software would automatically link the 5' and 3' exons, resulting in\na post-splicing tRNA product, which does not correspond to the pre-tRNA substrate state our study\naims to represent. 3. To resolve this, we ultimately modeled the 5' exon and the 3' exon as two\nseparate chains. Their sequences were arranged strictly according to the secondary structure\ndescribed in the manuscript, thereby ensuring that all critical numbering at the exon-intron\nboundaries is perfectly consistent with the descriptions in the text.\nWe believe that the current modeling strategy accurately reflects the pre-tRNA substrate state of\ninterest while faithfully adhering to the spirit of the reviewer's recommendation.\nResponse: Done.\nResponse: We sincerely thank the reviewer for the valuable suggestion. We have adjusted the\nplayback speed of the supplementary movie to slow down the transitions between structural states,\nin order to facilitate clearer visualization and interpretation of the conformational changes.\nResponse: We thank this reviewer’s helpful suggestion regarding the title. We have revised the\ntitle to: “Pre-Splicing Conformation and Stepwise Circularization of a Group I Intron in Azoarcus\nPre-tRNA.”\nResponse: We sincerely thank the reviewer for their insightful comments and valuable suggestions,\nwhich prompted us to conduct a more rigorous investigation into the formation mechanism of the\nC′ intron. We fully appreciate the concern that robust experimental evidence is required to\ndistinguish between a sequential mechanism (L → C → C′) and a parallel reaction pathway (where\nL → C and L → C′ occur independently).\nTo address this question directly, we designed and performed two complementary sets of\nexperiments.\nIn the first set of experiments, we sought to validate the sequential mechanism by using purified\nC intron as the starting material. After optimizing the preparation protocol to obtain highly pure C\nintron, we performed a time-course assay. The results show a clear kinetic profile (Fig R11&\nrevised Fig 4e): as the reaction progressed, the band intensity of the C intron gradually decreased,\naccompanied by a concurrent increase in its hydrolytic product (the L-3 intron) and a synchronous\nenhancement in the intensity of the C′ intron band. This observation, which captures the\nintermediate state of the reaction, provides direct evidence that the C′ intron is derived from the\nconversion of the C intron.\nIn the second set of experiments, we used a 5′-end labeling strategy to test for the possibility of\ndirect C′ intron formation from the linear L intron. Following the reviewer's suggestion, we\nconstructed an intron labeled with a FAM fluorophore at its 5′ end. Since the full-length 205-nt\nintron could not be directly synthesized, we split it at the P7 loop into two fragments (A and B),\nwhich were shown to co-refold and undergo circularization ligation in vitro. It is noteworthy that,\ndue to two additional guanines introduced at the 5′ end of our construct, the observed cleavage\nproducts were GGAU and GGAUUU, rather than the theoretical GAU and GAUUU.\nA key technical aspect of this experiment involved two independent imaging modalities performed\non the same denaturing gel. First, FAM fluorescence imaging was used to specifically visualize\nthe small, cleaved 5′ fragments. The results from this image (Fig R11& revised Fig 4f, lower) were\nunequivocal: only the expected 4-nt product (GGAU) was detected, with no band corresponding\nto the 6-nt product (GGAUUU). Subsequently, the same gel was stained with Gel-Red and imaged\nunder UV to visualize the total RNA. The upper Fig. R11 clearly shows an increasing ~200 nt\nband over time, confirming successful circularization ligation between Fragments A and B.\nTogether, these results lead us to two main conclusions. First, Experiment 1 positively confirms\nthe C → C′ conversion pathway. Second, Experiment 2 rules out the direct L → C′ pathway under\nour specific in vitro experimental conditions. We wish to emphasize that our inability to detect\ndirect L → C′ conversion is based on the constraints of our in vitro assay system, and does not\npreclude the possibility that this pathway might occur in vivo with additional cellular factors or\nunder different conditions. Nevertheless, based on the current evidence, the sequential\ncircularization mechanism (L → C → C′) remains the best-supported model. We are again grateful\nto the reviewer, as their input has significantly enhanced the rigor of our study and the robustness\nof our conclusions.\nFig. R11: Circularization analysis using split L intron fragments. FAM-labeled Fragment A (1-69\nnt, with two 5′ Gs) was co-refolded with Fragment B (70-205 nt). The top panel shows GelRed\nstaining for longer RNA fragments, and the bottom panel shows FAM imaging for short drop-off\nfragments. Only the 4-nt drop-off fragment (GGAU) from C intron circularization was observed,\nwhile the 6-nt fragment (GGAUUU) from direct C′ intron formation was absent.\nResponse: The same reply as the second question.\nResponse: We sincerely appreciate the reviewer's valuable suggestion. To help readers better\nunderstand the relevant content, we have incorporated the schematic diagram of splicing reactions\n(Previously Fig. S1a) into Fig. 1a as recommended. This modification aims to provide clearer\nillustration of the key concepts discussed in the manuscript.\nResponse: We thank the reviewer for this insightful suggestion. Comparative analysis of the local\nexon-intron junction in the Pre-1S_a and Pre-1S_b conformations revealed that the junction\nnucleotide a (t40) exhibits significant conformational flexibility between the two states. The\npronounced swing of a (t40) likely provides the structural basis for driving the rotation of the pre-\ntRNA body. We have now provided a more detailed visualization of this conformational change\nat the key junction in the revised Fig. R12 (Revised Fig. S2d) and the movie.\nFig. R12: Conformational shift at the exon-intron junction. The nucleotide a (t40) undergoes a\nsubstantial rotation, resulting in an overall ~45° rotation of the exon between the Pre-1S_a and\nPre-1S_b states.\nResponse: We sincerely appreciate the reviewer’s careful attention to this detail regarding the\n“extra G” nucleotides. We apologize for not clarifying this point in the original text and figure\nlegends. In Fig. 2f, the labeled \"ExoG\" refers to the exogenous guanosine derived from RNA\ndegradation during the splicing/cyclization process. In Fig. 2a and Fig. 3b-c, the “extra Gs”\nrepresent the two additional guanosines intentionally incorporated during in vitro transcription of\nthe intron. We have revised the figure legends and main text to explicitly explain the origin and\nidentity of these nucleotides. Thank you for bringing this important clarification to our attention,\nwhich will help readers better interpret our structural data.\nResponse: We fully agree that additional numerical labels in key regions would significantly aid\nreaders in tracing the structural connectivity and understanding the reaction mechanisms. As\nsuggested, we have added numerical labels to the key nucleotides flanking the 5' splice site to\npinpoint their locations. We also have highlighted the guanosine-binding site within the catalytic\ncore domain (Fig. 1e, Fig. S7).\nResponse: We sincerely thank the reviewer for raising this insightful question regarding the\nformation mechanism of the C' intron. The distinction between RNase action and hydrolysis is\nindeed crucial.\nAs the reviewer astutely noted, the generation of the C' intron in our system is consistent with the\nspecific reopening mechanism of group I intron circles first reported by Zaug et al. (Science, 1984).\nThat seminal work clearly demonstrated that circular introns can undergo specific reopening at the\noriginal circularization site, yielding linear molecules with 5'-phosphate and 3'-hydroxyl termini—\ncharacteristic products of enzymatic reopening rather than random RNase hydrolysis.\nTo directly test the potential involvement of solution RNases, we designed a specific experiment:\nadding Recombinant RNase Inhibitor (RRI) to the circularization reaction system. Our results\nclearly show that C' intron formation occurs with comparable efficiency both in the presence and\nabsence of RRI (Fig. R12 & revised Fig. S8). This key evidence indicates that C' intron formation\nprimarily depends on the specific reopening mechanism mediated by the intrinsic catalytic activity\nof the intron itself, rather than random cleavage by solution RNases.\nBased on this evidence, we conclude that the generation of the C' intron aligns with a specific,\nwater-driven hydrolysis mechanism, which is fully consistent with the inherent reversible catalytic\nproperty of group I introns.\nFig. R13: Circularization of the L intron in the presence (+) or absence (-) of Recombinant RNase\nInhibitor (RRI). The C′ intron is produced irrespective of Recombinant RNase Inhibition,\nconfirming it is a specific product of the two-step circularization pathway and not an artifact of\nrandom RNase cleavage.\nRegarding the potential advantages of producing the C′ intron compared to the C intron. At present,\nwe do not have direct evidence indicating a specific functional or structural advantage of the C′\nintron. We speculate that its formation may represent a thermodynamically more stable or end-\nstate configuration of the circularization process, achieved through further relaxation of the P1ext\nhelix. Alternatively, the existence of two circular species might reflect an intrinsic flexibility of\ngroup I introns that allows adaptive regulation under different physiological conditions. These\npossibilities remain open, and we plan to investigate the biochemical stability, dynamics, and\npotential biological implications of the C′ intron in future studies.\nResponse: Thanks very much.\nResponse: Thanks very much."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have done very well to address the points raised in our initial review of their manuscript. The revised manuscript\nhas been significantly improved with more detailed biochemical experiments and has been further strengthened by structural insights into the C’\nintron structure. We have several minor remaining comments towards the manuscript prior to publication:\n1. Regarding metal ion modeling: can the authors confirm that no density corresponding to catalytic metal Mc was visible in\nthe electron density map? It was clearly visualized in prior Azoarcus structure (Science 2005). The authors should\nacknowledge the fact that group I intron uses the two-metal catalysis mechanism while mentioning potential limitations of\nmetal ion visualization in the manuscript.\n2. Added RRI experiment (pg. 6. Line 178-180) does not necessarily prove that C’ intron generation is water driven. It is\nunclear which RRI was used since its identity is not mentioned in the method section. Regardless, recombinant RRI is only\ngood for inhibiting a certain subset of RNases. To really prove that the event is water-driven, a better experiment is to\nconduct the experiment at acidic pH (eg. 6.0), which would significantly reduce the C’ intron production if it is water-driven.\nRNases are rather insensitive to pH change and C’ intron production won’t change much if it is RNase-driven.\n3. “Register shifts” in introduction (pg. 2, line 54) is vague without further context. The author should be more specific about\nP1/P1ext dynamic changes here. Related, there are many interesting observations across multiple structures regarding\nP1/P1ext conformational changes through the splicing/circularization pathway, the authors should consider adding a brief\nsummary to the discussion section.\n4. Line 135, G37-G11 is described as “Watson-Crick-like base pair” but in Fig. 3c the model shows that G37 interacts with\nG11 with its Hoogsteen face, hence making the base pairing as a Hoogsteen pair instead.\n5. Line 241, remember to change “(cite paper)” into real citation.\n6. Line 276, it would be helpful to introduce the PIE acronym as ‘permuted intron-exon’.\n7. In the data availability statement (ln 452), it is recommended to annotate the data entries for which conformation/state they\nare in (pre-1S_a, pre-1S_b, L, C, C’) due to the number of structures involved in this paper.", "reviewer_2": "This is now a much improved manuscript describing an important contribution to the fields of RNA catalysis.\nMinor comments:\n1) on line 241 the citation is missing\n2) if authors wish to put the discussion of metal ions ME and M2 in more functional perspective , a long forgotten paper on\nthe closely related Anabaena ribozyme showed that the positions coordinating these metal ions show strong\nphosphorothioate interference to splicing but not global folding (NAR 2004 DOI: 10.1093/nar/gkh548)", "reviewer_3": "In my view the authors have done a great job addressing the reviewer comments carefully and effectively, including\nperforming additional clarifying experiments. These efforts have substantially strengthened and improved the manuscript. I\nrecommend its publication in its current form.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts.", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts."}, "author_response": "Response: Thank you for this important and helpful comment. We carefully examined\nthe cryo-EM density maps and confirm that density corresponding to the catalytic metal\nion Mc is indeed observable in our reconstructions (Fig. R1). The presence of this\ndensity is consistent with the established two-metal-ion catalytic mechanism reported\npreviously for the Azoarcus group I intron (Science, 2005). Following the reviewer’s\nsuggestion, we have revised the manuscript to explicitly acknowledge the two-metal-\nion catalytic model and to clarify the visualization of catalytic metal ions in our\nstructures. We also note that, as with many cryo-EM studies of RNA, metal ion density\ncan vary in strength depending on local coordination, occupancy, and resolution, which\nmay affect interpretability. We sincerely appreciate this insightful feedback, which has\nhelped us improve both the clarity and completeness of the discussion.\nFig. R1: Coordination of catalytic metal ions in the group I intron active site during\nsplicing and circularization.\nResponse: Thank you for this critical and constructive comment regarding the RRI\nexperiment. We agree with the reviewer that the initial use of RNase inhibitor alone is\nnot sufficient to definitively demonstrate that C′ intron generation is water driven,\nparticularly given that recombinant RNase inhibitors act on only a limited subset of\nRNases. We clarify here that the RNase inhibitor used in our experiments was the\nTaKaRa Recombinant RNase Inhibitor (Cat. No. 2313A), and this information has now\nbeen explicitly added to the Methods section.\nAs suggested, we performed additional experiments under acidic conditions to directly\ntest whether C′ intron formation is water driven. We compared reactions carried out at\nneutral pH (pH 7.5) and acidic pH (pH 6.0) (Fig. R2). After incubation at 37 °C for 6\nhours, hydrolysis of the C intron and subsequent generation of the C′ intron were readily\nobserved at pH 7.5. In contrast, under acidic conditions, no detectable C′ intron was\nobserved. Because RNase activity is generally insensitive to moderate pH changes,\nwhereas water mediated transesterification reactions are known to be suppressed under\nacidic conditions, these results support a water driven mechanism rather than an RNase\ndependent process. We have revised the manuscript accordingly to incorporate these\nnew data and to clarify the mechanistic interpretation (Page 6).\nFig. R2: Second-step circularization of the C intron performed at pH 7.5 or pH 6.0.\nResponse: Thank you for raising this important point. As suggested, we have revised\nboth the Introduction (Page 2) and Discussion (Page 8) to explicitly highlight the\nnucleotide register shift that occurs during the transition between functional states, and\nto better contextualize its role in coordinating splicing and circularization.\nResponse: Thank you for this careful observation. You are correct that the interaction\nbetween G37 and G11 involves the Hoogsteen face of G37, and therefore corresponds\nto a cis Watson–Crick/Hoogsteen base pair rather than a canonical Watson–Crick pair,\nas shown in Fig. 3c. We have corrected the description accordingly. We appreciate the\nreviewer’s attention to this detail.\nResponse: Thank you for the reminder. We have now updated it with the correct citation.\nResponse: Done.\nResponse: Thank you for this helpful suggestion. In the revised data availability\nstatement, we have now explicitly annotated each deposited entry with its\ncorresponding conformational states (Pre-1S_a: EMD-64818, PDB 9V7N; Pre-1S_b:\nEMD-64819, PDB 9V7O; L intron: EMD-64820, PDB 9V7P; C intron: EMD-64821,\nPDB 9V7Q; C’ intron: EMD-66618, PDB 9X6B).\nResponse: Thank you for the reminder. We have now updated it with the correct citation.\nResponse: Thank you for this helpful suggestion. We have added a brief discussion\nreferencing the Anabaena group I intron study (NAR 2004, DOI: 10.1093/nar/gkh548)\nto provide a more functional perspective on the potential roles of M and M in catalysis\nE 2\nrather than global folding (Pages 8-9).\nResponse: Thanks very much.\nResponse: Thanks very much.\nResponse: Thanks very much."}]

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