Structural basis of circularly permuted group II intron self-splicing

doi:10.21203/rs.3.rs-3888028/v1

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Structural basis of circularly permuted group II intron self-splicing

https://doi.org/10.21203/rs.3.rs-3888028/v1

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The group II introns are self-splicing ribozymes intervened between two exons that undergo excision and exon ligation through lariat formation, which are considered as ancestral forms of spliceosomes. Circularly permuted (CP) group II introns comprise of rearranged structural domains separated by two tethered exons have been artificially designed long ago to generate branched intron and circular exon products via back-splicing, whose natural occurrence has also been recently identified. Although extensive studies have elucidated mechanisms of both forward and reverse splicing processes of canonical group II introns, structural and mechanistic understandings of circular RNA (circRNA) generation by CP introns remain elusive. Here we resolve cryo-electron microscopy structures of a natural CP group II intron with 3'- and 5'-exons at 2.65-2.97 Å resolution, which represent different states associated to two steps of the back-splicing process. Novel tertiary interactions are identified to play allosteric effects on catalytic activity. The branching helix D6 undergoes a 65º conformational change enabled by tertiary interactions with D6 terminal T-loop after the branching reaction to allow docking of 3'-exon for ligation. Local shifts of peripheral domains, exon binding sites, and metal ions in the catalytic core, including a novel metal ion M₃₅ that stabilizes the 5'-exon, are also observed between two steps of splicing. These results elucidate the dynamics of CP group II intron back-splicing process and mechanism of circRNA generation with potential applications in circRNA research and therapeutics.

Biological sciences/Structural biology/Electron microscopy/Cryoelectron microscopy

Biological sciences/Molecular biology/Ribozymes

Biological sciences/Chemical biology/RNA

Group II introns are large structured RNA enzymes ranging from 400-900 nucleotides (nts) that carry out two transesterification reactions to self-splice themselves and ligate the exons^1,2. Most introns also encode a maturase protein with helicase and reverse transcriptase activity that facilitates the reverse splicing process called retrotransposition to invade DNA as selfish mobile genetic elements^2,3, which likely account for their widespread distribution in prokaryotes, mitochondria and chloroplasts of fungi, algae, plants, and lower eukaryotes^4,5. Similarity in structures and splicing mechanisms between group II introns and spliceosomes suggest that they are likely ancestors of nuclear pre-mRNA introns and spliceosomes^6,7.

The group II intron consists of six conserved structural domains (D1-D6) from 5' to 3' end (Fig. 1a)^1,4: Starting from 5' end is the 5'-exon and 5'-splice site (5'-SS), followed by intron’s largest D1 domain that serves as the structural scaffold and contains exon binding sites (EBS) specifically recognizing exons by complementary base-pairing; D2 and D3 form numerous interactions with D1, D5 and D6 to organize the catalytic core and to position D6 in place for catalytic reactions. Importantly, the conserved junction between D2 and D3 (J2/3) is essential to catalytic site formation in D5; D4 carries the intron encoded protein (IEP) that facilitates the forward and reverse splicing processes; D5 is the most conserved domain, analogous to U6 small nuclear RNA (snRNA) in spliceosome, that contains the catalytic triad stabilized by a 2-nt bulge and J2/3 to shape the intact catalytic core; D6 contains the conserved bulged A as the branch point facilitating the nucleophilic attack with its 2'-OH in the first transesterification reaction to cleave the 5'-SS and form the lariat with 2'-5' phosphodiester linkage; In the second reaction, the 3'-SS of 3'-exon is attacked by the 5'-exon to remove the lariat intron and ligate the exons, where D6 undergoes large conformational change assisted by IEP in both forward and reverse splicing ^8,9.

Circularly permuted (CP) group I and II introns with rearranged structural domains have been artificially designed to produce circular RNAs (circRNAs)^10,11. CircRNAs have been playing increasingly important roles in regulations of fundamental biological processes and applications in therapeutics such as vaccines^12,13. Recently, natural CP group II introns have been found in bacterial chromosomes and plasmids, with D5, D6 and 3'-exon swapping to the 5' end of 5'-exon analogous to those previously designed^11,14. Two steps of splicing reactions analogous to canonical group II introns are carried out in the absence of protein as D4 has no coding region. In the first step (1S), bulged A in D6 cleaves the 5'-SS to form a branching intermediate (BI) containing both exons and the 3'-SS, which is subsequently cleaved by the 5'-exon in the second step (2S) to remove the branched product (BP) and generate the circular exons (Fig. 1b). Although the circular exon lengths generated by the identified natural CP group II introns were generally below 100-nt¹⁴, recent study has reported production of longer than 700-nt circRNAs that could be robustly and persistently translated into proteins, including those from pathogens to trigger immune response¹⁵. Despite the promising therapeutic applications of circRNA vaccine produced by CP intron catalysis, structural insights into the CP introns remain elusive, precluding our mechanistic understanding of the dynamic process of circRNA production.

Single particle cryo-electron microscopy (cryo-EM) has been increasingly used to study structures and dynamics of large ribozymes undergoing catalysis in the absence of proteins^16-19. In this work, we used cryo-EM to study the catalytic process of a natural CP group II intron previously identified from Comamonas testosteroni KF-1 (Cte 1, 814-nt)¹⁴, and determined four structures revealing different conformations associated to the first branching step (pre-1S, 1S) and second ligation step (2S, post-2S) of back-splicing at 2.65, 2.97, 2.90 and 2.90 Å resolution, respectively (Fig. 1c). The core region adopts homologous architecture including the catalytic triad (A60, G61, C62) and conserved tertiary interactions compared to canonical group II introns, whereas novel tertiary interactions are observed in the peripheral regions that allosterically modulate catalytic activity. The 5'-SS is in close proximity to bulged A127 and ready for cleavage in 1S state upon binding to EBS1 (nts 540-545), which is unexpectedly stabilized by the major-groove interaction consists of three base triples from the adjacent nucleotides in the same EBS loop (nts 535-537). Prior to 1S (pre-1S), bulged A127 flips away from the 5'-SS while D6 maintains similar position as in 1S. In the 2S state in the absence of IEP, a 65º conformational change of D6 containing the branched 2'-5' phosphodiester linkage (A127-G220) facilitates local shift of peripheral domains and docking of 3'-exon recognized by EBS3 (G539) in the catalytic site, aligned for nucleophilic attack by 5'-exon to generate the ligated circular exons. After release of the circRNA, the EBS loop is stabilized via local rearrangement to form two internal base triples, and the 3'-SS coordinates with a network of interactions analogous to lariat forms of canonical group II introns poised for transposition. A total of more than 60 metal ions was found in each structure, with two metal ions consistently found in the catalytic site directly participating in the splicing process, suggesting that both reactions occurred through the conventional two-metal-ion mechanism²⁰. Interestingly, a novel metal ion M₃₅ was identified close to the conserved heteronuclear metal ion center to coordinate with the 5'-exon for both splicing reactions. Collectively, these structures illustrate snapshots of the dynamic back-splicing process and elucidate the mechanism of circular exon generation by a natural CP group II intron, providing structural basis for circRNA production with potential applications in research and therapeutics.

Cryo-EM structures of different catalytic states in the Cte 1 intron splicing process

The Cte 1 RNA construct was prepared by in vitro transcription (IVT), during which the catalytic reactions have already occurred after cotranscriptional folding in the presence of Mg²⁺ (Extended Data Fig. 1a)^17,21,22. We did not observe the BI band in denatured gel electrophoresis suggesting that both first and second catalytic reactions happened almost simultaneously, analogous to canonical group II intron splicing^8,23. Cryo-EM analysis of the IVT product yielded reconstruction of the branched product at 2.90 Å resolution with the A127-G220 2'-5' phosphodiester linkage and D6 pointing towards D2, corresponding to the post-2S state with circular exons released (Fig. 1c, Extended Data Fig. 1b, Extended Data Table 1).

To capture the branching (1S) and ligation (2S) states, we purified the Pre RNA and replaced Mg²⁺ with Ca²⁺ in catalytic reactions that has previously been shown to inhibit splicing ^8,23-25. Denatured gel electrophoresis showed accumulation of BI as splicing reaction proceeded, the 5-minute and 4-hour time points were subjected to cryo-EM analysis (Extended Data Fig. 2a). Three-dimensional (3D) classification and refinement yielded three reconstructions at 2.65 Å, 2.97 Å and 2.90 Å resolution, representing pre-1S, 1S and 2S states, respectively (Fig. 1c, Extended Data Fig. 2b, Extended Data Table 1). The 2S structure adopts almost identical conformation as the post-2S with extra density of 5'-exon bound to EBS1 and 3'-exon bound to EBS3. The pre-1S and 1S structures have the D6 helix translocated 65º away from the 2S and post-2S conformations, positioning the 2'-OH of bulged A127 in close proximity to the scissile phosphate at the 5'-SS. The 1S structure is distinguished from the pre-1S because bulged A127 in 1S is ready to cleave the docked 5'-exon.

Overall architecture and novel tertiary interactions that allosterically affect intron catalysis

The Cte 1 intron consists of novel P1 and P2 domains at the 5' end followed by D5 and D6 that establish the catalytic core, the intervened 3'- and 5'-exon await to be excised, and the scaffold D1-D3 with D4 at the 3' end forming a closing stem (Fig. 1d, Extended Data Fig. 3). P1 and P2 are found generally conserved and predicted to form pseudoknot (PK) interactions based on comparative genomics analysis^14,26. Our Cte 1 cryo-EM structures reveal that P1 forms a PK with the D2 terminal loop, whereas P2 forms another PK with a terminal loop at the 3' end of D3 (Γ'-Γ and Δ'-Δ) (Fig. 2a-b).

The following D5 contains the conserved AGC triad and 2-nt bulge that collectively form a catalytic triplex core homologous to the spliceosomal U2/U6 small nuclear RNA^1,27. Multiple tertiary interactions are identified between D5 and D1 (ψ'-ψ, λ'-λ, ζ'-ζ)^28,29, D3 (κ'-κ, μ'-μ) and D4 (φ'-φ) in order to organize the catalytic core ^30,31, consistent with those previously observed in canonical group II introns (Extended Data Fig. 4)^9,22,32,33. D6 is downstream of D5 that contains the branchpoint A127 and a few previously identified tertiary interactions with D1 (π'-π) and D2 (η'-η, γ'-γ) that facilitates D6 dynamics between different catalytic states (Extended Data Fig. 5a-c)^8,9,32,34. The 3'- and 5'-exon are presented next with intron binding sites (IBS3 and IBS1) that recognize EBS in D1^35,36.

The subsequent D1 (including D1a-D1d subdomains) forms numerous interactions with all domains of the intron to serve as the scaffold for correct folding, exon recognition, and catalytic core organization. Novel interactions have been observed in the peripheral regions, including three GAAA tetraloop/tetraloop receptor (TL/TLR) interactions between D1 and D1c (ξ-ξ') and D1b and D1c (Α-Α' and Β-Β'), a base triple between D1c and D2 (υ-υ'), and a PK between D1d and D3 (ο-ο') (Fig. 2c-g). Mutations that disrupted individual novel tertiary interactions in the peripheral regions resulted in significantly reduced catalytic activity compared to the wild-type (WT) sequence except for Δ'-Δ, suggesting that most of these tertiary interactions in the peripheral regions have allosteric effects on CP intron catalysis (Fig. 2h, Extended Data Fig. 5).

Additional interactions conserved in group II introns include D1c and D2 (θ-θ') interaction in order to secure the critical ε-ε' region in position for 5'-SS selection and catalysis^8,37, the peripheral interaction between D1b and D1d (α-α')³⁸, and interactions within D1d (ω-ω' and σ-σ') to reinforce positioning of D5 catalytic core (Extended Data Fig. 5d-h)^9,22. D2 and D3 next to D1 are crucial for intron catalysis efficiency by forming the aforementioned interactions with D5 and D6^32,39. Moreover, junction J2/3 directly interacts with the catalytic triad in D5 to form the catalytic triplex, which is reinforced by an interaction between D2 and D3 (ρ-ρ') (Extended Data Fig. 5i)^32,40,41. The 3' end of Cte 1 intron forms a short D4 as the closing stem with no IEP sequence. All interactions have been summarized in Extended Data Table 2.

Conformational dynamics of branchpoint A127 and the branching helix D6

Cryo-EM structures reveal substantial conformational changes and local shifts from branching to exon ligation of Cte 1 intron back-splicing. Different states are designated based on the connection of 5'-SS to IBS1 (pre-1S and 1S), the presence of both EBS1-IBS1 and EBS3-IBS3 ready for exon ligation (2S), and the A127-G220 2'-5' phosphodiester linkage density (2S and post-2S) (Extended Data Fig. 6). Previous studies have reported that branchpoint A could undergo dynamics either forming a 1-nt or 2-nt bulge ^42,43. In 1S state, we observed the 1-nt bulged A127 pointing outside of D6 with 2'-OH in close proximity of 2.62 Å to 5'-SS scissile phosphate ready for the branching reaction, and further stabilized by stacking with 5'-SS G220 and forming a base triple with G101-C125, analogous to the bulged A recognition pattern recently reported in prebranching structures of both canonical group II introns and spliceosomes (Fig. 3a-c)^8,9,44,45. In pre-1S state, the inactive A127 flips inside D6 and forms a non-canonical base-pair with C538 from the EBS loop while remaining stacking with G220, leading to local shifts of surrounding nucleotides in the bulge (Fig. 3d). In 2S and post-2S, A127 branched with G220 is translocated 20 Å away, facilitated by a 65º conformational change of D6, in order to allow docking of the 3'-exon in the catalytic core ready for ligation (Fig. 3e-f).

The branching helix D6 has been previously reported to undergo ~90º translocation enabled by IEP between two transesterification reactions in both self-splicing and transposition processes ^8,9. In Cte 1 intron, the conformational change of D6 between first and second reaction is mostly facilitated by the η' interaction motif of D6 terminal T-loop. In pre-1S and 1S, we observed tertiary interaction anchored by a G111-A451 non-canonical base-pair between D6 T-loop and the terminal loop of Β' interaction motif in D1c, designated as η'-η'' interaction (Fig. 3g). After branching reaction (2S and post-2S), D6 translocates to allow 3'-exon docking enabled by interactions with D1c (π'-π) and D2 (η'-η) as previously observed in canonical group II introns (Fig. 3h-i), which also leads to local dynamics of the peripheral regions (Extended Data Fig. 7)^{8,9,23,32,33,46,47}.

Unexpected major groove interaction that stabilizes EBS1-IBS1

Three exon binding sites (EBS1-3) have been previously identified to complementary base-pair with IBS1-3 in exons (EBS-IBS) in different classes of canonical group II introns³⁵. The Cte 1 intron contains only EBS1 and EBS3 that is identical to group IIC introns¹. EBS1 is found in a terminal loop in D1d that forms the 6-bp EBS1-IBS1 including a A543-A216 sheared pair in all structures except for post-2S (Extended Data Fig. 6a-d). In 2S when 5'- and 3'-exon are aligned for ligation, the single base-paired EBS3-IBS3 (G539-C134) that positions the 5' end of 3'-exon for transesterification is observed, also designated as δ-δ' interaction in group IIA introns ¹ (Extended Data Fig. 6c). Intriguingly, a 3-nt strand (C535-G537) upstream of EBS3 in the same EBS loop forms a major-groove interaction of three base triples reinforcing the EBS1-IBS1, which is usually facilitated by IEP in canonical group II introns (Fig. 3j-l)^8,9,23,33,47. After release of the circular exon product in post-2S, the EBS loop undergoes local rearrangement for stabilization by forming two internal base triples, C535-G537-C545 and U536-G539-A543 (Fig. 3m).

Metal ions enable folding and catalysis in Cte 1 intron

Metal ions are essential for ribozyme folding and catalysis^48,49, and previous studies have systematically evaluated 32 monovalent metal ion-binding sites (K⁺) and 34 divalent metal ion-binding sites (M²⁺) in group IIC intron crystal structures^25,50. More than 60 metal ions were identified in each cryo-EM structure of different states in Cte 1intron splicing, including designation of 7 K⁺ and 8 M²⁺ ions analogous to those previously identified in the group IIC intron⁵⁰. The rest metal ions are numbered M₃₅-M₈₆ as K⁺ and M²⁺ are indistinguishable in cryo-EM density under such resolution (Extended Data Table 3). Similar to other group II introns, the majority of metal ions resides in D1, suggesting that correct folding of D1 is critical to precede proper folding of the entire Cte 1 intron (Extended Data Fig. 8a)^51,52. A metal ion M₆₄ is found to reside in the major groove of the newly identified ο-ο' interaction (Fig. 2g). In addition, we observed M₅, K₃ and K₄ close to the catalytic core but not directly interacting with the reaction residues, which potentially have allosteric effects in catalysis by stabilizing the catalytic core as previously described (Extended Data Fig. 8b)⁵⁰.

The catalytic core of Cte 1 intron is organized in D5 with J2/3 and a 2-nt bulge that crucially binds to divalent metal ions M₁ and M₂^53,54. In pre-1S prior to the branching reaction where bulged A127 flips away from the catalytic site, M₁ and M₂ coordinate with non-bridging oxygens of A60 and G61 in the catalytic triad, G83 in the 2-nt bulge, and C81 adjacent to λ'-λ interaction to establish the catalytic core. In the meantime, coordinations of M₁ and M₂ with G220 pro-R_p, and M₂ with U219 bridging oxygen stabilize the leaving group (Fig. 4a). Additionally, K₁ coordinates with A60, G61, G83 and G696 to further stabilize the catalytic triad, whereas K₂ coordinates with G224 in D1 and A695 of the γ-γ' interaction that is critical for catalytic activity ³⁴. An unexpected metal ion that has not been previously described, termed M₃₅, is clamped by non-bridging oxygens of A422 and U423 next to λ-λ' interaction and coordinates with U218 to hold 5'-exon in place. Together these ions establish the heteronuclear metal ion center that is present in all states of Cte 1 intron catalysis and also conserved in canonical group II introns and spliceosomes (Fig. 4, Extended Data Fig. 6g-i)^6,22,25.

In 1S state ready for branching, A127 flips toward the catalytic site and the 2'-OH nucleophile is coordinated with M₁, whereas M₂ remain coordinating with U219 and G220 to prepare for the nucleophilic attack (Fig. 4b). After branching in 2S, the A127-G220 linkage moves away from the catalytic site to allow 3'-exon docking, in which the C134 leaving group is stabilized by M₁, while the 5'-exon U219 3'-OH nucleophile is coordinated by M₂ to prepare for the second transesterification reaction (Fig. 4c). In post-2S after the ligation, M₁, M₂ and K₁ remain in position and K₂ shifts 4.3 Å to coordinate with C223 (Fig. 4d). The A695-U133 γ-γ' interaction is stacked by G221, G220 and A127 that brings the 3'-SS U133 closer to the catalytic core to coordinate with M₁ and M_35* (Fig. 4d, Extended Data Fig. 6d). M_35* is another new metal ion unexpectedly appears in the catalytic core 17.1 Å from M₃₅ to coordinate with U133 2'-OH, whereas M₃₅ is no longer observed as 5'-exon is already released in post-2S. The interaction network of U133 in the catalytic core of post-2S is similar to 3'-SS in previous group II intron lariat forms ready for reverse splicing (Extended Data Fig.6 d-f)^8,42.

Permutation occurs in DNA, RNA and protein that alters their sequence order while retaining their functions and structures⁵⁵. Circular permutation is the most common rearrangement that swaps the sequence order by ligation of two original termini and cleavage at a new site to generate new termini ^56,57. Naturally occurring circular permutations have been previously found in catalytic RNAs^14,58-61, rRNAs⁶², and tRNAs⁶³. For self-cleaving catalytic RNAs such as twister and hammerhead ribozymes that carry out cleavage of a single RNA strand within themselves, permutations have minimal effects on their functions and catalysis products as long as the catalytic core structure of permuted ribozymes remains intact ^58,64. However, large self-splicing ribozymes such as group I and group II introns undergo both cleavage and ligation reactions, and the ligation reactions of CP introns with swapped 5' and 3' domains lead to circular instead of linear exon products ^14,61.

Circular RNAs have played emerging roles in regulation of gene expression, sequestration of proteins and microRNAs, and modulation of signaling pathways and innate immune responses^12,65,66. CircRNAs can also serve as translation template if embedded with internal ribosome entry site (IRES) or m⁶A modifications^67-70. The abovementioned characteristics and superior stability of circRNAs have promoted numerous potential biomedical applications, including development of circRNA biomarkers⁷¹, circRNA interference of disease-related proteins and RNAs¹², and circRNA vaccines supplementing the widely used mRNA vaccines¹³. However, efficient generation of circRNAs remains to be challenging¹².

Although artificially designed CP introns have been demonstrated to generate circRNA products decades ago ^10,11, structural information of CP introns carrying out the back-splicing process remains unknown, which greatly limits our understanding of the circular exon generation mechanism. In this study we elucidate the splicing mechanism of a recently discovered natural CP group II intron from C. testosteroni by determination of four cryo-EM structures associated to the first step of branching and second step of ligation during the catalytic process (Fig. 1). Canonical group II introns have been divided into several families based on their exon recognition patterns and intron scaffold architecture¹. Although the Cte 1 intron is greater than 700-nt that is close to the size of group IIB introns, the exon recognition pattern only contains EBS1 and EBS3, identical to group IIC introns. Our structures also reveal distinguished tertiary interactions unique to this CP intron that allosterically modulate catalytic activity (Fig. 2, Extended Data Table 2)¹⁴.

The entire dynamic splicing process and substantial conformational changes of Cte 1 CP intron largely resemble canonical group II introns and spliceosomes, except there is no protein involved (Fig. 3): D6 containing the conserved branchpoint A127 undergoes 65º translocation after branching reaction facilitated by η'-η'' and η'-η tertiary interactions instead of IEP; EBS1 is stabilized by a novel major-groove interaction whose role is normally played by maturase DNA binding or thumb-X domains in canonical group II introns (Fig. 5); a heteronuclear metal center is established in the catalytic core and both transesterification reactions are carried out via the general two-metal-ion mechanism (Fig. 4, Extended Data Table 3)²⁰.

In conclusion, these results reveal the overall architecture of a novel CP group II intron and conformational changes and dynamics involved in the back-splicing process, which elucidate the mechanism of circular exon generation via two consecutive transesterification reactions, and potentially serves as the foundation for future applications in circRNA research and therapeutics.

Cte 1 intron self-splicing directly following in vitro transcription (IVT)

The full-length C. testosteroni KF-1 (Cte 1) group II intron sequence was derived from NCBI (GenBank: NZ AAUJ02000001.1). DNA templates in pUC-19 plasmid were amplified using the primers listed in Extended Data Table 4, in which ‘Me’ refers to methoxyl modification that was introduced to ensure sequence homogeneity at 3' end of the amplified DNA template and subsequent RNA product (Kao et al., 1999).The resulting DNA templates were isolated by ethanol precipitation for subsequent IVT reactions. Splicing reactions were carried out by IVT containing 0.3 μM DNA templates, 10 mM NTP mixture, 40 mM Tris-HCl (pH 7.9), 20 mM MgCl₂, 2 mM spermidine, 0.01% TritonX-100, 10 mM dithiothreitol (DTT), 1 U μl^–1 RNase inhibitor (Thermo Scientific), 0.1 U μl^–1 pyrophosphatase (Thermo Scientific) and 7.5 U μl^–1 T7 RNA polymerase (in-house prepared) and incubated at 37 °C for 30 mins (Luo et al., 2023). The IVT product was mixed with 2× denaturing gel loading buffer containing 95% formamide, 0.025% SDS, 10 mM EDTA, 0.025% xylene cyanol, and 0.025% bromophenol blue, and loaded on a 6% 29:1 acrylamide:bis, 7 M urea polyacrylamide gel. The gel was run at 25 W for 2 h and stained for 5 min in SYBR-Gold (Invitrogen) and visualized using an ultraviolet transillumination molecular imager (Bio-Rad ChmiDoc XRS+).

RNA preparation of different Cte 1 constructs

Mutated Cte 1 constructs were prepared by PCR site-directed mutagenesis using the primers listed in Extended Data Table 4. To inhibit the self-splicing during IVT and purify the full-length precursor Cte 1 intron, the IVT condition was modified as following, 0.3 μM DNA templates, 40 mM NTP mixture, 40 mM Tris-HCl (pH 7.9), 100 mM MgCl₂, 2 mM spermidine, 0.01% TritonX-100, 10 mM DTT, 1 U μl^–1 RNase inhibitor, 0.1 U μl^–1 pyrophosphatase and 7.5 U μl^–1 T7 RNA polymerase and incubated at 37 °C for 2 h (Inoue et al., 1986). After DNase I digestion followed by proteinase K treatment to discard DNA templates and proteins, the RNA product was mixed with 2× denaturing gel loading buffer and loaded on a 6% 29:1 acrylamide:bis, 7 M urea polyacrylamide gel. The gel was run at 25 W for 4 h, then visualized briefly with a 254 nm UV lamp. RNA was excised from the gel and eluted overnight in RNase-free water with 500 mM NaOAc and 20 mM EDTA at 4 ℃, then purified by ethanol precipitation. The RNA sample quality was examined by electrophoresis on a denaturing 6% Urea-PAGE gel and stored in -80 ℃ until further use.

CP group II intron in vitro self-splicing assay

Purified Pre RNA at a final concentration of 1 μM was heated to 80 ℃ for 1 min in 10 mM Tris-HCl pH 7.5 and 100 mM KCl, then slowly cooled to 60 ℃ and incubated at 37 ℃ for the designated time in the presence of 20 mM CaCl₂. Reactions were terminated with 2× denaturing gel loading buffer containing 95% formamide, 0.025% SDS, 10 mM EDTA, 0.025% xylene cyanol, and 0.025% bromophenol blue. Products were loaded on a 6% 29:1 acrylamide:bis, 7 M urea polyacrylamide gel. The gel was run at 25 W for 2 h and stained for 5 min in SYBR-Gold (Invitrogen) and visualized using an ultraviolet transillumination molecular imager (Bio-Rad ChmiDoc XRS+).

For other CP group II intron splicing activity and quantification of splicing efficiency of Cte 1 mutants compared to WT Cte 1, splicing reactions were carried out in the abovementioned conditions in the presence of 20 mM MgCl₂ instead of CaCl₂.

RNA sample preparation for cryo-EM

For IVT reaction mixture, 200 μL was buffer-exchanged with 1× transcription buffer using concentrator columns with a 50 kDa cutoff (Ultrafiltration Centrifugal Tube, Millipore) and concentrated to a final volume of 20 μL.

For splicing reaction products in the presence of CaCl₂, 200 μL reaction products at 5 min and 4 h time points were concentrated using pre-chilled concentrator columns with 50 kDa cutoff (Ultrafiltration Centrifugal Tube, Millipore) to 20 μL. RNA concentration was quantified using a Nanodrop spectrophotometer (Thermo Scientific) and RNA quality was checked on a 6% 29:1 acrylamide:bis, 7 M urea polyacrylamide gel.

Cryo-EM sample preparation

A total of 3 μl of the abovementioned RNA samples (10 μM) was applied onto glow-discharged (10 s) 200-mesh R1.2-1.3 Quantifoil Cu grids with 2 nm continuous carbon film. After holding for 30 s, the grids were blotted for 1.5 s in 100% humidity at 4 °C with no blotting offset and rapidly frozen in liquid ethane using a Vitrobot Mark IV (Thermo Fisher).

Cryo-EM single particle data acquisition and data processing

Three datasets were collected using the same cryo-EM microscope settings. Frozen grids were loaded into a Titan Krios (Thermo Fisher) operated at 300kV, condenser lens aperture 50 μm, spot size 6, parallel beam with illuminated area of 1.03 μm in diameter. Microscope magnification was at 165,000x (corresponding to a calibrated sampling of 0.85 Å per pixel).

For Cte 1 Pre RNA splicing product for 5 min, 9,449 movie stacks were collected automatically using EPU software on a K2 direct electron camera equipped with a Bioquantum energy filter with an energy slit of 20 eV (Gatan), operating in counting mode at a recording rate of 5 raw frames per second and a total exposure time of 6 s, yielding 30 frames per stack, and a total dose of 59 e^-/Å². All movie stacks were collected with defocus values ranging between -0.8 and -1.5 μm. These movie stacks were motion-corrected using MotionCor2. After CTF correction by CTFFIND4, 9,402 micrographs were subjected to EMAN2.31 for neural network particle picking. A total of 3,484,858 particles were extracted with a box size of 320 × 320 pixels and were subjected to two rounds of 2D classification in Relion 4.0, then 1,020,874 particles were subjected to cryoSPARC v.4.3.1. A total of 150,000 particles were selected for initial model creation, generating one reconstruction as template for further classification. Two rounds of 3D classification resulted in 124,886 particles and 100,513 particles corresponding to the pre-1S and 1S states. Homogeneous refinement and CTF refinement of 1S yielded a final reconstruction at 2.97 Å resolution, respectively.

For Cte 1 Pre RNA splicing product for 4 h, 5,131 movie stacks were collected and processed as aforementioned. Three rounds of 3D classification resulted in 129,948 particles and 77,976 particles corresponding to the pre-1S and 2S states. Homogeneous refinement and CTF refinement of 2S yielded a final reconstruction at 2.90 Å resolution, whereas particles of pre-1S from two datasets were combined to 254,834 particles for homogeneous refinement and CTF refinement that yielded a final reconstruction at 2.65 Å resolution.

For IVT product, 3,591 movie stacks were collected and processed as aforementioned. After two rounds of 3D classification, 59,466 particles were selected for refinement, yielding reconstruction of 2.98 Å. After CTF refinement, the final resolution of post-2S reconstruction is 2.90 Å.

All global resolution was estimated by the 0.143 criterion of the Fourier shell correlation (FSC) curve. Local resolution maps were determined, and low-pass filtered accordingly. All final maps were displayed in UCSF Chimera v1.6 using local resolution low-pass filtered maps unless noted otherwise.

Cryo-EM model building and refinement

The Cte 1 models were built de novo with the assistance of auto-DRRAFTER and CryoREAD (Kappel et al., 2018; Wang et al., 2023). Then manually adjusted in Coot as needed. These models were refined with Phenix.real_space_refine, yielding an averaged model-map correlation coefficient (CCmask) of 0.81, 0.83, 0.80 and 0.90, respectively. Final models were validated with MolProbity and Q-score analysis.

Figure preparation

Figures and illustrations were prepared using Chimera v1.16, ChimeraX v1.6, GraphPad Prism 10.0. and Adobe Illustrator.

Data availability

The cryo-EM maps and associated atomic coordinate models of Comamonas testosteroni KF-1 CP-group II intron pre-1S, 1S, 2S, and post-2S have been deposited in the wwPDB OneDep System under EMD accession codes of EMD-38649, EMD-38647, EMD-38648 and EMD-38646 and PDB ID codes of 8XTS, 8XTQ, 8XTR and 8XTP, respectively.

Acknowledgements

Cryo-EM data were collected on Can Cong at SKLB West China Cryo-EM Center and processed at SKLB Duyu High Performance Computing Center in West China Hospital of Sichuan University. This work was supported by National Key Research and Development Program of China (2022YFC2303701, 2021YFA1301900), Natural Science Foundation of China (NSFC 32070049, 32222040, 32300030 and 81970936), Tianjin Synthetic Biotechnology Innovation Capacity Improvement Action (TSBICIP-KJGG-008), and the 1.3.5 Project for Disciplines Excellence of West China Hospital (ZYYC21006).

Author contributions

Z.S. conceived the project; L.W. and C.Z. performed RNA preparation and gel electrophoresis; L.W., C.Z. and Y.Y. collected cryo-EM data; L.W., D.H. and Z.S. processed cryo-EM data; J.X., L.W., J.Z. and Z.S. generated RNA atomic coordinates; Z.H., Y.Y. and X.C. prepared figure illustrations. All authors contributed to the preparation of the manuscript.

Competing interests

The authors declare no competing interests.

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Structural basis of circularly permuted group II intron self-splicing

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