The group I self-splicing introns, like other RNAs and RNA–protein complexes, undergo multiple conformational changes in completing two transesterification reactions that cleave the intron and ligate the exons, but the detailed mechanism remains largely unknown. Here we use cryogenic electron microscopy to reveal six conformations associated with Tetrahymena intron self-splicing at 2.84–3.73 Å resolution directly following transcription, in which the RNAs can fold and splice cotranscriptionally. We identify two states with the dynamically undocked P1 helix in addition to the P1 docked conformation positioned for the first step, and three states associated with the second step, with one state carrying an unforeseen pseudoknotted structure collectively formed by the 5′-exon, 5′-intron and 3′-exon, providing an example of exons modulating splicing activity that is conserved among group IC1 introns. Translocations of nucleotides are observed in helix docking and intron splicing, whereas identification of metal ions validates the general two-metal-ion-splicing mechanism.
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The cryo-EM maps and associated atomic coordinate models of Tetrahymena group I introns have been deposited in the wwPDB OneDep System under EMD accession codes EMD-33134 and 7XD3 for relaxed pre-Tet-S1; EMD-33135 and 7XD4 for intermediate pre-Tet-S1; EMD-33136 and 7XD5 for Tet-S2a; EMD-33137 and 7XD6 for Tet-S2b; EMD-33138 and 7XD7 for pre-Tet-C; EMD-34670 and 8HD6 for relaxed pre-TetG264A-S1; EMD-34671 and 8HD7 for intermediate pre-TetG264A-S1; and EMD-35223 and 8I7N for TetG264A-S1, respectively. Full-length WT and mutated Tetrahymena intron sequences were used according to NCBI (GenBank, no. JN547815.1). Raw data for sequence and structure conservation analyses are included in Supplementary Data 1. All other data are available from the authors on reasonable request. Source data are provided with this paper.
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We thank R. N. Sengupta and D. Herschlag for helpful discussions. Cryo-EM data were collected at the SKLB West China Cryo-EM Centre (Sichuan University) and the Cryo-EM Centre at the Southern University of Science and Technology, and processed at SKLB Duyu High Performance Computing Centre at Sichuan University. This work was supported by the Ministry of Science and Technology of China (nos. MoST 2022YFC2303700 and 2021YFA1301900), the Natural Science Foundation of China (nos. NSFC 32222040 and 32070049) and Sichuan University start-up funding (no. 20822041D4057 to Z.S.) J.M.B., E.F.B. and S.M. were supported by the Polish National Science Centre (nos. NCN 2017/26/A/NZ1/01083 and 2021/43/D/NZ1/03360). E.F.B. was supported by EMBO (no. ALTF 525-2022). Computational resources for SimRNA simulations were provided by the Poznań Supercomputing and Networking Centre at the Institute of Bioorganic Chemistry, Polish Academy of Sciences through the Polish Grid Infrastructure (grant: plgsimcryox).
The authors declare no competing interests.
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Extended Data Fig. 1 Cryo-EM workflow of Tetrahymena intron self-splicing from transcription, related to Fig. 1.
(a) Denatured gel analysis of transcription mixture showed all intermediate and final self-spliced products, representative of three independent experiments. (b) Cryo-EM single-particle workflow yielded three conformations corresponding to Tet-S2a, Tet-S2b and pre-Tet-C states, coloured according to local resolution maps with angular distribution and FSC curves indicating resolutions according to the 0.143 cutoff.
Extended Data Fig. 2 Cryo-EM density in the catalytic site and different J1/2 conformations, related to Fig. 1.
(a) Superposition of J1/2 in different states reveals conformational changes. (b) Superposition of all conformations with previous apo L-21 ScaI ribozyme reveals almost identical global architecture and apparent conformational changes of P1/P1′ helix. IGS of apo L-21 ScaI ribozyme, P1 helix of relaxed pre-Tet-S1 and P1′ helix of pre-Tet-C are in the same relaxed position. (c) nucleotides of J1/2 are stacked in pre-Tet-C at 4.0σ threshold. (d-e) Cryo-EM density connectivity of Tet-S2a (d) and Tet-S2b (e) at 3.0σ and 4.0σ threshold reveals connection of ωG with u(+1) in 3′-exon and cleaved 5′-exon, indicating that these conformations are prior to the second step of splicing.
Extended Data Fig. 3 Spermidine binding in Tetrahymena intron and 70S ribosome, related to Fig. 1m.
(a) Spermidine observed in Tet-S2b. (b) Electrostatic potential map of the spermidine and surrounding area in Tet-S2b. (c) Spermidine observed in 70S ribosome. (d) Electrostatic potential map of the spermidine and surrounding area in 70S ribosome. Black dashed line indicates hydrogen bond.
Extended Data Fig. 4 Splicing activity of WT and mutated Tetrahymena introns, related to Fig. 1F-1G.
(a-d) Gel electrophoreses of splicing reactions of WT (a), mutant 1 with disrupted P0 (b), mutant 2 with disrupted P0′ (c), and mutant 3 with disrupted P0 and P0′ (d). (e-f) Quantifications of the first (e, data size n = 252), and second (f, data size n = 252), splicing reaction product fractions over time reveal increased rate of second step of splicing for mutant 1 and 3. The fractions of each band in the WT and mutants were quantified by Bio-Rad Image Lab software. Curves of the first and second splicing reaction are fit to the one-phase association model \(Y = Y_0 + (Plateau - Y_0) \ast (1 - exp( - K \ast x))\) by GraphPad Prism 7.0. The value of plateau has been indicated. Data are presented as mean values ± standard deviation (SD) from three independent experiments, n = 3.
Extended Data Fig. 5 Sequence and structure conservation analyses of the Tetrahymena 5′-exon, 5′-intron and 3′-exon that forms the novel pseudoknot structure.
The conservation analyses of the novel pseudoknot region were performed against a library of 310 group IC1 introns of rRNAs. The raw data of the sequence and structure conservation analysis is in Supplementary Data 1.
Extended Data Fig. 6 Cryo-EM workflow of Tetrahymena intron before self-splicing.
(a) Denatured gel analysis of transcription mixture showed inhibited self-splicing, representative of three independent experiments. (b) Cryo-EM single-particle workflow yielded three conformations, two of which corresponded to relaxed and intermediate pre-Tet-S1, and one with flexible single-stranded 5′-exon and 5′-intron since no P1 density was observed. All maps are coloured according to local resolution maps with angular distribution and FSC curves indicating resolutions according to the 0.143 cutoff.
Extended Data Fig. 7 Cryo-EM workflow of Tetrahymena intron G264A mutation self-splicing from transcription, related to Fig. 2.
(a) Denatured gel analysis of transcription mixture showed all intermediate and final self-spliced products, representative of three independent experiments. (b) Cryo-EM single-particle workflow yielded three conformations corresponding to relaxed and intermediate pre-TetG264A-S1, and docked TetG264A-S1 states, coloured according to local resolution maps with angular distribution and FSC curves indicating resolutions according to the 0.143 cutoff.
Extended Data Fig. 8 Superposition reveals that relaxed and intermediate conformations of pre-Tet-S1 and pre-TetG264A-S1 are identical, related to Fig. 2a-b.
(a) Cryo-EM maps and model comparisons of the relaxed pre-Tet-S1 (green) and pre-TetG264A-S1 (pink). (b) Cryo-EM maps and model comparisons of the relaxed pre-Tet-S1 (grey) and pre-TetG264A-S1 (cyan).
Extended Data Fig. 9 The metal ion identification of all constructs associated to the first splicing reaction and comparison with Tet-S2a.
(a-e) Metal ions identifications of (a) relaxed pre-TetG264A-S1 (pink), (b) intermediate pre-TetG264A-S1 (cyan), (c) TetG264A-S1 (dark green), (d) relaxed pre-Tet-S1 (green) and (e) intermediate pre-Tet-S1 (grey) compared to Tet-S2a (purple). The missing ions are marked in red.
Extended Data Fig. 10 Cryo-EM maps and models show metal ion compositions of different states in Tetrahymena group I intron splicing, Related to Fig. 4.
(a) Relaxed pre-TetG264A-S1 at 1.0σ threshold. (b) Intermediate pre-TetG264A-S1 at 1.0σ threshold. (c) TetG264A-S1 at 1.0σ threshold. (d) Tet-S2a at 1.5σ threshold. (e) Tet-S2b at 2.0σ threshold. (f) Pre-Tet-C at 1.0σ threshold without applying the local resolution low-pass filter.
Supplementary Tables 1 and 2.
Supplementary Data 1
Raw data from sequence and structure conservation analyses of the novel pseudoknotted structure formed by exons and the 5′-intron of group IC1 introns.
Video demonstration of Tetrahymena intron self-splicing process.
Source Data Extended Data Figs. 1, 4, 6 and 7.
Unprocessed gels of Extended Data Figs. 1a, 4a–d, 6a and 7a.
Source Data Extended Data Fig. 4
Statistical Source Data for Extended Data Fig. 4e–f.
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Luo, B., Zhang, C., Ling, X. et al. Cryo-EM reveals dynamics of Tetrahymena group I intron self-splicing. Nat Catal 6, 298–309 (2023). https://doi.org/10.1038/s41929-023-00934-3
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