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Structural basis of circularly permuted group II intron self-splicing

Nature Structural & Molecular Biology volume 32, pages 1091–1100 (2025)Cite this article

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Abstract

Circularly permuted group II introns (CP introns) consist of rearranged structural domains separated by two tethered exons, generating branched introns and circular exons via back-splicing. Structural and mechanistic understanding of circular RNA (circRNA) generation by CP introns remains elusive. We resolve cryo-electron microscopy structures of a natural CP intron in different states during back-splicing at a resolution of 2.5–2.9 Å. Domain 6 (D6) undergoes a conformational change of 65° after branching, to facilitate 3′-exon recognition and circularization. Previously unseen tertiary interactions compact the catalytic triad and D6 for splicing without protein, whereas a metal ion, M35, is observed to stabilize the 5′-exon during splicing. While these unique features were not observed in canonical group II introns and spliceosomes, they are common in CP introns, as demonstrated by the cryo-EM structure of another CP intron discovered by comparative genomics analysis. Our results elucidate the mechanism of CP intron back-splicing dynamics, with potential applications in circRNA research and therapeutics.

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Fig. 1: Cryo-EM structures of Cte 1 intron self-splicing process.
Fig. 2: Conformational change of D6 facilitates rearrangement of 5′-exon-IBS3 base pairing for novel 3′-SS recognition.
Fig. 3: Cryo-EM density and models of novel tertiary interactions of Cte 1 intron.
Fig. 4: Metal ion composition in the catalytic core of different states reveal mechanism of Cte 1 intron self-splicing.
Fig. 5: Schematic illustration of Cte 1 intron self-splicing dynamics.

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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, and Paracandidimonas lactea strain Q2-2 CP group II intron 2S have been deposited in the wwPDB OneDep System under EMD accession codes EMD-38649, EMD-38647, EMD-38648, EMD-38646 and EMD-60833 and PDB codes 8XTS, 8XTQ, 8XTR, 8XTP and 9IS7, respectively. The NCBI nucleotide dataset (https://ftp.ncbi.nlm.nih.gov/blast/db/FASTA/nt.gz) was used for comparative analysis. Full-length WT and mutated Comamonas testosteroni KF-1 sequences were used according to NCBI (GenBank: NZ_AAUJ02000001.1). Full-length WT Paracandidimonas lactea strain Q2-2 sequence was used according to NCBI (GenBank: NZ_JAJJOZ000000000.1). 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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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 (2022YFC2303700 to Z.S., 2021YFA1301900 to H.D.), Natural Science Foundation of China (NSFC 32222040 and 32070049 to Z.S., 32300030 to Y.Y. and 81970936 to D.H.) and the 1.3.5 Project for Disciplines Excellence of West China Hospital (ZYYC21006 to Z.S.).

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  1. These authors contributed equally: Liu Wang, Jiahao Xie, Chong Zhang.

Authors and Affiliations

  1. The State Key Laboratory of Biotherapy, National Clinical Research Center for Geriatrics, West China Hospital; The State Key Laboratory of Oral Diseases, National Clinical Research Center for Oral Diseases, National Center for Stomatology, West China Hospital of Stomatology, Sichuan University, Chengdu, China

    Liu Wang, Jiahao Xie, Chong Zhang, Jian Zou, Zirui Huang, Xingyu Chen, Yang Yang, Haohao Dong, Dingming Huang & Zhaoming Su

  2. Department of Cariology and Endodontics, West China Hospital of Stomatology, Sichuan University, Chengdu, China

    Liu Wang & Dingming Huang

  3. Mingle Scope (Chengdu), Chengdu, China

    Jiahao Xie

  4. The Key Laboratory for Bio-resources and Eco-environment of Ministry of Education, College of Life Sciences, Sichuan University, Chengdu, China

    Sitong Shang & Jianquan Liu

  5. Department of Prosthodontics, West China Hospital of Stomatology, Sichuan University, Chengdu, China

    Yang Yang

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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. L.W., J.X., J.Z., S.S. and Z.S. generated RNA atomic coordinates. L.W., Z.H., Y.Y., X.C., H.D. and J.L. prepared figure illustrations. All authors contributed to the preparation of the manuscript.

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Correspondence to Dingming Huang or Zhaoming Su.

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Extended data

Extended Data Fig. 1 Cte 1 CP intron back-splicing compared to canonical group II intron splicing.

a. Schematic representation of canonical group II intron self-splicing to generate linear exon product. b. Representative denaturing gel electrophoresis of Cte 1 IVT product from three independent experiments with consistent results. c. Representative denaturing gel electrophoresis of Cte 1 Pre RNA splicing in the presence of Ca2+ at different time points from three independent experiments with consistent results. Red boxes highlight samples subjected to cryo-EM analysis.

Source data

Extended Data Fig. 2

Cryo-EM workflow for the Cte 1 RNA splicing products. Cryo-EM data collection and processing of Cte 1 RNA splicing products in the presence of Ca2+ at 5 minutes (left) and 4 hours (right).

Extended Data Fig. 3

Cryo-EM workflow for the Cte 1 IVT product. Cryo-EM data collection and processing of cotranscriptionally folded Cte 1 RNA splicing product directly after IVT.

Extended Data Fig. 4 The heteronuclear metal ion center in Cte 1 intron and comparison to canonical group II introns and spliceosome.

a-c. The cryo-EM maps and models depicting the heteronuclear metal ion center showing EBS-IBS interactions in a. pre-1S, b. 1S, c. 2S, d-f. 3′-SS interaction network in d. post-2S, e. chimeric O.i intron lariat (5J02), f. E.r. intron RNP post-2F (8T2T), and homology to g. E.r. intron RNP pre-1F (8T2S), h. E.r. intron RNP pre-2F (8T2R), i. yeast S. cerevisiae spliceosome C complex (7B9V). Black dashed lines indicate metal ion coordination, blue dashed lines indicate hydrogen bonds, arrows indicate nucleophilic attacks, and base stackings are highlighted in red.

Extended Data Fig. 5 Tertiary interactions of D5 and D6 in Cte 1 intron compared to canonical group II introns.

a. Projected secondary structure with designated tertiary interactions of Cte 1 intron based on 3D structures. b. Tertiary interactions of D5 (cyan) with D1, D3 and D4 (gray). c-h. Cte 1 (cyan and gray) tertiary interactions of c. ψ‘-ψ compared to 6ME0 (tan), d. λ‘-λ compared to 8T2T (forest green), e. ζ‘-ζ compared to 4R0D (gold), f. κ‘-κ compared to 8T2T, g. μ‘-μ compared to 8T2T, h. φ‘-φ compared to 8T2T. i-k. Cte 1 (gray) tertiary interactions of i. π-π‘ compared to 8T2T (forest green), j. η-η‘ compared to 6ME0 (tan), k. γ-γ‘ compared to 8T2T.

Extended Data Fig. 6 Local shifts of bulged A between pre-1S and 1S.

a. Secondary structure, cryo-EM density map and model of D6 showing a 2-nt bulge (G126 and A127) in pre-1S, in which two base triples C100-A127-C538 and G101-C125-G220 were formed. b. Secondary structure, cryo-EM density map and model of D6 with bulged A127 interacting with G101-C125 in 1S. c-d. Analogous bulged A structure in c. canonical group II intron E.r. intron pre-1F (8T2S) and d. yeast S.c. spliceosome (7B9V). Blue dashed lines indicate hydrogen bonds.

Extended Data Fig. 7 Other tertiary interactions in Cte 1 intron compared to canonical group II introns.

a. Projected secondary structures of Cte 1 intron. Mutations that disrupt novel tertiary interactions are denoted. Cte 1 intron (gray) tertiary interactions of b. θ-θ‘ compared to 8T2T, c. ε-ε‘ compared to 6ME0, d. α-α‘ compared to 4E8K (magenta), e. ω-ω‘ compared to 4E8K, f. σ-σ‘ compared to 6ME0, g. ρ-ρ‘ compared to 4R0D (gold). Mutations that disrupt novel tertiary interactions are denoted in the secondary structure on top.

Extended Data Fig. 8 Metal ion distribution in Cte 1 intron and comparison with O.i. group IIC intron (4E8Q).

a. Metal ion distribution shows that most metal ions reside in D1 domain. b. The metal core in D1d domain. c. Comparison with O.i. group IIC intron reveals analogous metal ion-binding sites in the catalytic core. Black dashed lines indicate metal ion coordination.

Extended Data Fig. 9 Covariation analysis facilitates discovery of novel CP group II introns in other bacteria.

a. Consensus sequence and secondary structure model of CP group II intron, highlighting over one hundred covarying mutation pairs (green boxes). b. In vitro splicing assay demonstrates catalytic activity of five newly identified CP group II introns from Ape, Glu, Kpn, Pla, and Pni with incubation at 37 °C for 30 minutes. This is a representative gel electrophoresis from three independent experiments with consistent results. c. 2.9 Å cryo-EM map of the Pla CP-GII intron.

Source data

Extended Data Fig. 10 The Pla CP intron structure reveals conserved features as those in Cte 1 CP intron.

a. The overall structure of Pla CP intron. b. 5′- and 3′-exon base-pairing interaction. c. The 2-bp EBS3-IBS3 interaction motif. d. M35 stabilizing the 5′-exon. Black and blue dashed lines indicate metal ion coordination and hydrogen bonds, respectively.

Supplementary information

Supplementary Information

Supplementary Tables 1–3 and Data 1.

Reporting Summary

Supplementary Video 1

Dynamics of Cte 1 CP intron back-splicing.

Supplementary Data 1

The multiple sequence alignment of CP group II intron in Stockholm format.

Source data

Source Data Fig. 3

Unprocessed gels.

Source Data Fig. 3

Statistical source data.

Source Data Extended Data Fig. 1

Unprocessed gels.

Source Data Extended Data Fig. 9

Unprocessed gels.

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Wang, L., Xie, J., Zhang, C. et al. Structural basis of circularly permuted group II intron self-splicing. Nat Struct Mol Biol 32, 1091–1100 (2025). https://doi.org/10.1038/s41594-025-01484-x

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