Crystal structure of a eukaryotic group II intron lariat


The formation of branched lariat RNA is an evolutionarily conserved feature of splicing reactions for both group II and spliceosomal introns. The lariat is important for the fidelity of 5′ splice-site selection and consists of a 2′-5′ phosphodiester bond between a bulged adenosine and the 5′ end of the intron. To gain insight into this ubiquitous intramolecular linkage, we determined the crystal structure of a eukaryotic group IIB intron in the lariat form at 3.7 Å. This revealed that two tandem tetraloop-receptor interactions, η–η′ and π–π′, place domain VI in the core to position the lariat bond in the post-catalytic state. On the basis of structural and biochemical data, we propose that π–π′ is a dynamic interaction that mediates the transition between the two steps of splicing, with η–η′ serving an ancillary role. The structure also reveals a four-magnesium-ion cluster involved in both catalysis and positioning of the 5′ end. Given the evolutionary relationship between group II and nuclear introns, it is likely that this active site configuration exists in the spliceosome as well.

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Figure 1: A comparison of the tertiary structures of O. iheyensis and group II introns.
Figure 2: Tertiary interactions in a IIB intron.
Figure 3: The position of DVI within the intron structure.
Figure 4: The core of the of intron.
Figure 5: Model for DVI as the conformational switch for splicing.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Coordinates and structure factors have been deposited in the Protein Data Bank under accession code 4R0D.


  1. 1

    Grabowski, P. J., Padgett, R. A. & Sharp, P. A. Messenger RNA splicing in vitro: an excised intervening sequence and a potential intermediate. Cell 37, 415–427 (1984)

    CAS  PubMed  Google Scholar 

  2. 2

    Padgett, R. A., Konarska, M. M., Grabowski, P. J., Hardy, S. F. & Sharp, P. A. Lariat RNA’s as intermediates and products in the splicing of messenger RNA precursors. Science 225, 898–903 (1984)

    CAS  PubMed  ADS  Google Scholar 

  3. 3

    Konarska, M. M., Grabowski, P. J., Padgett, R. A. & Sharp, P. A. Characterization of the branch site in lariat RNAs produced by splicing of mRNA precursors. Nature 313, 552–557 (1985)

    CAS  PubMed  ADS  Google Scholar 

  4. 4

    Peebles, C. L. et al. A self-splicing RNA excises an intron lariat. Cell 44, 213–223 (1986)

    CAS  PubMed  Google Scholar 

  5. 5

    van der Veen, R. et al. Excised group II introns in yeast mitochondria are lariats and can be formed by self-splicing in vitro. Cell 44, 225–234 (1986)

    CAS  PubMed  Google Scholar 

  6. 6

    Di Leo, E. et al. A point mutation in the lariat branch point of intron 6 of NPC1 as the cause of abnormal pre-mRNA splicing in Niemann-Pick type C disease. Hum. Mutat. 24, 440 (2004)

    PubMed  Google Scholar 

  7. 7

    Galej, W. P., Oubridge, C., Newman, A. J. & Nagai, K. Crystal structure of Prp8 reveals active site cavity of the spliceosome. Nature 493, 638–643 (2013)

    CAS  PubMed  PubMed Central  ADS  Google Scholar 

  8. 8

    Fica, S. M. et al. RNA catalyses nuclear pre-mRNA splicing. Nature 503, 229–234 (2013)

    CAS  PubMed  PubMed Central  ADS  Google Scholar 

  9. 9

    Toor, N., Keating, K. S., Taylor, S. D. & Pyle, A. M. Crystal structure of a self-spliced group II intron. Science 320, 77–82 (2008)

    CAS  PubMed  PubMed Central  ADS  Google Scholar 

  10. 10

    Lambowitz, A. M. & Zimmerly, S. Group II introns: mobile ribozymes that invade DNA. Cold Spring Harb. Perspect. Biol. 3, a003616 (2011)

    PubMed  PubMed Central  Google Scholar 

  11. 11

    Toor, N., Hausner, G. & Zimmerly, S. Coevolution of group II intron RNA structures with their intron-encoded reverse transcriptases. RNA 7, 1142–1152 (2001)

    CAS  PubMed  PubMed Central  Google Scholar 

  12. 12

    Michel, F., Umesono, K. & Ozeki, H. Comparative and functional anatomy of group II catalytic introns–a review. Gene 82, 5–30 (1989)

    CAS  PubMed  Google Scholar 

  13. 13

    Costa, M., Fontaine, J. M., Loiseaux-de Goër, S. & Michel, F. A group II self-splicing intron from the brown alga Pylaiella littoralis is active at unusually low magnesium concentrations and forms populations of molecules with a uniform conformation. J. Mol. Biol. 274, 353–364 (1997)

    CAS  PubMed  Google Scholar 

  14. 14

    Rest, J. S. & Mindell, D. P. Retroids in archaea: phylogeny and lateral origins. Mol. Biol. Evol. 20, 1134–1142 (2003)

    CAS  PubMed  Google Scholar 

  15. 15

    Toor, N., Robart, A. R., Christianson, J. & Zimmerly, S. Self-splicing of a group IIC intron: 5′ exon recognition and alternative 5′ splicing events implicate the stem-loop motif of a transcriptional terminator. Nucleic Acids Res. 34, 6461–6471 (2006)

    CAS  PubMed  PubMed Central  Google Scholar 

  16. 16

    Boudvillain, M. & Pyle, A. M. Defining functional groups, core structural features and inter-domain tertiary contacts essential for group II intron self-splicing: a NAIM analysis. EMBO J. 17, 7091–7104 (1998)

    CAS  PubMed  PubMed Central  Google Scholar 

  17. 17

    Jacquier, A. & Michel, F. Base-pairing interactions involving the 5′ and 3′-terminal nucleotides of group II self-splicing introns. J. Mol. Biol. 213, 437–447 (1990)

    CAS  PubMed  Google Scholar 

  18. 18

    Fedorova, O. & Pyle, A. M. A conserved element that stabilizes the group II intron active site. RNA 14, 1048–1056 (2008)

    CAS  PubMed  PubMed Central  Google Scholar 

  19. 19

    Fedorova, O., Mitros, T. & Pyle, A. M. Domains 2 and 3 interact to form critical elements of the group II intron active site. J. Mol. Biol. 330, 197–209 (2003)

    CAS  PubMed  Google Scholar 

  20. 20

    Chanfreau, G. & Jacquier, A. An RNA conformational change between the two chemical steps of group II self-splicing. EMBO J. 15, 3466–3476 (1996)

    CAS  PubMed  PubMed Central  Google Scholar 

  21. 21

    Adams, P. L., Stahley, M. R., Kosek, A. B., Wang, J. & Strobel, S. A. Crystal structure of a self-splicing group I intron with both exons. Nature 430, 45–50 (2004)

    CAS  PubMed  ADS  Google Scholar 

  22. 22

    Li, C. F., Costa, M. & Michel, F. Linking the branchpoint helix to a newly found receptor allows lariat formation by a group II intron. EMBO J. 30, 3040–3051 (2011)

    CAS  PubMed  PubMed Central  Google Scholar 

  23. 23

    Klein, D. J., Moore, P. B. & Steitz, T. A. The contribution of metal ions to the structural stability of the large ribosomal subunit. RNA 10, 1366–1379 (2004)

    CAS  PubMed  PubMed Central  Google Scholar 

  24. 24

    Fica, S. M., Mefford, M. A., Piccirilli, J. A. & Staley, J. P. Evidence for a group II intron-like catalytic triplex in the spliceosome. Nature Struct. Mol. Biol. 21, 464–471 (2014)

    CAS  Google Scholar 

  25. 25

    Marcia, M. & Pyle, A. M. Visualizing group II intron catalysis through the stages of splicing. Cell 151, 497–507 (2012)

    CAS  PubMed  PubMed Central  Google Scholar 

  26. 26

    Query, C. C., Moore, M. J. & Sharp, P. A. Branch nucleophile selection in pre-mRNA splicing: evidence for the bulged duplex model. Genes Dev. 8, 587–597 (1994)

    CAS  PubMed  Google Scholar 

  27. 27

    Anokhina, M. et al. RNA structure analysis of human spliceosomes reveals a compact 3D arrangement of snRNAs at the catalytic core. EMBO J. 32, 2804–2818 (2013)

    CAS  PubMed  PubMed Central  Google Scholar 

  28. 28

    Parker, R. & Guthrie, C. A point mutation in the conserved hexanucleotide at a yeast 5′ splice junction uncouples recognition, cleavage, and ligation. Cell 41, 107–118 (1985)

    CAS  PubMed  Google Scholar 

  29. 29

    Lesser, C. F. & Guthrie, C. Mutations in U6 snRNA that alter splice site specificity: implications for the active site. Science 262, 1982–1988 (1993)

    CAS  PubMed  PubMed Central  ADS  Google Scholar 

  30. 30

    Roitzsch, M. & Pyle, A. M. The linear form of a group II intron catalyzes efficient autocatalytic reverse splicing, establishing a potential for mobility. RNA 15, 473–482 (2009)

    CAS  PubMed  PubMed Central  Google Scholar 

  31. 31

    Yang, J., Zimmerly, S., Perlman, P. S. & Lambowitz, A. M. Efficient integration of an intron RNA into double-stranded DNA by reverse splicing. Nature 381, 332–335 (1996)

    CAS  PubMed  ADS  Google Scholar 

  32. 32

    Lynch, M. & Richardson, A. O. The evolution of spliceosomal introns. Curr. Opin. Genet. Dev. 12, 701–710 (2002)

    CAS  PubMed  Google Scholar 

  33. 33

    Tseng, C. K. & Cheng, S. C. Both catalytic steps of nuclear pre-mRNA splicing are reversible. Science 320, 1782–1784 (2008)

    CAS  PubMed  ADS  Google Scholar 

  34. 34

    Otwinowski, Z. & Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997)

    CAS  PubMed  PubMed Central  Google Scholar 

  35. 35

    Schneider, T. R. & Sheldrick, G. M. Substructure solution with SHELXD. Acta Crystallogr. D 58, 1772–1779 (2002)

    PubMed  Google Scholar 

  36. 36

    Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010)

    CAS  PubMed  PubMed Central  Google Scholar 

  37. 37

    Sheldrick, G. M. A short history of SHELX. Acta Crystallogr. A 64, 112–122 (2008)

    CAS  MATH  ADS  Google Scholar 

  38. 38

    Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004)

    PubMed  PubMed Central  Google Scholar 

  39. 39

    Keating, K. S. & Pyle, A. M. RCrane: semi-automated RNA model building. Acta Crystallogr. D 68, 985–995 (2012)

    CAS  PubMed  Google Scholar 

  40. 40

    Blanc, E. et al. Refinement of severely incomplete structures with maximum likelihood in BUSTER-TNT. Acta Crystallogr. D 60, 2210–2221 (2004)

    CAS  PubMed  Google Scholar 

  41. 41

    Schroder, G. F., Levitt, M. & Brunger, A. T. Super-resolution biomolecular crystallography with low-resolution data. Nature 464, 1218–1222 (2010)

    PubMed  PubMed Central  ADS  Google Scholar 

  42. 42

    Chou, F. C., Sripakdeevong, P., Dibrov, S. M., Hermann, T. & Das, R. Correcting pervasive errors in RNA crystallography through enumerative structure prediction. Nature Methods 10, 74–76 (2013)

    CAS  PubMed  Google Scholar 

  43. 43

    Morin, A. et al. Collaboration gets the most out of software. eLife 2, e01456 (2013)

    PubMed  PubMed Central  Google Scholar 

  44. 44

    Chanfreau, G. & Jacquier, A. Interaction of intronic boundaries is required for the second splicing step efficiency of a group II intron. EMBO J. 12, 5173–5180 (1993)

    CAS  PubMed  PubMed Central  Google Scholar 

  45. 45

    Parker, R. & Siliciano, P. G. Evidence for an essential non-Watson-Crick interaction between the first and last nucleotides of a nuclear pre-mRNA intron. Nature 361, 660–662 (1993)

    CAS  PubMed  ADS  Google Scholar 

  46. 46

    Basu, S. et al. A specific monovalent metal ion integral to the AA platform of the RNA tetraloop receptor. Nature Struct. Biol. 5, 986–992 (1998)

    CAS  PubMed  Google Scholar 

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We thank S. Banerjee and the staff of the NE-CAT beamlines at the Advanced Photon Source (APS) of Argonne National Laboratory. We thank P. Ghosh, S. Joseph, G. Ghosh, R. Doolittle, Y. Tor, D. Donoghue and T. Wiryaman for comments on the manuscript. We thank R. Das and F.-C. Chou for assistance with phenix.erasser for structure refinement and G. Bricogne for advice on Buster refinement. We also thank N. T. Schirle for preliminary biochemical characterization of the intron. R.T.C. was supported by the Cell, Molecular, and Genetics Training Program funded by NIH predoctoral training grant 5T32GM007240. J.K.P. was supported by the UCSD Molecular Biophysics Training Program funded by NIH predoctoral training grant 5T32GM008326. NE-CAT is supported by NIH grant 8P41GM103403-10 and APS is supported by the US DOE under contract number DE-AC02-06CH11357. This work was supported by a Hellman Foundation Fellowship and NIH grant 5R01GM102216 awarded to N.T.

Author information




A.R.R. and J.K.P. performed the experiments. A.R.R., R.T.C., J.K.P. and N.T. designed the experiments. A.R.R., R.T.C., J.K.P., K.R.R. and N.T. analysed the data. A.R.R., R.T.C. and N.T. wrote the manuscript with input from all authors.

Corresponding author

Correspondence to Navtej Toor.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Secondary structure of intron crystallization construct.

Tertiary interactions are indicated with Greek letters and domains are labelled with Roman numerals. Colouring of the individual domains is consistent with the overall view of the tertiary structure shown in Fig. 1.

Extended Data Figure 2 The Yb-MAD experimental, density-modified map of the portion of DV containing the catalytic triad contoured at 1.8σ.

Extended Data Figure 3 The path of the 5′ exon through the intron structure.

The EBS1–IBS1 and EBS2–IBS2 interactions position the 5′ exon. They do not form a continuous binding interface with the presence of a highly distorted backbone at the junction between these two motifs. As a result, the helical axes of the EBS1–IBS1 and EBS2–IBS2 pairings are positioned 90° relative to each other. The EBS3–IBS3 interaction places the 3′ exon in the active site.

Extended Data Figure 4 Overall tertiary structure of the intron.

Individual domains and subdomains are depicted in different colours. Domain names are labelled with Roman numerals. a and b show different rotations of the intron structure.

Extended Data Figure 5 Companion to Fig. 2 showing the location of the individual tertiary interactions relative to the overall structure.

a, κ–κ′. b, ε–ε′. c, ρ–ρ′.

Extended Data Figure 6 Splicing assays for the DVI mutants showing the proportion of branched product.

Blue and orange bars correspond to lariat 3′ exon and lariat, respectively. The Δη′ and Δπ mutants accumulate large amounts of lariat 3′ exon, thus indicating a second-step splicing defect. The Δη′Δπ double mutant is almost completely blocked before the second step. The A620C mutant shows 2.4-fold greater accumulation of lariat 3′ exon compared to the wild-type intron, indicating that the interaction between G1 and A620 is important for the second step. In the yeast aI5γ intron, G1 instead interacts with the penultimate residue44, indicating a certain degree of flexibility for this pairing. There is evidence for a similar interaction between the termini of nuclear introns45 involving nearby (but not exactly equivalent) residues, which also has a significant effect upon the second step of splicing. Therefore, the 5′ and 3′ ends of nuclear introns may have a similar arrangement within the spliceosome.

Extended Data Figure 7 Detailed view of the lariat bond.

a, The lariat 2′-5′ phosphodiester bond in wall-eyed stereo format. b, Stereo version of Fig. 3c. See Fig. 3c legend for details. Fo − Fc density for the lariat bond contoured at 3σ.

Extended Data Figure 8 Anomalous maps identifying core metal ions.

a, b, Depiction of the RNA ligands surrounding metals M3 and M4, respectively. Yb3+ anomalous map contoured at 9σ. c, Yb3+ anomalous map for wild type contoured at 9σ. d, Compared with the wild-type intron, the Yb3+ anomalous map for the G79A mutant (contoured at 4σ) is lacking the peaks corresponding to M3 and M4, even at a lower contour level. e, Tl+ was used as a probe for monovalent ions in the RNA structure46. The Tl+ anomalous map (purple mesh contoured at 5.5σ) revealed a strong peak located 3.8 Å from M1 that coordinates to the nucleobase of J2/3 residue G421 and the backbone of DV nucleotide G550. This sodium ion Na1 (purple sphere) is significantly closer to M1 than the equivalent K+ ion found in O. iheyensis25. Otherwise, this monovalent ion binding site is relatively conserved between these two introns.

Extended Data Figure 9 2Fo − Fc density for DVI in the pre-catalytic structure contoured at 1σ.

The η–η′ interaction persists throughout the splicing reaction and is visible in the pre-catalytic state. The weaker density for the central region of DVI suggests a partially disordered, dynamic region with possible helical remodelling in the conserved internal loop during splicing. The general pattern of side-by-side packing of domains II and VI persists between the two steps. Catalytic triad mutation consisted of an AGC→GAU substitution.

Extended Data Table 1 X-ray and kinetic data for

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Robart, A., Chan, R., Peters, J. et al. Crystal structure of a eukaryotic group II intron lariat. Nature 514, 193–197 (2014).

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