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Sub-3-Å cryo-EM structure of RNA enabled by engineered homomeric self-assembly

Nature Methods volume 19, pages 576–585 (2022)Cite this article

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Abstract

High-resolution structural studies are essential for understanding the folding and function of diverse RNAs. Herein, we present a nanoarchitectural engineering strategy for efficient structural determination of RNA-only structures using single-particle cryogenic electron microscopy (cryo-EM). This strategy—ROCK (RNA oligomerization-enabled cryo-EM via installing kissing loops)—involves installing kissing-loop sequences onto the functionally nonessential stems of RNAs for homomeric self-assembly into closed rings with multiplied molecular weights and mitigated structural flexibility. ROCK enables cryo-EM reconstruction of the Tetrahymena group I intron at 2.98-Å resolution overall (2.85 Å for the core), allowing de novo model building of the complete RNA, including the previously unknown peripheral domains. ROCK is further applied to two smaller RNAs—the Azoarcus group I intron and the FMN riboswitch, revealing the conformational change of the former and the bound ligand in the latter. ROCK holds promise to greatly facilitate the use of cryo-EM in RNA structural studies.

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Fig. 1: Conceptualization and workflow of ROCK.
Fig. 2: Construct engineering of the Tetrahymena group I intron (TetGI) as a case study for ROCK.
Fig. 3: Sub-3-Å-resolution cryo-EM map of TetGI-DS, a dimeric pre-2SΔ5′ex construct of the TetGI, enabled by ROCK.
Fig. 4: Assembly, activity and cryo-EM analyses of TetGI-D and TetGI-T.
Fig. 5: Structural insights gained from the cryo-EM structures of the TetGI.
Fig. 6: The homomeric self-assembly strategy applied to two smaller structured RNAs.

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Data availability

The data supporting the findings of this study are principally within the figures and the associated Supplementary Information. Atomic coordinates and cryo-EM maps have been deposited with the Protein Data Bank and the Electron Microscopy Data Bank under the accession codes: 7R6L and EMD-24281 for TetGI-DS, 7R6M and EMD-24282 for TetGI-D, 7R6N and EMD-24283 for TetGI-T, EMD-24284 for AzoGI-T and EMD-24285 for FMNrbsw-T. In this study, the following structures from the PDB were utilized: 2BJ2, 1X8W, 1U6B and 3F2Q.

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Acknowledgements

We thank E. Westhof for providing the computer model of the complete TetGI and information on the peripheral domains of group I introns, T. Cech and F. Guo for information on the X-ray model of the TetGI core, M. Dai and Y. Shao for helpful discussions, S. Stoilova-McPhie for the help in pilot EM experiments, and M. Ziegler for proofreading. D.L. is a Merck Fellow of the Life Sciences Research Foundation. This work was supported by NSF grants (CMMI-1333215, CMMI-1344915 and CBET-1729397), AFOSR grant (MURI FATE, FA9550-15-1-0514), NIH grant (5DP1GM133052) and Molecular Robotics Initiative fund from the Wyss Institute to P.Y., a NIH grant (R01GM122797) to M.L. and a NIH grant (R01GM102489) to J.A.P.

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Author notes

  1. These authors contributed equally: Di Liu, François A. Thélot.

Authors and Affiliations

  1. Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, MA, USA

    Di Liu & Peng Yin

  2. Department of Systems Biology, Harvard Medical School, Boston, MA, USA

    Di Liu & Peng Yin

  3. Department of Cell Biology, Blavatnik Institute, Harvard Medical School, Boston, MA, USA

    François A. Thélot & Maofu Liao

  4. Department of Chemistry, the University of Chicago, Chicago, IL, USA

    Joseph A. Piccirilli

  5. Department of Biochemistry and Molecular Biology, the University of Chicago, Chicago, IL, USA

    Joseph A. Piccirilli

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  1. Di Liu
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Contributions

D.L. conceived and designed the study, designed and prepared the constructs, performed the functional assays, conducted pilot EM experiments, built and refined the atomic models and drafted the manuscript. F.A.T. collected the EM data, performed the reconstruction and generated the maps, assisted in building atomic models and drafted the manuscript. J.A.P. advised the experiments and drafted the manuscript. M.L. conceived, designed and supervised the study, performed the reconstruction and generated the maps. P.Y. conceived, designed and supervised the study and drafted the manuscript. All authors analyzed the data and commented on the manuscript.

Corresponding authors

Correspondence to Maofu Liao or Peng Yin.

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Competing interests

A provisional patent related to this work has been filed with D.L., F.A.T., M.L. and P.Y. listed as coinventors. J.A.P. declares no competing interests.

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Nature Methods thanks the anonymous reviewers for their contribution to the peer review of this work. Rita Strack was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 The benefits of RNA oligomerization-enabled cryo-EM via installing kissing-loops (ROCK) and the process of screening the lengths of connector helices for ring closure.

a, The folding wild-type monomeric RNA leads to correctly folded RNA and species of misfolded or alternative conformations: the former is a difficult subject for cryo-EM structural determination due to small size and structural flexibility and the latter are difficult to eliminate. b, The engineered RNA construct with kissing-loop sequences installed folds and assembles into the desired oligomer from the correctly folded RNA that has a larger size and mitigated structural flexibility, while the species of misfolded or alternative conformations assembled into other assemblies of undesired oligomeric states that can be readily eliminated by native purification methods such as native PAGE. Therefore, besides offering RNA constructs more amenable to high-resolution cryo-EM reconstruction, the self-assembled system also facilitates the experimental procedures of RNA folding optimization and native RNA purification. This helps eliminate the misfolding and conformational heterogeneity that are well known to complicate the functional and structural studies of RNA. c, The lengths of the connector helices between the RNA core and kissing-loop motif need to be optimized for ring closure. The computer model with optimized lengths of connector helices is shown in the middle, and the removal (left) or addition (right) of one base-pair (bp) would lead to the formation of open spiral structures. The screening process can be performed by NanoTiler as described in Methods.

Extended Data Fig. 2 Designing the 5′ and 3′ fragments for the pre-2SΔ5′ex TetGI complex.

a, Sequences of the RNA mimicking the 5′ fragment of the TetGI (5′ mimic) as the reverse transcription (RT) control and the 56-FAM-labelled RT primer. Underlined nucleotides in 5′ mimic were mutated to facilitate the synthesis by IVT; the nucleotides marked by grey line above are the primer-binding region. b, Analysis of the 5′ sequences of the TetGI by RT. Lanes 1 to 5 are the dideoxynucleotide sequencing results of 5′ mimic with a single dideoxynucleoside triphosphate (ddNTP; indicated under the gel) added (lanes 1 to 4) or no ddNTP added (lane 5); lanes 6 and 7 are the results of the constructs with wild-type 5′ sequence (same as the post-2S complex in Fig. 2c) or mutated 5′ sequence (same as the pre-2SΔ5′ex complex in Fig. 2d). While the RNA used in the post-2S complex is cleaved after U20, the RNA in the pre-2SΔ5′ex complex is not cleaved at this site. c, The likely formation of hairpin structures (two possibilities shown) of the wild-type 5′ sequence accounting for the self-cleavage. We note that the reaction of the wild-type leads to a L-19 product that is cleaved after U19 (ref. 87) and we attribute the difference of cleavage products to the different 5′ sequences. d, Two RNA constructs transcribed with a self-cleaving ribozyme (rbz; VS or HDV) for producing a homogeneous 3′-end. The G14-a+9 RNA was originally designed to have 3′-exon, which was almost completely cleaved in the absence of the 5′-exon. Green and red scissors mark the cleavage sites for the appended ribozyme and the group I intron itself, respectively. e, Structures of the 3′-ends produced by the cleavage of the appended ribozyme or the self-cleavage of the intron. While the latter can be extended by yeast poly(A) polymerase (PAP), the former cannot. f, PAP extension assay demonstrates that the majority products of both G14-A386 (lanes 1 and 2) and G14-a+9 (lanes 3 and 4) RNA preparation are extended by PAP (lanes 2 and 4), indicating that they are mostly the products from the intron’s self-cleavage. g, h, Sequences of the 5′ IVT RNA (g; G14-A386, from nucleotides 14 to 386) and the 3′ fragments (h; dTetCIRC with two deoxynucleotides flanking the 3′ splice site, and rTetCIRC with all-RNA nucleotides) for the pre-2SΔ5′ex TetGI complex. i, TetGI-catalyzed hydrolysis at the 3′ splice site is inhibited by the modification of DNA bases flanking the scissile phosphate of the 3′ splice site. Black arrow points to the band of the 3′ fragments of (dTetCIRC or rTetCIRC); grey arrows point to the bands of the cleaved 3′ fragments. j, Assembly assays for the monomeric (lanes 1 to 3) and dimeric (lanes 4 to 6) constructs of 5′ IVT G14-A386 RNA without 3′ fragment (lanes 1 and 4), with dTetCIRC (lanes 2 and 5) or with rTetCIRC (lanes 3 and 6). The annealing buffer contains 3 mM Mg2+, which is chosen from the assembly assay of the post-2S constructs (TetGI-M, -D, and -T; see Fig. 3a). Lanes 7 and 8 are control assemblies of TetGI-M and -D. Without 3′ fragment (lanes 1 and 4), there are trailing smears for the 5′ IVT RNA, indicating incorrect folding of the intron if P9.2, P9a and P9.0 are not formed. In the presence of dTetCIRC (lanes 2 and 5) or rTetCIRC (lanes 3 and 7), especially for dTetCIRC, some slower-migrating bands emerged, probably due to domain swapping, that is a 3′ fragment simultaneously binds to a 5′ IVT RNA molecule to form P9.2 and another 5′ IVT RNA molecule to form P10.

Extended Data Fig. 3 Cryo-EM imaging, processing and validation for TetGI-DS.

a, Representative cryo-EM image of TetGI-DS. The scale bar represents 20 nm. b, 2D class averages of TetGI-DS. Box size is 264 Å. c, Processing flowchart for the TetGI-DS dataset. d, Angle distribution for the particles included in the final 3D reconstruction. e, Fourier Shell Correlation (FSC) curves of the final TetGI-DS reconstruction. Half map #1 vs. half map #2 for the entire monomer is shown in black. The remaining FSC curves were calculated for the core domains only: half map #1 vs. half map #2 (red), model vs. refined map (blue), model refined in half map #1 vs. half map #1 (green), and model refined in half map #1 vs. half map #2 (orange).

Extended Data Fig. 4 Cryo-EM imaging, processing and validation for TetGI-D.

a, Representative cryo-EM image of TetGI-D. The scale bar represents 20 nm. b, 2D class averages of TetGI-D. Box size is 264 Å. c, Processing flowchart for the TetGI-D dataset. 3D classification of the symmetry-expanded monomers results in classes according to the conformations with double-stranded P1 (green arrows) or with single-stranded IGS (red arrows). The ratio of the two conformations is calculated and shown. For the conformation with double-stranded P1, green and blue boxes show the details of tertiary contacts of P1 on the P4-P6 side and P3-P8 side, respectively. The contacts agree well with the previous biochemical studies48,49,50 and the crystal structure of AzoGI40. The final cryo-EM map for TetGI-D was refined from two classes, and exhibits a stronger map intensity of double-stranded P1 than single-stranded IGS; therefore, the atomic model was built with double-stranded P1. d, Angle distribution for the particles included in the final 3D reconstruction. e, Fourier Shell Correlation (FSC) curves of the final TetGI-D reconstruction. Half map #1 vs. half map #2 for the entire monomer is shown in black. The remaining FSC curves were calculated for the core domains only: half map #1 vs. half map #2 (red), model vs. refined map (blue), model refined in half map #1 vs. half map #1 (green), and model refined in half map #1 vs. half map #2 (orange).

Extended Data Fig. 5 Cryo-EM imaging, processing and validation for TetGI-T.

a, Representative cryo-EM image of TetGI-T. The scale bar represents 20 nm. b, 2D class averages of TetGI-T. Box size is 317 Å. c, Processing flowchart for the TetGI-T dataset. Similar to the case of TetGI-D, 3D classification of the symmetry-expanded monomers of TetGI-T also results in classes according to the conformations with double-stranded P1 (green arrows) or with single-stranded IGS (red arrows). The ratio of the two conformations is calculated and shown, which is close to the dimeric construct TetGI-D shown in Extended Data Fig. 4c. The final cryo-EM map for TetGI-T was refined from two classes, and exhibits a stronger map of single-stranded IGS than double-stranded P1, probably due to the stabilization of the peripheral domains by the trimer construct; therefore, the atomic model was built with the single-stranded IGS. d, Angle distribution for the particles included in the final 3D reconstruction. e, Fourier Shell Correlation (FSC) curves of the final TetGI-T reconstruction. Half map #1 vs. half map #2 for the entire monomer is shown in black. The remaining FSC curves were calculated for the core domains only: half map #1 vs. half map #2 (red), model vs. refined map (blue), model refined in half map #1 vs. half map #1 (green), and model refined in half map #1 vs. half map #2 (orange).

Extended Data Fig. 6 Newly visualized structural elements involving the peripheral domains of the TetGI.

a, Structural details of the tertiary interaction P13. P13 is a 6-bp duplex formed by the base-pairings of U75 through U80 (p13′) and A352 through A347 (p13′), and stacks coaxially between the G73:C81 pair of P2.1 and the G346:C353 pair of P9.1a, bearing a conformational resemblance to some other 6-bp kissing-loop complexes88,89. A 4-nt bulge consisting of A69 through A72 is present near the tip of P2.1, allowing for the bent shape at the junction of P2.1 and P13. b, Structure of the four-way junction (4WJ) at P9a-P9b and P9.1-P9.2. Top-right inset shows the strand directions of the continuous strands of the 4WJ, indicating the flanking helices stacked in a left-handed parallel configuration90. Bottom-right inset shows interaction details at the crossover site. Though the overall connectivity and other long-range tertiary interactions may be the major determinant for the configuration of this 4WJ, the sugar-phosphate interactions of the nucleotides from the exchanging strands at the crossover site may contribute to the configuration of this 4WJ. c, Structure of the complex multiway junction at P1, P3-P8 and P2-P2.1. Insets show the details of two tertiary contacts centered by A95 (top-right) and A97 (bottom-right), respectively, stabilizing the juxtaposition of the pseudo-continuous helices of P2-P2.1 and P3-P8. are two tertiary interactions between the P2-P2.1 and P3-P9 domains. Within the A97-centered tertiary interaction (right), U300 forms a base-triple with A97:U277 pair, corroborating the previous biochemical evidence91. d, A similar contact is observed in the crystal structure of the Twort group I intron51 (TwoGI; PDB code: 1y0q) formed between P7 and the internal loop in P7.2. e, f, Comparing the tertiary interactions observed in TetGI and TwoGI (overlayed by P7). g, Sequence alignment of different class IC1 and class IE group I introns reveals a conserved purine-rich loop (highlighted in yellow) at J9.1/9.1a. The extracts of the alignments of 14 sequences are from Lehnert et al. 35, where all the sequences were regarded as subgroup IC1, but later some of them were categorized as subgroup IE92. TtLSU (intron in the large ribosomal RNA precursor of Tetrahymena thermophila) is the TetGI studied in this work. We note that the internal loop of J9.1/9.1a of the TetGI, which has the sequence of 5′-AUGA-3′/5′-GGAG-3′, is reminiscent of but not exactly the loop E motif93, which is normally 5′-AGUA-3′/5′-GAA-3′, though the sequence of the abovementioned internal loop in P7.2 of the TwoGI is the same as loop E motif.

Extended Data Fig. 7 AzoGI-T sequence design, assembly and activity.

a, Two RNA designs, G4-G206 and G4-a+7, transcribed with a self-cleaving ribozyme (rbz) for producing a homogeneous 3′ end. Green and red scissors mark the cleavage site for the appended ribozyme or the intron itself. AzoLEM is the ligated exon mimic for the AzoGI, and its complexing with G4-G206 RNA forms the post-2S state of the intron. b, The PAP extension assay of various constructs for the AzoGI. The products from the preparation of G4-G206 RNA constructs cannot be extended by PAP (lanes 2 and 8), indicting a 2′,3′-cyclic phosphate generated by rbz at the 3′ end (this is different from the G14-G414 RNA of TetGI, the majority of which can be extended by PAP; see lane 2 of Extended Data Fig. 2f). For G4-a+7 constructs, the products generated by the cleavage of rbz and intron itself can be directly distinguished based on the different electrophoretic mobilities in the presented analytical gel because the former is longer due to the appendage of a 7-nt 3′ exon (the small size difference is not noticeable in the preparative gel for RNA purification). As shown in lanes 3 and 9, about half of G4-a+7 RNA of either construct is cleaved at the 3′ splice site (whereas, in the case of the TetGI G14-a+9 RNA, more than 90% is cleaved at the 3′ splice site, indicating a substantially higher activity of the TetGI; see lane 4 of Extended Data Fig. 2f). Only the shorter products, which is produced by intron cleavage, can be extended by PAP. After folding, the intron-cleaved shorter products increase to ~70% (lanes 5 and 11), and some portion of these shorter products cannot be extended by PAP, probably due to the lower accessibility of the 3′ end after RNA folding. c, Assembly assay of the trimeric construct AzoGI-T (lanes 2 to 5) and the monomer control AzoGI-M (lane 1). Similar to the TetGI constructs, the optimal condition for folding/assembly was determined to be 3 mM Mg2+. Interestingly, monomer control AzoGI-M runs into two major bands, indicating conformational heterogeneity that is likely due to the open and closed conformations of the post-2S construct. d, Activity assay to test the activity of the second step of splicing. The reactions were conducted with 1 µM monomer units of either intron construct and 2 µM substrate (S, reacting as the 5′ exon) at 25 °C. The assay takes the advantage of the fact that there is still about 30% of AzoGI*-M and AzoGI*-T constructs containing 5′ exon after IVT preparation, purification and folding due to presumably lower splicing activity of the AzoGI.

Extended Data Fig. 8 Cryo-EM imaging, processing and validation for AzoGI-T.

a, Representative cryo-EM image of AzoGI-T. The scale bar represents 20 nm. b, 2D class averages of AzoGI-T. Box size is 236 Å. c, Processing flowchart for the AzoGI-T dataset. d, Angle distribution for the particles included in the final 3D reconstruction. e, Gold-standard Fourier Shell Correlation (FSC) for the final AzoGI-T reconstruction.

Extended Data Fig. 9 FMNrbsw-T assembly and activity.

a, b, Assembly assay. FMNrbsw-M is the monomer control. Three factors to increase the trimer yield: the presence of FMN ligand; high Na+; and increasing RNA concentration. c, Native PAGE (6%, in the presence of 2 mM free Mg2+) analysis of preassembled FMNrbsw-T trimer (lane T) under different temperatures ranging from 23 °C to 58 °C. Disassembly of the trimer starts to occur at 45 °C. The high thermal stability of the kissing-loop motif may contribute to the formation of the kinetic assembly of dimer because the kissing-loop formation may precede the complete folding of the riboswitch during annealing as suggested by its weaker ligand binding as shown in e. Lane M contains the low-molecular weight DNA ladder (NEB). d, Derivation of the equation to analyze the ligand binding (1:1 stoichiometry) based on fluorescence quenching. Data were fitted using a two-parameter (Kd and fc) quadratic Equation (4). e, Fluorescent binding assay of FMN (60 nM) with the riboswitch constructs conducted in 100 mM KCl and 2 mM MgCl2. Each data point is represented as mean ± s.d from three independent measurements. FMNrbsw-T dimer is the alternate dimeric assembly of the FMNrbsw-T RNA. The calculated Kd (nM, mean ± s.d.), fc (mean ± s.d.), and R2 are: monomer control, 30.1 ± 2.2, 0.254 ± 0.006, 0.9961; FMNrbsw-T dimer, 91.6 ± 21.9, 0.273 ± 0.017, 0.9944; FMNrbsw-T trimer, 17.4 ± 7.6, 0.197 ± 0.020, 0.9881. The dashed blue line for is fitted FMNrbsw-T trimer taking [L]t as 20 nM, which assumes that each monomeric subunit in the trimer can locally sense only one third of the ligand, and calculated values of Kd (nM, mean ± s.d.), fc (mean ± s.d.), and R2 are: 28.8 ± 6.3, 0.188 ± 0.015, 0.9937. This adjustment results in an improved fitting as reflected by an improved R2.

Source data

Extended Data Fig. 10 Cryo-EM imaging, processing and validation for FMNrbsw-T.

a, Representative cryo-EM image of FMNrbsw-T. The scale bar represents 20 nm. b, 2D class averages of FMNrbsw-T. Box size is 236 Å. c, Processing flowchart for the FMNrbsw-T dataset d, Angle distribution for the particles included in the final 3D reconstruction. e, Gold-standard Fourier Shell Correlation (FSC) for the final FMNrbsw-T reconstruction.

Supplementary information

Supplementary Information

Supplementary Notes 1 and 2, Supplementary Figures 1–10, Supplementary Tables 1–3 and Supplementary References

Reporting Summary

Supplementary Video 1

Two conformations of the TetGI refined from the cryo-EM reconstruction of the construct TetGI-D

Supplementary Video 2

Two conformations of the AzoGI corresponding to the closed and open states after the second step of splicing

Supplementary Video 3

Different classes of the 3D classification of FMNrbsw-T particles showing the relatively larger structural motion of the peripheries

Source data

Source Data Extended Data Fig. 9

Statistical Source Data.

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Liu, D., Thélot, F.A., Piccirilli, J.A. et al. Sub-3-Å cryo-EM structure of RNA enabled by engineered homomeric self-assembly. Nat Methods 19, 576–585 (2022). https://doi.org/10.1038/s41592-022-01455-w

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