Cryo-EM structure of a natural RNA nanocage

Abstract

Long (>200 nucleotides) non-coding RNAs (lncRNAs) play important roles in diverse aspects of life. Over 20 classes of lncRNAs have been identified in bacteria and bacteriophages through comparative genomics analyses, but their biological functions remain largely unexplored^1-3. Owing to the large sizes, the structural determinants of most lncRNAs also remain uncharacterized. Here, we report the structures of two natural RNA nanocages formed by the ROOL (rumen-originating, ornate, large) lncRNA found in bacterial and phage genomes. The cryo-electron microscopy (cryo-EM) structures at 2.9-Å resolution reveal that ROOL RNAs form an octameric nanocage with a diameter of 28 nm and an axial length of 20 nm, in which the hollow inside features poorly ordered regions. The octamer is stabilized by numerous tertiary and quaternary interactions, including triple-strand A-minors, for which we propose the term 'A-minor staples'. The structure of an isolated ROOL monomer at 3.2-Å resolution indicates that nanocage assembly involves a strand-swapping mechanism resulting in quaternary kissing loops. Finally, we show that ROOL RNA fused to an RNA aptamer, transfer RNA or microRNA retains its structure, forming a nanocage with radially displayed cargoes. Our findings, therefore, may enable engineering of novel RNA nanocages as delivery vehicles for research and therapeutic applications.

Extended Data Fig. 1.. Biochemical analyses of ROOL RNAs.

a, Denaturing PAGE analyses demonstrate that the RNAs are intact full-length molecules. RNAs were analyzed at least once. b, Mass photometry analysis of ROOL_Efa and ROOL_Firm at 50–100 nM in 240 mM K⁺ and 20 mM Mg²⁺ shows that monomers and octamers are the two most abundant species. The RNA size of each peak was calibrated using the Millennium RNA Marker and labeled. c, Size exclusion chromatography (SEC) analyses of ROOL_Efa and ROOL_Firm coupled with denaturing PAGE (d), mass photometry (e), and negative-stain EM (f) showed three major states (aggregates, octamer, and monomer) in the cryo-EM samples. Peak 1 (void volume) contained aggregated ROOL of different oligomerization states; peaks 2 and 3 correspond to predominantly octamers and monomers, respectively. Experiments in c and e were repeated at least once with similar results; experiments in d and f were performed once; three EM micrographs were analyzed in f. g, Dynamic light scattering shows a diameter range of 10–30 nm for ROOL RNAs, consistent with its oligomerization states. A representative result from two independent technical replicates is shown. For original RNA gel images and negative-stain micrographs, see Supplementary Figure.

Extended Data Fig. 2.. Cryo-EM data processing workflow for ROOL_Efa.

a, Maximum-likelihood classification of cryo-EM data using cryoSPARC. b, Example of a micrograph with ROOL_Efa particles. 6,966 micrographs were obtained and analyzed. c, Fourier shell correlation curve as a function of resolution for the final map shown in panels a and e. d, Euler angle distribution of particles contributing to the final reconstruction. e, Cryo-EM maps colored according to local resolution.

Extended Data Fig. 3.. Cryo-EM data processing workflow for ROOL_Firm.

a, Maximum-likelihood classification of cryo-EM data using cryoSPARC. b, Example of a micrograph with ROOL_Firm particles. 6,390 micrographs were obtained and analyzed. c, Fourier shell correlation curves as a function of resolution for the final octamer and tetramer maps shown in panels e and f. d, Euler angle distribution of particles contributing to the final reconstructions. e, f, Cryo-EM maps of the octamer (e) and tetramer (f), colored according to local resolution.

Extended Data Fig. 4.. Cryo-EM data processing workflow for the individual monomer of ROOL_Efa.

a, Maximum-likelihood classification of cryo-EM data using cryoSPARC. b, Example of a micrograph with ROOL_Efa particles. 5,253 micrographs were obtained and analyzed. c, Cryo-EM maps colored according to local resolution. d, Fourier shell correlation curves as a function of resolution for the final maps shown in panels a and c. e, Euler angle distribution of particles contributing to the final reconstruction.

Extended Data Fig. 5.. Tertiary interactions stabilize each ROOL_Firm monomer in the nanocage.

a, Molecular model of a ROOL_Firm monomer within the octameric structure. Close-up views of examples of tertiary interactions stabilizing the monomer are shown in the corresponding colored boxes in panels b–g with map rendered as transparent surface (σ = 4.16). Panels b and f show different views for the orange box in panel a. h, Secondary structure annotation of ROOL_Efa within the octamer, with tertiary interactions labeled by colored boxes that match panels a–g. Nucleotides in panels b–h are colored according to helix colors in a.

Extended Data Fig. 6.. Projected secondary structure with tertiary and quaternary interactions of ROOL_Efa shown based on the cryo-EM structure.

H1–H16 of monomers 1–4, H7, and H13, H15, H16 of monomers 5–8 are shown to indicate tertiary and quaternary interactions in ROOL_Efa. In ROOL_Firm, G-A stacking replaces G-C Hoogsteen base pairing.

Extended Data Fig. 7.. Quaternary interactions in ROOL_Firm and inter-tetramer interactions in ROOL_Firm and ROOL_Efa.

a, Front view of the ROOL_Firm dimer stabilized by four key interactions, the close-up views of which are shown in panels c–f with map rendered as transparent surface (σ = 4.16). b, Side view of the inter-tetramer interface of ROOL_Firm, the close-up views of which are shown in panels g and h with map rendered as transparent surface (σ = 4.16). i, Side view of the inter-tetramer interface of ROOL_Efa (viewed similarly to ROOL_Firm in b), the close-up views of which are shown in panels j and k with map rendered as transparent surface (σ = 2.88).

Extended Data Fig. 8.. Mutational analysis of H12 supports the strand-swapping mechanism.

a, Predicted secondary structures (by UNAFold) of the WT or mutated H12 sequences of ROOL_Efa. Nucleotides that participate in new kissing-loop interactions with H2 and H11 in the octamer are circled in red and green boxes, respectively. The purple shade indicates the disorder region in the octamer structure. In mut1, a G is inserted after U356 to pair with C423; in mut2, AAU (417–419) is changed to a C to pair with G360; in mut3, the stem-loop structure in the disordered region was replaced by a more stable hairpin (GC-rich with a GAAA tetra loop); mut4 combines the mutations in mut1 and mut2; mut5 combines mut4 and an additional removal of a bulge shown in dark blue on the WT structure diagram. b, c, Negative-stain EM analysis of the WT and mutant ROOL_Efa. Representative micrographs are shown in b and quantifications from three images for each sample are shown in c. Data are presented as mean ± SD. For original negative-stain micrographs, see Supplementary Figure. d, SEC analyses of WT and mutant ROOL_Efa constructs. Two independent experiments were performed yielding similar results, and a representative chromatogram is shown for each construct. e, Mass photometry analysis of ROOL_Efa H12 mutants. Two technical replicates were performed, yielding similar results.

Extended Data Fig. 9.. Analysis of ROOL_Efa oligomerization at varying salt concentrations.

a, Negative-stain EM analysis of ROOL_Efa at different K⁺ and Mg²⁺ concentrations. b, Quantification of ROOL_Efa nanocage formation using three different full images from the negative-stain EM analysis of each condition shown in a. Data are presented as mean ± SD. For original negative-stain micrographs, see Supplementary Figure. c, SEC analyses of ROOL_Efa show that different K⁺ and Mg²⁺ concentrations shift the distributions among three major peaks/states (aggregates, octamer, and monomer). Two independent experiments were performed yielding similar results, and a representative chromatogram is shown for each condition. d, Mass photometry analysis of ROOL_Efa at different K⁺ and Mg²⁺ concentrations. Two technical replicates were performed, yielding similar results.

Fig. 1.. Cryo-EM analyses of ROOL_Efa and ROOL_Firm.

a, ROOL-encoding bacteria are associated with human diseases. Circle sizes and colors indicate enrichment scores of each bacteria species or group for certain diseases available from the Human Gut Microbiome Atlas. The two ROOL RNA-bearing bacteria species or groups used in this study and their potentially associated human diseases are labeled red. b, c, Cryo-EM maps of ROOL_Efa (σ = 2.88) and ROOL_Firm (σ = 4.16) determined at 2.9 Å resolution; top, side, and bottom views are shown, with ROOL monomers individually colored. d, Alignment of ROOL_Efa (σ = 3.88) and ROOL_Firm (σ = 3.97) maps demonstrates similar architectures. ROOL_Efa and ROOL_Firm are blue and brown, respectively.

Fig. 2.. Tertiary interactions stabilize each ROOL_Efa monomer in the nanocage.

a, Molecular model of a ROOL_Efa monomer within the octameric structure. Close-up views of examples of tertiary interactions stabilizing the monomer are shown in the corresponding colored boxes in panels b–g with map rendered as transparent surface (σ = 2.88). h, Secondary structure annotation of ROOL_Efa within the octamer, with tertiary interactions labeled by colored boxes that match panels b–h. Nucleotides in panels b–h are colored according to helix colors in a.

Fig. 3.. Quaternary interactions stabilize ROOL_Efa octamer.

a, Top front view of the ROOL_Efa tetramer looking down the four-fold axis. Close-up views of two key interactions are shown in panels c and d with map rendered as transparent surface (σ = 2.88). b, The ROOL_Efa dimer is stabilized by four key interactions, the close-up views of which are shown in panels c–f with map rendered as transparent surface (σ = 2.88). This inside view was obtained by flipping the molecule in panel a (top view). Colored boxes in panels c and d show close-up views of those in panels a and b; residues are colored to match panel b. g, Molecular model of a ROOL_Firm monomer within the octameric structure. h, The alignment of ROOL_Efa and ROOL_Firm structures of monomers within the corresponding octamers shows highly similar architecture. ROOL_Efa and ROOL_Firm are colored blue and brown, respectively.

Fig. 4.. Analyses of ROOL_Firm variants.

a, Poorly resolved (disordered) regions are located in the cavities of ROOL_Firm and ROOL_Efa nanocages. These regions (D1, D2 in ROOL_Firm and D, D′ in ROOL_Efa) are colored in low-pass-filtered maps. The Gaussian filter with width 1.64 for sharpened cryo-EM map is displayed at the following contours: 2.74 σ for ROOL_Firm, 2.49 σ for ROOL_Efa. b, Diagrams of ROOL_Firm wild-type (WT) and deletion mutants tested in this work, in which the disordered regions were removed. Each colored box represents a helix scaled to length in the structure. c, Negative-stain analysis indicates that ROOL_Firm deletion mutants without disordered regions can form nanocages similar to those formed by WT ROOL_Firm. This experiment was performed once with two micrographs collected for each RNA. d, Design of ROOL-cargo RNA fusions. e, When fused to a Mango-III aptamer, pre-tRNA, or primary microRNA (pri-miRNA) at the 3′ end, ROOL_Firm forms stable nanocages with radially displayed cargos demonstrated by negative-stain EM (micrographs shown) and corresponding 2D averages (examples of 12 classes are shown for each construct). This experiment was performed once with >70 micrographs collected for each RNA. For original and additional negative-stain micrographs, see Supplementary Figure and data deposited to Figshare.

Fig. 5.. Structure of the individual ROOL_Efa monomer.

a, Cryo-EM map of ROOL_Efa monomer at 3.25 Å resolution (σ = 12.02). b, Comparison of ROOL_Efa individual monomer with the monomer within the octameric assembly. c, Comparison of the individual monomer with the dimer within the octamer details the rearrangements required to form two kissing-loop interactions shown in panels d and e. The individual monomer in b–e is colored dark blue, the monomer within an octamer is colored in green in b, in helix color in c–e, and the neighboring monomer within an octamer is colored in light blue in c–e.