Naturally ornate RNA-only complexes revealed by cryo-EM

Key results

1. 01

   ShownThe 659-nt ROOL env-120 RNA, discovered in cow rumen, self-assembles into a closed, hollow octameric nanocage with D4 symmetry. It is about 280 Å across, wider than a bacterial ribosome (about 250 Å).

   3.1 Å map; two half-shells of four chains each; within a half-shell each chain makes eight bridges (B1-B4), and each chain reaches two chains of the opposite half-shell (B5, B6); P7 loops form an inner ring ∼36 Å wide; the modeled fold matches the covariation-predicted stems P1-P19, including pseudoknot P10. · ROOL assembles into an atomically ordered…; Cryo-electron Microscopy Data Processing; Figure legends

2. 02

   ShownGOLLD env-38 RNA, from a marine metagenome, forms a larger, completely empty 14-mer nanocage with D7 symmetry and a 380 Å diameter. Its 5′ and 3′ regions fold as separate domains that form the cap and the ring of each half-shell.

   3.0 Å reconstruction, with models for all 14 chains at Q-scores consistent with resolution; each of seven cap monomers forms eight quaternary bridges; half-shells are joined by self-complementary kissing loops B8 and B9 plus A-minor B10; the ROOL-like daisy-chain spacing shrinks from 9 nt to 4 nt. · GOLLD assembles into a distinct atomically…; Cryo-electron Microscopy Data Processing; Figure legends

3. 03

   ShownThe 577-nt Clostridium acetobutylicum OLE RNA folds without protein into a two-fold symmetric dimer of parallel A-form helices welded by three intermolecular bridges. Putative OapC and RpsU binding sites are preformed and solvent exposed.

   2.9 Å map with C2 symmetry; 308 nt of the 5′ region modeled per chain with Q-scores above expectation; bridges are four inter-chain A-A pairs (L4), L5/L6/L7 pairing and stacking, and an L9.3 kissing loop; the kink-turn at J4a/6 forms with J7/8, not J5/6; 343-nt Limnospira maxima HEARO RNA was disordered without IsrB. · OLE forms an RNA-only dimer presenting protein…; Cryo-electron Microscopy Data Processing; Figure legends

4. 04

   ShownThe oligomeric states hold far below cryo-EM concentrations and survive heating, while another large RNA, raiA, imaged under similar conditions stays monomeric. This argues that multimerization is not a high-concentration artifact.

   Mass photometry: GOLLD 14 and ROOL 8 copies at 50 nM, OLE dimer at 12.5 nM (∼10 molecules per cell); OLE also dimerized without magnesium when monovalent salt was present; DLS: no detectable monomer for GOLLD (110 nM) or ROOL (140 nM) up to 55 °C, melting at 65 °C; raiA was solved as a 2.9 Å monomer. · Biological relevance of homo-multimerization; Mass photometry; Dynamic Light Scattering of RNA Nanocages; Figure legends

5. 05

   SuggestedComparative and covariation analysis of genomic homolog alignments indicates that intermolecular bridge sites are evolutionarily conserved. This is consistent with selection for RNA-only quaternary assembly.

   Nonredundant hits: 806 GOLLD, 1,596 ROOL, 8,585 OLE; R-scape covariation (E < 0.05) supports the intramolecular stems; palindromic loops OLE L9.3 (GGCC to GAUC or AGCU) and GOLLD L21a (GC/CG) covary at adjacent nucleotides, implying cross-chain pairing (bridges B3, B8); A positions in OLE B1 and GOLLD B6 are highly conserved. · Biological relevance of homo-multimerization; Bioinformatic analysis; section 19