Cryo-EM structures reveal a conserved architecture for raiA noncoding RNA.

RaiA motif RNA is a family of bacterial noncoding RNAs (ncRNAs) found in over 2700 bacterial species. Although its cellular abundance is comparable to that of rRNAs and tRNAs in the human pathogen Clostridioides difficile and its knockout results in pronounced phenotypes, its function remains unknown. Sequence conservation analysis predicted a consensus secondary structure of raiA motif RNA with several major subtypes that differ in the number and composition of stems. Here, we present cryogenic electron microscopy (cryo-EM) structures of three raiA motif RNAs from three bacterial species, one from each subtype, at 3.0-3.5 Å resolution, as well as a minimal variant with 113 nucleotides at ∼8 Å resolution. Comparison of the structures reveals a conserved architecture, with a compact core comprising stems P3a-P3b bent by an asymmetric internal loop, P4, pseudoknot 1 (PK1), and PK2 with unusual tertiary interactions. While most of the peripheral stems vary, the length, structure, and tertiary interactions of the closing P1 are remarkably conserved, suggesting an essential role. Our study defines the conserved structural framework of raiA motif RNAs and provides a foundation for structure-based functional studies. This work also highlights the utility of cryo-EM for de novo structure determination of ncRNAs.