Group I introns are catalytic RNAs capable of self-splicing, yet structural insights into full-length precursor RNAs and post-splicing circularization have been limited. Here, we present cryo-EM structures of the Azoarcus pre-tRNAIle system across key catalytic stages, including the full-length precursor in Pre-1S conformations, the linear intron, and the circular introns, at 2.8-3.3 Å resolution. These structures reveal a preformed P1 helix, register shifts during splicing, and a conformational flip of G37 that stabilizes the circularization site. Biochemical analyses confirm a two-step circularization mechanism, generating a secondary circular product via an alternative ligation site. Together, our results provide an atomic-level view of a group I intron system through splicing and circularization. This work uncovers structural principles governing RNA conformational dynamics, catalysis, and circular RNA formation, with broad implications for ribozyme engineering. Cryo-EM structures of the Azoarcus group I intron capture its progression from pre-splicing to circular RNA products, revealing coordinated RNA rearrangements that drive catalysis and a two-step circularization pathway.
Hong et al. (2026) studied this question.