Self-splicing and cyclization mechanisms of the full-length Anabaena pre-tRNA.

Group I introns are catalytic RNAs capable of self-splicing and generating circular RNAs, processes central to RNA metabolism and biotechnology. Yet, full-length ribozyme structures containing entire exon sequences and the structural basis of postsplicing circularization have remained limited. Using cryo-electron microscopy, we resolved multiple conformational states of the full-length Anabaena tRNA(Leu) precursor, capturing key intermediates of splicing and cyclization. In the apo state, the exons preassemble into a mature tRNA-like conformation that promotes P1 helix formation. Transitions through the splicing states involve substantial rearrangements essential for catalysis. Unlike other group I introns, the Anabaena intron circularizes without sequence loss, using its guanosine-binding site as the catalytic center. Mutational analyses confirm that G37 reorientation and a conserved wobble receptor motif precisely position the circularization site, driving efficient cyclization even in engineered PIE systems. These findings uncover unique mechanisms of RNA catalysis and establish structure-based optimization for advancing RNA circularization technologies.