Structural basis of circularly permuted group II intron self-splicing

The group II introns are self-splicing ribozymes intervened between two exons that undergo excision and exon ligation through lariat formation, which are considered as ancestral forms of spliceosomes. Circularly permuted (CP) group II introns comprise of rearranged structural domains separated by two tethered exons have been artificially designed long ago to generate branched intron and circular exon products via back-splicing, whose natural occurrence has also been recently identified. Although extensive studies have elucidated mechanisms of both forward and reverse splicing processes of canonical group II introns, structural and mechanistic understandings of circular RNA (circRNA) generation by CP introns remain elusive. Here we resolve cryo-electron microscopy structures of a natural CP group II intron with 3'- and 5'-exons at 2.65-2.97 Å resolution, which represent different states associated to two steps of the back-splicing process. Novel tertiary interactions are identified to play allosteric effects on catalytic activity. The branching helix D6 undergoes a 65º conformational change enabled by tertiary interactions with D6 terminal T-loop after the branching reaction to allow docking of 3'-exon for ligation. Local shifts of peripheral domains, exon binding sites, and metal ions in the catalytic core, including a novel metal ion M ₃₅ that stabilizes the 5'-exon, are also observed between two steps of splicing. These results elucidate the dynamics of CP group II intron back-splicing process and mechanism of circRNA generation with potential applications in circRNA research and therapeutics.