Structures of a natural circularly permuted group II intron reveal mechanisms of branching and backsplicing

Circularly permuted (CP) group II introns, identified in various bacteria phyla, swap domains D5 and D6 near the 5′ end and have reversed splice sites (SSs), leading to backsplicing and circular RNA formation. In this study, we present multiple high-resolution cryo-electron microscopy structures of a natural CP group II intron from Comamonas testosteroni KF-1 (Cte 1), elucidating the molecular mechanisms of branching and backsplicing. During branching, the 5′ SS is positioned by an auxiliary sequence (AUX)-enhanced interaction between the exon-binding site and intron-binding site (IBS) and stacks on the branch-site adenosine within D6, allowing the attacking 2′-OH group to coordinate with a metal ion in the active center. In backsplicing, the 3′ SS is aligned with the branching step, leaving IBS in the active center, stabilized by base pairing with the AUX, which enables the free 3′-end hydroxyl group to directly attack the scissile phosphate of 3′ SS. Furthermore, a groove in Cte 1 may stabilize the circular RNA. These findings highlight a conserved catalytic mechanism for canonical group II introns, albeit facilitated by the versatile AUX, opening avenues for designing potent ribozymes producing circular RNAs. This study reports six cryo-electron microscopy structures of a natural group II intron, uncovering backsplicing dynamics, auxiliary sequence roles and intron space critical for circular RNA (circRNA) formation. The findings might guide improvements in circRNA production for various applications.