Structural basis for hydrolytic splicing of a circularly permuted group II intron

Group II introns are self-splicing ribozymes that are considered the ancestors of the eukaryotic spliceosome. Unlike canonical group II introns that self-splice to generate linear exons and lariats, circularly permuted (CP) group II introns identified in various bacterial phyla perform back-splicing, resulting in the production of circular RNAs and branched products via branching pathway. Furthermore, CP introns may switch to a hydrolysis pathway resulting in distinct products to differentially regulate retrotransposition. In this study, we present biochemical data and high-resolution cryogenic electron microscopy (cryo-EM) structures of a CP group II intron from Comamonas testosteroni KF-1 (Cte 1), allowing mechanistic dissection of the switch from the branching pathway to the hydrolysis pathway, and enabling reconstruction of both steps of the hydrolysis pathway. The structures reveal that CP group II intron undergoes the hydrolysis pathway upon mutations of the branch point or splice sites (SS) due to rearrangements in the active site. Here, the branching nucleotide in domain D6 is retracted from its catalytically competent conformation, giving way to the nucleophilic water molecule to attack the 5' splice site. Furthermore, we visualized the intermediates of the second splicing step, which reveal the movement of domain D6 out of the way for the 3'-splicing to occur, closely resembling the second-step structures of the branching pathway. Finally, our structures provide direct evidence for domains D1-D3 acting as a scaffold in group II introns. Together, these findings visualize the complete hydrolysis pathway and offer a new strategy to engineer CP group II introns for circular RNA production, with potential applications in both basic research and therapeutic development.