LAUSR

Abstract Group II introns can self-splice from RNA transcripts through branching, hydrolysis and circularization, being released as lariats, linear introns and circles, respectively. In contrast to branching, the circularization pathway is mostly based on assumptions and has been largely overlooked. Here, we address the molecular details of both transesterification reactions of the group II intron circularization pathway in vivo. We show that free E1 is recruited by the intron through base pairing interactions and that released intron circles can generate free E1 by the spliced exon reopening reaction. The first transesterification reaction was found to be induced inaccurately by the 3′OH of the terminal residue of free E1 at the 3′ splice site, producing circularization intermediates with heterogeneous 3′ ends. Nevertheless, specific terminal 3′OH, selected by a molecular ruler, was shown to precisely attack the 5′ splice site and release intron circles with 3′–5′ rather than 2′–5′ bonds at their circularization junction. Our work supports a circularization model where the recruitment of free E1 and/or displacement of cis-E1 induce a conformational change of the intron active site from the pre-5′ to the pre-3′ splice site processing conformation, suggesting how circularization might initiate at the 3′ instead of the 5′ splice site.