Abstract
The self-splicing activity of group II introns relies on forming complex three-dimensional structures. While crystallographic and cryogenic electron microscopy studies provide snapshots of the group II intron tertiary folds during splicing, they fall short in capturing the inherent dynamics of group II introns. To address this, we illuminate the critical structure-function relationship of group II introns by employing single-molecule Förster Resonance Energy studies and fluorescent cleavage assays. We first elucidate the dynamic folding trajectory of group II introns during splicing. This involves exploring how the RNA molecule rearranges itself during each splicing stage. Secondly, we dissect the roles played by the individual exon-intron binding sites in the folding and splicing of group II introns. The splicing of the Sc.ai5γ group II intron in baker's yeast depends on the DEAD-box RNA helicase Mss116, its sole known protein cofactor. In comparison, the yeast nuclear spliceosome requires approximately 90 proteins for the same function. Given the evolutionary relationship between these systems, we hypothesise the existence of additional group II intron cofactors. To investigate this, we first explore the localisation of recently identified potential group II intron binding proteins. Subsequently, we delve deeper into the RNA-protein interaction between Sc.ai5γ and its key cofactor, Mss116.
Fazliji, Besim