Dual roles for the Mss116 cofactor during splicing of the ai5g group II intron

The autocatalytic group II intron ai5γ from Saccharomyces cerevisiae self-splices under high-salt conditions in vitro, but requires the assistance of the DEAD-box protein Mss116 in vivo and under near-physiological conditions in vitro. We recently showed that Mss116 influences the folding mechanism in several ways (1). By comparing intron precursor RNAs with long (∼300 nt) and short (∼20 nt) exons, we observe that long exon sequences are a major obstacle for self-splicing in vitro. Kinetic analysis indicates that Mss116 not only mitigates the inhibitory effects of long exons, but also assists folding of the intron core. Moreover, a mutation in conserved Motif III that impairs unwinding activity (SAT → AAA) only affects the construct with long exons, suggesting helicase unwinding during exon unfolding, but not in intron folding. Thus, Mss116 displays a repertoire of diverse activities for resolving the folding problems of large RNAs. Here, we show further data that illustrate the complicated kinetics of protein assisted group II intron splicing.