Do DEAD-Box proteins promote group II intron splicing without unwinding RNA?

The DEAD-box protein Mss1 16p promotes group 11 intron splicing in vivo and in vitro. Here we explore two hypotheses for how Mss1 16p promotes group 11 intron splicing: by using its RNA unwinding activity to act as an RNA chaperone or by stabilizing RNA folding intermediates. We show that an Mss1 16p mutant in helicase motif III (SAT/AAA), which was reported to stimulate splicing without unwinding RNA, retains ATP-dependent unwinding activity and promotes unfolding of a structured RNA. Its unwinding activity increases sharply with decreasing duplex length and correlates with group 11 intron splicing activity in quantitative assays. Additionally, we show that Mss1 16p can promote ATP-independent RNA unwinding, presumably via single-strand capture, also potentially contributing to DEAD-box protein RNA chaperone activity. Our findings favor the hypothesis that DEAD-box proteins function in group 11 intron splicing as in other processes by using their unwinding activity to act as RNA chaperones.