The DExH-box NTPase/helicase Prp22p plays two important roles in pre-mRNA splicing. It promotes the second transesterification reaction and then catalyzes the ATP-dependent release of mature mRNA from the spliceosome. Evidence that helicase activity is important emerged from the analysis of Prp22p motif III (SAT) mutations that uncouple the NTPase and helicase activities. We find that S635A and T637A hydrolyse ATP, but are defective in unwinding duplex RNA and releasing mRNA from the spliceosome. The S635A mutation is lethal in vivo at </=30 degrees C and results in slow growth at 34-37 degrees C. Further insights into helicase action during splicing were gleaned by isolating and characterizing intragenic suppressors of prp22-S635A. Biochemical analysis of the S27 suppressor protein showed that a second mutation of Val539 to Ile in motif Ia revived the helicase activity of the S635A mutant together with the ability to catalyze mRNA release. These findings underscore the tight correlation of RNA unwinding and spliceosome disassembly and demonstrate how suppressor analysis can be used to dissect the subtle internal domain dynamics of helicase action.