Prp22, a DExH-box RNA helicase, plays two distinct roles in yeast pre-mRNA splicing

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Abstract
In order to assess the role of Prp22 in yeast pre‐mRNA splicing, we have purified the 130 kDa Prp22 protein and developed an in vitro depletion/reconstitution assay. We show that Prp22 is required for the second step of actin pre‐mRNA splicing. Prp22 can act on pre‐assembled spliceosomes that are arrested after step 1 in an ATP‐independent fashion. The requirement for Prp22 during step 2 depends on the distance between the branchpoint and the 3′ splice site, suggesting a previously unrecognized role for Prp22 in splice site selection. We characterize the biochemical activities of Prp22, a member of the DExH‐box family of proteins, and we show that purified recombinant Prp22 protein is an RNA‐dependent ATPase and an ATP‐dependent RNA helicase. Prp22 uses the energy of ATP hydrolysis to effect the release of mRNA from the spliceosome. Thus, Prp22 has two distinct functions in yeast pre‐mRNA splicing: an ATP‐independent role during the second catalytic step and an ATP‐requiring function in disassembly of the spliceosome.