| Summary: | Eukaryotic genes are frequently interrupted by non-coding introns, which are removed from precursor mRNAs by the spliceosome through two sequential transesterification reactions. The spliceosome is a dynamic ribonucleoprotein machine that catalyzes the two chemical steps of splicing using a single catalytic core. Importantly, the catalytic core of the spliceosome is not formed in the absence of an intron and instead assembles de novo on a splicing substrate through a series of rearrangements that are dependent on members of the DExD/H-box ATPase family. These ATPases promote splicing by coupling the energy of ATP hydrolysis to spliceosomal remodeling events and several of these factors contribute to the fidelity of splicing by discriminating against suboptimal splicing substrates, though relatively little is known about the physical rearrangements promoted by DExD/H-box ATPases.
Here, we investigate the function and mechanism of spliceosomal DExD/H-box ATPases at the catalytic stage of splicing through a combination of ensemble and single molecule techniques. We demonstrate a novel function for two ATPases, Prp16p and Prp22p, in enabling the spliceosome to search for alternative branch sites and 3' splice sites, respectively. Prp16p and Prp22p separate candidate splice sites to both promote usage of optimal sites and antagonize usage of suboptimal sites. Additionally we provide evidence that Prp16p and Prp22p function through a mechanism involving translocation along the splicing substrate from downstream of the catalytic core of the spliceosome. Importantly, Prp16p and Prp22p need not translocate through interactions targeted for disruption, suggesting that they may act as molecular winches that pull on the substrate. Finally, we show that a third ATPase Prp43p acts irreversibly dissociate unreactive splicing substrates from the spliceosome through a pathway that parallels release of a genuine intron following its excision.
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