The DEAH Box ATPases Prp16 and Prp43 Cooperate to Proofread 5′ Splice Site Cleavage during Pre-mRNA Splicing

SummaryTo investigate the mechanisms underlying accurate pre-mRNA splicing, we developed an in vitro assay sensitive to proofreading of 5′ splice site cleavage. We inactivated spliceosomes by disrupting a metal-ligand interaction at the catalytic center and discovered that, when the DEAH box ATPase Prp16 was disabled, these spliceosomes catalyzed 5′ splice site cleavage but at a reduced rate. Although Prp16 does not promote splicing of a genuine substrate until after 5′ splice site cleavage, we found that Prp16 can associate with spliceosomes before 5′ splice site cleavage, consistent with a role for Prp16 in proofreading 5′ splice site cleavage. We established that Prp16-mediated rejection is reversible, necessitating a downstream discard pathway that we found requires the DEAH box ATPase Prp43, a spliceosome disassembly factor. These data indicate that spliceosomes distinguish slow substrates and that the mechanisms for establishing the fidelity of 5′ splice site cleavage and exon ligation share a common ATP-dependent framework.

Graphical AbstractFigure optionsDownload high-quality image (179 K)Download as PowerPoint slideHighlights
► DEAH box ATPase Prp16 competes with and thereby proofreads 5′ splice site cleavage
► Prp16 antagonizes a substrate when the substrate splices slowly
► Prp16-mediated rejection is reversible
► DEAH box ATPase Prp43 discards stalled pre-mRNA from the spliceosome