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

• Published in: Molecular cell 2010-08, Vol.39 (3), p.385-395
• Main Authors: Koodathingal, Prakash, Novak, Thaddeus, Piccirilli, Joseph A., Staley, Jonathan P.
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphatases - genetics; Adenosine Triphosphatases - metabolism; Adenosine Triphosphate - genetics; Adenosine Triphosphate - metabolism; Adenosinetriphosphatase; DEAD-box RNA Helicases - genetics; DEAD-box RNA Helicases - metabolism; PROTEINS; RNA; RNA Helicases - genetics; RNA Helicases - metabolism; RNA Precursors - genetics; RNA Precursors - metabolism; RNA Splice Sites - physiology; RNA Splicing - physiology; RNA Splicing Factors; RNA, Fungal - genetics; RNA, Fungal - metabolism; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - metabolism; Spliceosomes - genetics; Spliceosomes - metabolism
• ISSN: 1097-2765; 1097-4164
• DOI: 10.1016/j.molcel.2010.07.014

To 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. [Display omitted] ► 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