The DEAH box ATPases Prp16 and Prp43 cooperate to proofread 5' splice site cleavage during pre-mRNA splicing.

ABSTRACT: 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.

SUBMITTER: Koodathingal P 

PROVIDER: S-EPMC3722364 | biostudies-literature | 2010 Aug

REPOSITORIES: biostudies-literature