Constraining errors in splice site choice

Abstract

While even single nucleotide errors in pre‐mRNA splicing can lead to catastrophic consequences, our understanding of fidelity mechanisms in splicing is poor. Genetics studies have established the importance of fidelity mechanisms in splicing and implicated 3 of the 8 spliceosomal DExD/H box ATPases in competing with splicing to enable kinetic proofreading. However, due to a paucity of in vitro assays, it has remained unclear how specificity is established by kinetic proofreading and how rejected substrates are discarded. We have established in vitro that each chemical step in splicing is proofread by a DEAH box ATPase – 5′ splice site cleavage is proofread by Prp16 and exon ligation is proofread by Prp22. In a kinetic proofreading mechanism, specificity can be achieved if the rate of the ATPase and/or the rate of the proofread step varies with the authenticity of the substrate. We have found evidence that both Prp16 and Prp22 enhance specificity by discriminating against slowly splicing substrates. Both Prp16 and Prp22 reject substrates reversibly, necessitating an independent discard activity that we attribute to the DEAH box ATPase Prp43, which normally functions to discard a genuine intron after excision from the precursor. These findings establish that both chemical steps in splicing are proofread by a common ATP‐dependent framework and support a general role for DExD/H box ATPases in fidelity. Support: ACS, NIH.