Pre-mRNA splicing is an essential step in eukaryotic gene expression, wherein interrupting sequences (“introns”) are excised from newly transcribed precursor messenger RNA (pre-mRNA). At the same time, expressed sequences (“exons”) are ligated together to form mature mRNA that codes for a given polypeptide product. As errors in splicing may lead to insertion of aberrant intronic sequence or deletion of exonic sequence, fidelity in splicing is necessary to ensure proper gene expression. This fidelity is maintained by a set of ATPases belonging to the SF2 family of nucleic acid helicases. Here, I focus on two of these helicases: the DEAH-box ATPases Prp16 and Prp43. Through characterization of disease-correlated mutations in DHX38, the gene encoding human Prp16, I present evidence for a conserved motif that mediates an autoinhibitory interaction between the N-terminal and helicase domains of Prp16. I also present evidence for a novel role for Prp43 in discarding splicing intermediates that are deficient in transition between the first and second catalytic steps of pre-mRNA splicing. These data deepen our knowledge of how DEAH-box ATPases ensure faithful gene expression.