The KH-like CTD of Prp5 is conserved and essential for splicing

Abstract

Pre-mRNA splicing is a fundamental process in eukaryotic gene expression. The RNA helicase Prp5 bridges U1 and U2 snRNPs during spliceosome assembly and enhances splicing fidelity. While the N-terminal domain (NTD) of Prp5 is well characterized, the C-terminal domain (CTD) remains underexplored. Here, we show that CTD deletion is lethal in budding yeast. Structural predictions indicate the CTD adopts a KH-like fold, yet lacks the conserved GxxG loop and RNA-binding surface, and does not bind RNA in vitro. No other KH domain can functionally substitute the Prp5 CTD, indicating a unique role. Genetic analysis reveals that compromising both NTD and CTD simultaneously produces dominant-negative splicing defects, suggesting functional coupling between the two termini. In vitro pull-down assays show the CTD directly interacts with the spliceosomal protein Hsh155 via its HEAT repeat region. Together, our findings establish the Prp5 CTD as a non-canonical KH domain mediating protein-protein interactions critical for splicing. The C-terminal domain of the yeast splicing helicase Prp5 is essential for cell survival and, despite resembling a KH-like RNA-binding fold, it actually interacts with the key spliceosomal protein Hsh155.