The G-patch protein Cwf28 interacts with the RNA helicase Cdc28 in catalytically active spliceosomes in Schizosaccharomyces pombe.

G-patch proteins are emerging as key regulatory cofactors of DEAH-box RNA helicases involved in pre-mRNA splicing, yet the functions of many family members remain poorly understood. Here, we characterize the conserved G-patch domain-containing protein Cwf28 from Schizosaccharomyces pombe, an essential factor with a previously unclear molecular function, and define its role within the spliceosome. Tandem affinity purification coupled with mass spectrometry revealed that Cwf28 predominantly associates with components of the Prp19 complex (NTC) and factors involved in the catalytic activation and progression of the spliceosome, placing it within catalytically active spliceosomal assemblies. Gene ontology analysis showed enrichment of the Cwf28 interactome in factors involved in spliceosome assembly, activation, and catalytic remodeling. Notably, the DEAH-box RNA helicase Cdc28, the ortholog of human DHX16 and Saccharomyces cerevisiae Prp2, was identified as the most abundant interactor. Further analysis demonstrated that Cwf28 interacts with Cdc28 via its conserved G-patch domain, and domain mapping confirmed that this interaction is G-patch domain dependent. Together, these findings identify Cwf28 as a component of catalytically active spliceosomes and suggest a potential role for Cwf28 in modulating the activity of the RNA helicase Cdc28, providing insight into conserved mechanisms underlying RNA helicase regulation during splicing.