Abstract
RNA-binding protein EWS, a member of the FET (FUS, EWS, TAF15) family, contributes to mRNA biogenesis through roles in transcription, splicing, and RNA transport. Despite evidence linking EWS to spliceosomal complexes, its interactions with spliceosome-associated cyclophilins remain unclear. Here, we describe the first structural and biochemical characterization of the EWS low-complexity domain (EWSLCD) interaction with the spliceosomal cyclophilin PPIL1. Nuclear magnetic resonance (NMR) titration experiments reveal that the proline-rich PxxP motifs of EWSLCD engage the catalytic face of PPIL1, forming low-affinity “fuzzy” complexes. Notably, this interaction is undetected in an EWS construct containing the RNA recognition motif (RRM) and RGG2 domain, suggesting that PxxP accessibility or local context is critical for PPIL1 binding. Phase separation assays demonstrate that PPIL1 is recruited into EWSLCD condensates under physiological salt conditions, while altering condensation properties at lower salt concentrations. These findings support a model where EWS is recruited to spliceosomal cyclophilins, potentially influencing splicing and nascent mRNA processing. This study underscores the functional importance of proline-rich motifs within EWS and highlights the potential of spliceosomal cyclophilins as both catalytic and structural partners. Our work provides a foundation for exploring the mechanism by which cyclophilins modulate EWS biology and for developing novel therapeutic strategies targeting EWS-cyclophilin interactions in cancer.