The VCX/VCY gene family member VCX3A encodes an RNA-binding protein that modulates the proliferation and motility of spermatogonia GC-1 cells via pre-mRNA splicing

VCX3A , a member of the VCX / VCY gene family, is predominantly expressed in spermatogonia of the male testis. Emerging evidence implicates that mutations or duplication of VCX3A may contribute to nonobstructive azoospermia. Although VCX3A has been characterized as a nuclear RNA-binding protein, its functional role in spermatogonia remains poorly defined. Among the members of the VCX gene cluster, VCX3A showed the highest expression in human testicular tissue. Functionally, exogenous VCX3A promoted the proliferation and migration of the mouse spermatogonial cell line GC-1, whereas knockdown of endogenous VCX3A in NCCIT cells, a human embryonal carcinoma line, suppressed both behaviors. Biophysical analyses revealed VCX3A to be an intrinsically disordered protein that undergoes liquid–liquid phase separation in vitro as well as within the nuclei of GC-1 and NCCIT cells. irCLIP-seq profiling demonstrated that VCX3A binds preferentially to the intronic regions of protein-coding genes, with marked enrichment of an RRDGG motif. IP–MS and co-immunoprecipitation confirmed that VCX3A interacts with the splicing regulator KHDRBS1 and associates with pre-mRNAs transcribed from genes governing cell motility and proliferation, including Lpp and Frmd5 . VCX3A modulated the alternative splicing of these pre-mRNAs, thereby increasing the abundance of specific transcript isoforms. Notably, either deletion of the RGG-like domain of VCX3A or knockdown of Khdrbs1 disrupted VCX3A-mediated splicing regulation and abolished its stimulatory effects on GC-1 cell proliferation and migration. VCX3A is an RNA-binding protein that cooperates with KHDRBS1 in GC-1 cells to coordinately regulate the alternative splicing of pre-mRNAs derived from motility- and proliferation-related genes such as Lpp and Frmd5 , thereby enhancing the proliferative and migratory capacity of GC-1 cells. Our findings provide the direct mechanistic evidence linking VCX3A protein to the post-transcriptional control of spermatogonial cell behavior.