The RNA helicase DDX53 (CAGE) contributes to RNA metabolism in a human germ cell model

BackgroundDDX53 (DEAD-box helicase 53, known also as CAGE) is an intronless gene on the X chromosome, which expression shows strong testis specificity. It belongs to the group of cancer-testis (CT) antigens, with most studies to date focusing on its role in cancer, but the precise biological function of DDX53 remains unclear. Previous reports identifying rare DDX53 variants in infertile men provided the rationale for investigating the role of DDX53 in the context of human spermatogenesis. By using the human seminoma cell line (TCam-2) as an in vitro male germline model, we aimed to investigate the function and molecular targets of DDX53. MethodsIn our study, we used transcriptomic and proteomic approaches (RNA sequencing (RNA-seq), enhanced crosslinking and immunoprecipitation (eCLIP), and Co-immunoprecipitation coupled with Mass Spectrometry (Co-IP-MS)) to investigate the role of DDX53 in the context of human spermatogenesis. By using modified TCam-2 cells to express either DDX53-FLAG or GFP-FLAG, we identified regulated genes, RNA targets, and potential protein interactors of DDX53. In addition, we employed Western Blot, RT-qPCR, immunostaining, and confocal microscopy to gain deeper insight into the DDX53 protein. ResultsOur RNA-seq and eCLIP data provide evidence that DDX53 regulates gene expression changes and directly interacts with a broad spectrum of RNA transcripts. Moreover, for the first time, we described RNAs and protein interactors of DDX53 in the context of spermatogenesis. Subcellular localization analysis by confocal microscopy indicated a predominantly cytoplasmic distribution of DDX53, with partial nuclear presence in TCam-2 cells. We also identified DDX53-positive structures that may correspond to germ granule-like assemblies, although their precise nature remains to be determined. Additionally, we confirmed DDX53 presence in human testis using a specific, commercially available anti-DDX53 antibody. ConclusionsThis studys data indicate that DDX53 protein acts as a regulator of RNA metabolism in human cells. Collectively, we show that it participates in transcriptome regulation (including splicing) in male germ cells and exhibits transcriptome-wide RNA interactions, but its wider biological role remains to be clarified.