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Updated: May 29, 2026
Enhanced Crosslinking Immunoprecipitation (eCLIP) Method for Efficient Identification of Protein-bound RNA in Mouse Testis
Published on: May 10, 2019
Multi-omic screening identifies RBMXL3 as a primate-specific RNA-binding protein and candidate regulator of RNA
Agata J Barszcz1, Katarzyna Tutak1,2, Joanna Zyprych-Walczak3
1Institute of Human Genetics, Polish Academy of Sciences, Poznan, Poland.
Background:
RBMXL3 is a primate-specific gene localized on the X chromosome, of which the expression is detectable mainly in the male gonad. So far, very little is known about the RBMXL3 protein function and its molecular interactions. However, recent reports mention the RBMXL3 gene in the context of human spermatogenesis, cancer, and a breathing disorder that affects newborns. In this study, we investigate the RBMXL3's molecular network on a genome-wide scale using the human seminoma cell line (TCam-2) as a male germline in vitro model.
Methods:
By using transcriptomic (RNA sequencing [RNA-seq] and enhanced crosslinking and immunoprecipitation [eCLIP]) and proteomic (co-immunoprecipitation coupled with mass spectrometry, (Co-IP-MS)) approaches, we show RBMXL3's importance in RNA metabolism. In addition, western blot, quantitative polymerase chain reaction with reverse transcription (qRT-PCR), immunostaining, and confocal imaging were used to investigate the function of RBMXL3. Finally, we used a plasmid-based L1 retrotransposition assay to demonstrate the suppressive effect of RBMXL3 on human Long Interspersed Nuclear Element-1 (LINE-1, L1) retrotransposition.
Results:
Our RNA-seq data show that RBMXL3 expression drives gene expression changes and influence alternative splicing in human cells. Moreover, by performing eCLIP, we provide proof that RBMXL3 binds to a wide range of RNA transcripts. In addition, we confirmed the nuclear localization of RBMXL3 in TCam-2 cells and its presence in spermatogonia and spermatocytes within the human testis. Finally, we report for the first time that RBMXL3 restricts human LINE-1 retrotransposition.
Conclusions:
Our findings for the first time identify primate-specific RBMXL3 protein as a new upstream regulator of RNA metabolism, characterized by broad RNA-binding activity in human TCam-2 cells. Finally, we show that RBMXL3 expression heavily reduces LINE-1 retrotransposition in human cells, underlying RBMXL3 importance in maintaining genome integrity. Our data suggest that RBMXL3 may contribute to the regulation of transcriptome dynamics in male germ cells, while its broader functional implications remain to be determined.
