Identification and Functional Analysis of a Testis-specific E3 Ubiquitin Ligase Gene Rnf148 in Mouse

Objective?To investigate the temporal and spatial features of mouse Rnf148 gene expression and the function of RING finger domain of Rnf148 protein. Methods?The whole RNA was extracted from different tissues of adult mice, embryo in four developmental stages, and testes of postnatal mice respectively. RT-PCR and Northern blotting analysis were used to investigate the expression of Rnf148 gene in the above tissues. The in vitro expression vector for GST-Rnf148 fused protein was constructed, which encompassing the entire RING domain of Rnf148 protein. GST-Rnf148 fused protein was expressed in Escherichia coli. BL21(DE3) cells and purified with glutathione-sepharose 4B. In vitro ubiquitination assay was performed to analyze whether GST-Rnf148 fused protein possess the function of E3 ubiquitin ligase. Results?The Mice Rnf148 mRNA expression was only observed in testis, and Northern blotting confirmed that there was only one 1.2 kb mRNA band present in mice testis. Rnf148 mRNA started to appear in the testis of day 21 mice, and then increased dramatically and reached to the highest level in day 25, and continued to express thereafter. GST-Rnf148 fused protein was induced and purified, in vitro ubiquitination reaction showed that the recombinant protein has E3 ubiquitin ligase activity. Conclusion?Rnf148 gene is specifically expressed in mice testis.