小鼠睾丸组织特异表达基因Rnf148的鉴定及其编码E3泛素连接酶的功能分析

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

目的研究小鼠Rnf148基因表达的时空特异性及其环指结构域的E3泛素连接酶功能。方法提取不同成年小鼠组织、不同胚胎期组织和出生后小鼠睾丸组织的总RNA,通过实时荧光RT-PCR和Northern杂交分析小鼠Rnf148基因的表达谱。构建包含整个Rnf148蛋白的环指结构域与谷胱甘肽-S-转移酶(GST)的融合蛋白原核表达载体,在BL21细菌中诱导表达后,经GST琼脂糖凝胶纯化GST-Rnf148重组蛋白。体外泛素化反应试验检测GST-Rnf148重组蛋白的E3泛素连接酶功能。结果在小鼠13种不同器官组织中,Rnf148mRNA仅存在于睾丸组织中。进一步Northern杂交验证了只在小鼠睾丸组织表达一个1.2kb左右的Rnf148基因mRNA片段。小鼠Rnf148基因在胚胎期及出生后3周内不表达,出生后21d开始表达,25d后达到表达高峰并一直持续表达。实验成功诱导表达并纯化了GST-Rnf148重组蛋白,体外蛋白泛素化反应显示该重组蛋白具有E3泛素连接酶的功能。结论小鼠Rnf148基因特异地表达在出生3周后的睾丸组织中,Rnf148蛋白的环指结构域具有泛素连接酶活性。

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 Rnf148mRNA 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.