Identification and Functional Analysis of E3 Ubiquitin Ligase g2e3 in Chinese Tongue Sole, Cynoglossus semilaevis

Gametogenesis, the intricate developmental process responsible for the generation of germ cells (gametes), serves as a fundamental prerequisite for the perpetuation of the reproductive cycle across diverse organisms. The <i>g2e3</i> enzyme is a putative ubiquitin E3 ligase implicated in the intricate regulatory mechanisms underlying cellular proliferation and division processes. The present study delves into the function of G2/M phase-specific E3 ubiquitin protein ligase (<i>Cs-g2e3</i>) in gametogenesis in Chinese Tongue Sole (<i>Cynoglossus semilaevis</i>). Sequence analysis shows that the <i>Cs-g2e3</i> mRNA spans 6479 bp, encoding a 733 amino acid protein characterized by three conserved structural domains: PHD, RING, and HECT-typical of HECT E3 ubiquitin ligases. The predominant expression of <i>Cs-g2e3</i> in the gonad tissues is further verified by qPCR. The expression profile of <i>Cs-g2e3</i> in the gonads of the Chinese Tongue Sole is analyzed at different ages, and the results show that its expression peaks at 8 months of age and then begins to decline and stabilize. It is noteworthy that the expression level remains significantly elevated compared to that observed during the juvenile period. In situ hybridization shows that the mRNA of <i>Cs-g2e3</i> is mainly localized in the germ cells of the ovary and the testis. RNA interference experiments show that the knockdown of <i>Cs-g2e3</i> in ovarian and testicular germ cell lines significantly downregulates the expression of key genes involved in oogenesis (e.g., <i>sox9</i> and <i>cyp19a</i>) and spermatogenesis (e.g., <i>tesk1</i> and <i>piwil2</i>), respectively. Furthermore, the analysis of mutations in the transcription factor binding sites reveals that mutations within the Myogenin, YY1, and JunB binding sites significantly impact the transcriptional activity of the <i>Cs-g2e3</i> gene, with the mutation in the YY1 binding site exhibiting the most pronounced effect (<i>p</i> < 0.001). This study contributes to a deeper understanding of the tissue-specific expression patterns of <i>Cs-g2e3</i> across various tissues in <i>Cynoglossus semilaevis</i>, as well as the potential regulatory influences of transcription factors on its promoter activity. These findings may facilitate future research endeavors aimed at elucidating the expression and functional roles of the <i>Cs-g2e3</i> gene.