Eriocheir sinensis feminization-1c (Fem-1c) and Its Predicted miRNAs Involved in Sexual Development and Regulation

SIMPLE SUMMARY: The Fem-1c gene in Eriocheir sinensis (Henri Milne Edwards, 1854) (EsFem-1c) is related to sex differentiation; however, its functional mechanism is less reported. In this study, qRT-PCR results of EsFem-1c from the intersex crab implied that EsFem-1c plays a role in crab androgenic gland (AG) development. RNAi and scanning electron microscope observations revealed that EsFem-1c influenced sexual development in E. sinensis. A dual-luciferase reporter assay demonstrated that tcf-miR-315-5p binding to the 3′UTR sequence effectively inhibited the translation of the EsFem-1c gene, and tcf-miR-307 binding to the alternative splicing region in the 3′UTR sequence could increase EsFem-1c expression. In summary, this study provides a better understanding of the molecular regulation mechanism of EsFem-1c. ABSTRACT: Feminization-1c (Fem-1c) is important for sex differentiation in the model organism Caenorhabditis elegans. In our previous study, the basic molecular characteristics of the Fem-1c gene (EsFem-1c) in Eriocheir sinensis (Henri Milne Edwards, 1854) were cloned to determine the relationship with sex differentiation. In this study, the genomic sequence of EsFem-1c contained five exons and four introns, with an exceptionally long 3′UTR sequence. The qRT-PCR results of EsFem-1c demonstrated lower tissue expression in the androgenic gland of the intersex crab than the normal male crab, implying that EsFem-1c plays a role in crab AG development. RNA interference experiments and morphological observations of juvenile and mature crabs indicated that EsFem-1c influences sexual development in E. sinensis. A dual-luciferase reporter assay disclosed that tcf-miR-315-5p effectively inhibits the translation of the EsFem-1c gene, influencing male development. An intriguing finding was that miRNA tcf-miR-307 could increase EsFem-1c expression by binding to the alternative splicing region with a length of 248 bp (ASR-248) in the 3′UTR sequence. The present research contributes to a better understanding of the molecular regulation mechanism of EsFem-1c and provides a resource for future studies of the miRNA-mediated regulation of sexual development and regulation in E. sinensis.