Role of the nucleoplasmin 2 C‐terminal domain in the formation of nucleolus‐like bodies in mouse oocytes

Abstract

Nucleolus-like bodies (NLBs) are characteristic structures found in the germinal vesicles of mammalian oocytes. Although these structures are essential for embryonic development, their composition, precise function, and mechanism of formation have not been elucidated. Here, we used immunoblotting and EGFP fusion protein fluorescence to demonstrate that murine nucleoplasmin 2 (NPM2) is a component of mouse NLBs and that the targeting of NPM2 to NLBs is regulated by a lysine-rich, 16-aa C-terminal motif (K-rich motif). When the K-rich motif was fused to another nuclear protein, MafG, the resultant fusion protein accumulated in NLBs but not in the nucleoli of somatic cells, suggesting that the K-rich motif functions to target NPM2 specifically to NLBs. To investigate the role of the K-rich motif in NLB formation, we replaced the endogenous NPM2 in growing oocytes with a mutant NPM2 protein lacking the K-rich motif (NPM2(C16del)). Growing oocytes surrounded by granulosa layers were coinjected with NPM2(C16del) mRNA and with small-interfering RNA targeting NPM2 (siNpm2), which was used to degrade the endogenous NPM2 mRNA. After culture in vitro, the NLBs in the resulting full-grown oocytes were significantly smaller than those in control oocytes that had been coinjected with siNpm2 and NPM2 mRNA, indicating that the K-rich motif is necessary for NLB development. Together, these results suggest that NPM2 targeting of NLBs is regulated by the K-rich motif and is essential for the formation of NLBs.