Fbos, a Novel Oocyte-specific Protein, Interacts with Proteins Important for Oocyte Development in Rainbow Trout (Oncorhynchus mykiss).

Abstract

Oogenesis is characterized by a series of developmentally regulated events, which result in the matured oocyte that can give rise to a new organism after fertilization. Oocyte-specific genes play critical roles in oogenesis; however, the molecular details of oocyte-specific genes are poorly defined. Through analysis of expressed sequence tags (ESTs) from a rainbow trout oocyte cDNA library, we identified and characterized a novel oocyte-specific gene coding for an F-box protein (Fbos, F-box oocyte-specific). F-box proteins are components of ubiquitin-ligase complexes, which bind substrates for uiquitin-mediated proteolysis. F-box proteins have also been associated with cellular functions such as signal transduction and regulation of the cell cycle. To define the molecular mechanisms of Fbos-dependent biological processes during oogenesis in rainbow trout, we used Fbos as bait in a yeast two-hybrid screen system, along with a rainbow trout ovary cDNA library. More than 300 yeast colonies were able to grow on nutritional selective plates; 18 clones displayed strong LacZ activity as shown by colony lift filter assay for β-galactosidase activity. After verifying the interactions by reintroduction of the candidate prey plasmids into yeast reporter strain containing Fbos plasmid, 8 plasmids were rescued successfully and subjected to DNA sequence analysis. The sequencing data demonstrated that Fbos protein interacts with a number of proteins including histone H3.3, adenylate kinase 2, ribosomal protein L36, oocyte protease inhibitor 1, zona pellucida protein 2 (ZP2) and tissue inhibitor of metalloproteinase 2 (TIMP-2). All 5 proteins showed significant β-galactosidase activities at different levels compared to the control in a quantitative β-galactosidase assay, indicating different binding affinities of these proteins with Fbos protein. Some of these proteins like ZP2 and TIMP-2 are known to play critical roles in oocyte development in various species. Our results suggest that through protein-protein interactions, the novel oocyte-specific F-box protein may play an important role in early oocyte development by regulating other critical proteins involved in oogenesis in rainbow trout. (poster)