Fertilization and Early Embryogenesis
(M319) INVOLVEMENT OF AN OOCYTE-SPECIFIC NOVEL GENE, OOG1 , IN ZYGOTIC GENOME ACTIVATION VIA RAS SIGNALING PATHWAY.
Tsukamoto, Satoshi1, Ihara, Ryo1, Aizawa, Akira2, Imai, Hiroshi1, Minami, Naojiro1, 1 Kyoto University, Kyoto, Japan2 Maebashi Institute of Animal Science, Livestock Improvement Association JAPAN, Inc., Maebashi, Japan
ABSTRACT- We previously identified an oocyte-specific novel gene, Oogenesin (Oog1) that encodes 326 amino acids containing a leucine zipper and a leucine rich repeat which appears to be necessary for protein-protein interactions. More interestingly, Oog1 protein localized in nuclei at late one-cell and early two-cell stages, the time when the zygotic genome activation occurs in mice. The zygotic genome activation relies on transcripts and proteins stored in oocyte during oogenesis. However, the molecular mechanisms governing these events are largely unknown. Thus, we hypothesized that Oog1 plays some roles in zygotic genome activation during early preimplantation development. Recently, another group identified three additional Oog1-like genes (Oog2, 3, 4) containing a leucine rich repeat, speculating that this family function via mediating protein-protein interactions. In the present study, to identify the interacting proteins of Oog1, we carried out a yeast two-hybrid screening using a GV oocyte cDNA library and found that Ral guanine nucleotide dissociation stimulator (RalGDS) is potential binding partner of Oog1. RalGDS is one of the Ras effector proteins, exchanging a GDP-bound inactive state Ral to a GTP-bound active state Ral in a Ras-dependent manner. Interaction between RalGDS and Oog1 was confirmed by immunoprecipitation and co-localization using co-transfected mammalian cell lines. We also showed by RT-PCR that RalGDS transcript is detected in GV oocytes and preimplantation embryos until the end of the four-cell stage. Furthermore, we revealed that RalGDS protein was localized in the cytoplasm in oocytes and preimplantation embryos. Interestingly, the protein appeared in the nucleus rather than the cytoplasm between late one-cell and late two-cell stages. The patterns of RalGDS expression and localization are quite similar to that of Oog1, suggesting that RalGDS-Oog1 complex is formed in vivo. Additionally, in the same screening, we have found another potential binding partner of Oog1, Ras P21 protein activator 4 (Rasa4). Rasa4 has RasGAP activity that allows Ras to return to its GDP-bound inactive state. This is the first report concerning Ras/RalGDS/Ral pathway in mammalian embryos, and this finding may be a clue to elucidate the mechanisms of zygotic genome activation.
KEY WORDS: Oog1, Yeast two-hybrid, RalGDS, Zygotic genome activation